Effects of In Utero and Lactational Exposure to New Generation Green Plasticizers on Adult Male Rats: A Comparative Study With Di(2-Ethylhexyl) Phthalate

Effects of In Utero and Lactational Exposure to New Generation Green Plasticizers on Adult Male... Abstract Di(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, is a ubiquitous environmental contaminant and may act as an endocrine disruptor. Early life exposures to DEHP may result in anti-androgenic effects, impairing the development of the male reproductive tract. However, data on the long-lasting consequences of such DEHP exposures on adult male reproductive function are still rare and discrepant. Previously, we identified 2 novel plasticizers, 1,4-butanediol dibenzoate (BDB) and dioctyl succinate (DOS), as potential substitutes for DEHP that did not reproduce classically described endocrine disrupting phenotypes in prepubertal male offspring after maternal exposure. Here, we investigated the consequences of in utero and lactational exposure to BDB and DOS on adult male rat reproductive function in a comparative study with DEHP and a commercially available alternative plasticizer, 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH). Timed pregnant Sprague Dawley rats were gavaged with vehicle or a test chemical (30 or 300 mg/kg/day) from gestation day 8 to postnatal day 21. While DEHP exposure (300 mg/kg/day) significantly increased epididymal weight in the adult, exposure to DINCH, BDB, or DOS did not affect reproductive organ weights, steroid levels, or sperm quality. Using a toxicogenomic microarray approach, we found that adult testicular gene expression was affected by exposure to the higher dose of DEHP; transcripts such as Nr5a2, Ltf, or Runx2 were significantly downregulated, suggesting that DEHP was targeting estrogen signaling. Lesser effects were observed after treatment with either DINCH or BDB. DOS exposure did not produce such effects, confirming its potential as a responsible substitute for DEHP. endocrine disruptors, testis, epididymis, androgens, estrogens, sperm Phthalates are plasticizing agents used as emollients, matrices, solvents, and excipients in numerous industrial applications (Heudorf et al., 2007). Among them, di(2-ethylhexyl) phthalate (DEHP) is the most commonly used additive to provide flexibility to polyvinyl chloride (PVC) and is found in products such as construction materials, household products, toys, cosmetics, food packaging, and medical tubing (Schettler, 2006). Since phthalates are not covalently bound to their supports, they leach into the environment over time (Erythropel et al., 2014; Thomas and Thomas, 1984), resulting in widespread human exposure (Hauser and Calafat, 2005; Koch et al., 2003; Wittassek et al., 2011). Biomonitoring studies reveal their presence in several body fluids, including blood, urine (Silva et al., 2004a), semen (Duty et al., 2003), amniotic fluid (Silva et al., 2004b), umbilical cord blood (Latini et al., 2003), and breast milk (Calafat et al., 2004). Over the past few decades, phthalates, including DEHP, have been established to act as endocrine disrupting compounds, producing adverse effects in various in vitro and in vivo models in both males (Albert and Jégou, 2014) and females (Hannon and Flaws, 2015). Numerous studies have demonstrated the effects of in utero and lactational exposure to these agents on the developing fetus, a sensitive target for chemical insults (Skakkebaek et al., 2001). Pre- and perinatal exposure to DEHP in rodents results in decreased testosterone production (Parks et al., 2000) and anogenital index (Gray et al., 2000; Parks et al., 2000), increased nipple retention (Gray et al., 2000), hemorrhagic testes (Gray et al., 2000; Nardelli et al., 2017), and multinucleated gonocytes in the seminiferous chords (Gray et al., 2000; Nardelli et al., 2017; Parks et al., 2000) in male pups. Information on the impact of early life exposure to DEHP in the adult remains limited. In utero and lactational exposure to DEHP in rat models has been reported to result in either unchanged (Akingbemi et al., 2001), increased (Andrade et al., 2006) or decreased (Culty et al., 2008; Martinez-Arguelles et al., 2009) testosterone levels in the adult male. Such changes are sometimes associated with decreased sperm production (Andrade et al., 2006; Dorostghoal et al., 2012) and testicular histological changes and lesions (Andrade et al., 2006; Culty et al., 2008; Dorostghoal et al., 2012), but the underlying mechanisms are still unclear. The growing body of evidence in support of the deleterious effects of phthalates has prompted public health authorities to regulate their use in specific applications, triggering a search for safer replacements. For example, 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) was first introduced to the European market in 2002, received final approval from the European Food Safety Authority in 2006, and has been marketed as a safe replacement for DEHP ever since. Exposure to DINCH is now widespread, as assessed by increased urinary levels of DINCH metabolites (Giovanoulis et al., 2016; Silva et al., 2013). However, recent reports identify DINCH as a bioactive compound (Nardelli et al., 2015), a potent metabolic disruptor (Campioli et al., 2015), and a potential endocrine disruptor (Boisvert et al., 2016; Campioli et al., 2017; Nardelli et al., 2017), raising concerns about its possible impact on public health. In previous studies, we used a 4-step approach to identify greener replacements for DEHP with desirable plasticizing properties, biodegradability, minimal leaching, and fewer deleterious effects. We identified 2 candidate compounds, 1,4-butanediol dibenzoate (BDB) and dioctyl succinate (DOS), that did not cause significant physiological perturbations in several immortalized cell lines or after acute exposure in vivo (Albert et al., 2018; Boisvert et al., 2016; Nardelli et al., 2015). Recently, we demonstrated that in utero and lactational exposure to BDB and DOS did not produce the phenotypes described after exposure to DEHP from gestational day (GD) 8 to post-natal day (PND) 3, 8, or 21 (Nardelli et al., 2017). Here, we sought to investigate the consequences of in utero and lactational exposure to BDB and DOS on the adult male rat reproductive function, in a comparative study with DEHP and DINCH. MATERIALS AND METHODS Chemicals and reagents DEHP was purchased from Sigma-Aldrich Corporation (CAS #117-81-7; Cat #80030, St. Louis, MO). DINCH (CAS #474919-59-0 and 166412-78-8) was purchased from BASF Canada (Mississauga, ON). BDB (CAS #19924-27-2) and DOS (CAS #14491-66-8) were synthesized as previously described (Erythropel et al., 2013; Firlotte et al., 2009; Kermanshahi-pour et al., 2009), and their purity was determined to be 99% by NMR analysis. Chemicals were stored in a vacuum chamber with desiccant at room temperature until mixed with corn oil on each day of treatment (Catalogue #C8267; Lot#MKBN5383V, Sigma-Aldrich). Animals All manipulations and terminal procedures were approved by the McGill University Animal Care Committee (protocol #7317). Mating and treatments were done as described in Nardelli et al. (2017). In brief, virgin female and proven-breeder male Sprague Dawley rats were purchased from Charles River Laboratories (St-Constant, Quebec, Canada) and mated on the morning of proestrus. The next morning, sperm-positive females were placed in individual cages; this was considered GD 0. On GD 8, pregnant dams were randomly assigned to a treatment group, weighed and administered doses of 30 or 300 mg/kg of DEHP, DINCH, BDB, or DOS by gavage; control animals were administered 1 ml of vehicle (corn oil). The lower dose (30 mg/kg) is representative of high human exposure to DEHP (U.S. Food and Drug Administration, 2002) with an adjustment for interspecies metabolism (Nair and Jacob, 2016), and the higher dose (300 mg/kg) was selected based on previous literature that reported deleterious reproductive outcomes following exposure to DEHP during gestation and lactation (Gray et al., 2009). 15–19 dams per group were treated daily across 3 cohorts, except on the day of delivery, until pups were weaned at PND 21. Animals were maintained on a 12-h light/dark cycle and provided food and water ad libitum. At PND 90, 1 male per litter was euthanized by CO2 asphyxiation followed by cardiac puncture using a 10 ml syringe with 21 G 1-1/2-in. needle. Whole blood was collected in a BD Vacutainer SST tube (Becton, Dickinson and Company, Mississauga, ON) by negative pressure. The tubes were inverted several times and allowed to clot at room temperature for 30 min. To isolate serum, tubes were spun at 1000 × g in an Allegra-X 15R benchtop centrifuge (Beckman Coulter, Pasadena CA) at 4°C with a SX4750 swinging bucket rotor. Serum was aliquoted and kept at −80°C until further use. Organs were collected, weighed, flash frozen in liquid nitrogen, and stored at −80°C for further RNA extraction and sperm counts. Sperm head counts 5–10 mm3 portions of frozen testes were thawed on ice and weighed. Frozen epididymides were separated into 2 parts (caput-corpus and cauda) and weighed. Samples were homogenized in 5 ml of 0.9% sodium chloride, 0.1% thimerosal, and 0.05% Triton X-100 using the Polytron PT10-35GT (Kinematica Inc, Bohemia, NY) at 20 000 rpm for 2 intervals of 15 s separated by a 30-s interval. Sperm heads were counted using a hemocytometer (Hausser Scientific, Horsham, PA). Daily sperm production was calculated as described in Robb et al. (1978). Computer-assisted sperm analysis (CASA) The motility of spermatozoa was measured by CASA, as previously described (Zubkova and Robaire, 2004). In brief, immediately following euthanasia, the left cauda epididymidis was clamped, excised and minced in 3 ml of motility buffer [Hanks Balanced Salt Solution (Thermo Fisher Scientific, Waltham, MA), 0.35 g/l sodium bicarbonate (ACP Chemicals, Montreal, QC, CA), 4.2 g/l HEPES (Sigma-Aldrich), 0.9 g/l d-glucose (Sigma-Aldrich), 0.1 g/l sodium pyruvate (Thermo Fisher Scientific), 2 g/l bovine serum albumin (Sigma-Aldrich), 0.025 g/l soybean trypsin inhibitor (Sigma-Aldrich), pH 7.4]. Spermatozoa were allowed to disperse for 3 min and diluted 1:30 in motility buffer. 20 µl of this suspension were loaded onto 80 µm 2X-Cel Dual Sided Sperm Analysis Chambers and covered with 2X-Cel Cover Glass (Hamilton-Thorne Research, Beverly, MA). Movement characteristics were analyzed using the Hamilton-Thorne TOX Integrated Visual Optical System for sperm analysis (version 12). Settings were as follows: stage temperature: 37°C; frames acquired: 30; frame rate: 60 Hz; minimum contrast: 80; minimum cell size: 4 pixels; minimum static contrast: 15; cell intensity: 80: magnification: 0.82; static size limits: 0.29–8.82; static intensity: 0.18–1.8. A minimum of 350 spermatozoa and 5 fields were analyzed per sample. For each animal, data are expressed as the mean of the 5 fields with the highest percentage of motile sperm. Hormone assays Serum testosterone levels were assessed using enzyme-linked immunosorbent assay kits (IB79106; IBL America, Minneapolis, MN) according to the manufacturer’s instructions (dynamic range: 0.083–16 ng/ml; intra-assay variability: 3.2%–4.2%; inter-assay variability: 4.7%–9.9%). Luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations were assessed by the Ligand Assay & Analysis Core of the University of Virginia School of Medicine Center for Research in Reproduction using Millipore Pituitary Panel Multiplex kits (EMD Millipore, Saint Charles, MO). RNA extraction RNA was extracted from entire testes using the Qiagen RNEasy Mini Kit (Qiagen, Toronto, ON). In brief, frozen testes were pulverized to a thin powder using a chilled mortar and pestle. Approximately 30 mg of powder were placed in 1 ml of RLT buffer supplemented with ß-mercaptoethanol and mechanically disrupted using a rotor–stator homogenizer (Polytron PT10-35GT, Kinematica Inc.) at 20 000 rpm for 20 s. Homogenates were further processed with a QIAshredder column (Qiagen), and RNA was isolated as per the manufacturer’s instructions. RNA quality was measured using a NanoDrop 2000 Spectrophotometer (Thermo Fisher), with 260/280 and 260/230 absorbance ratios comprised between 1.9–2.1 and 2.0–2.2, respectively. Microarrays RNA (100 ng) was labeled with Cy3 using the Agilent One Color Low Input Quick Amp kit and hybridized on arrays using the SurePrint G3 Rat Gene Expression 8x60K microarray kit (Agilent Technologies, Santa Clara, CA) as per manufacturer’s instructions. All arrays were confirmed by the manufacturer to be from the same batch. Arrays were done using RNA from the testes of 6 animals from independent litters for each treatment group and read on an Agilent SureScan Microarray Scanner G2600D. The resulting data were analyzed using GeneSpring version 14.9 (Agilent Technologies). Data were normalized using percentile shift normalization to the 75th percentile. Following quality control using principal component scores on all arrays, 1 sample was removed in the DINCH 30 group. Transcripts affected by more than 1.5-fold were determined using a moderated t test and Benjamini–Hochberg FDR correction for each treatment. The microarray data have been uploaded to Gene Expression Omnibus (accession number GSE110553). The biological relationships between transcripts were analyzed using Ingenuity Pathway Analysis, version 01.12 (42012434). RT-qPCR We identified 6 genes of interest to be validated (Supplementary Table 1) from the microarray data. RNA from whole testis was converted to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen), as per the manufacturer’s instructions. Gene expression was quantified using the QuantiTect SYBR Green PCR Kit (Qiagen) using the StepOne Plus Real Time PCR System (Applied Biosytems, Foster City, CA) with the following thermal conditions: initial heat activation for 15 min at 95°C, followed by 50 cycles of denaturation for 15 s at 94°C, annealing for 30 s at 55°C, and extension for 30 s at 72°C. Serial dilutions of whole testis RNA pooled from animals from all treatment groups were used as an internal reference and to create standard curves for primer efficiency and template concentration optimization. Each gene was amplified in triplicate from 6 independent samples for each treatment group. Relative levels of gene amplification compared with the control group and housekeeping gene, Ppia, were calculated using StepOnePlus Software (version 2.1). A melt curve was systematically generated to ensure the specificity of the PCR reaction. Statistical analysis Significance was determined by ANOVA followed by Dunnett’s post hoc test. Outliers due to biological differences have not been removed from any of the data. Statistical calculations were generated using GraphPad Prism 6.07 (GraphPad Software, La Jolla, CA). RESULTS Overall Health and Androgen-Sensitive Organ Weights Maternal and offspring overall health data were previously published in Nardelli et al. (2017). In brief, there was no significant effect of the treatments on maternal weight gain or general health except for a significant decrease in heart weight after exposure to 300 mg/kg/day DEHP. Litter sizes, offspring viability, and postnatal growth remained unchanged by treatment. Animals were weighed and necropsied at PND 90 to assess whether exposure to the treatments altered general health. In utero and lactational exposure to DEHP, DINCH, BDB, and DOS did not alter body weight in adult males. This was also the case for the weights of the heart, lungs, liver, spleen, and kidneys (Supplementary Table 2). Exposure to 300 mg/kg/day DEHP produced a significant increase in paired epididymides weight (p = .02), but other androgen-responsive and reproductive tract organs, including paired testes and seminal vesicles, were unaffected by the treatment (Figure 1). Figure 1. View largeDownload slide Androgen-sensitive organ weights in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Data are standardized to 100 g body weight (bw) except for testes, which are encapsulated organs. Significance was determined by one-way ANOVA corrected by Dunnett’s multiple comparison test; n = 8–11 animals from independent litters per group; *p < .05. Figure 1. View largeDownload slide Androgen-sensitive organ weights in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Data are standardized to 100 g body weight (bw) except for testes, which are encapsulated organs. Significance was determined by one-way ANOVA corrected by Dunnett’s multiple comparison test; n = 8–11 animals from independent litters per group; *p < .05. Hormonal Measurements Serum from males from 7 to 11 independent litters was assessed for testosterone, LH, and FSH concentrations to determine whether early life exposures disrupt hormonal function in adult males (Figure 2). None of the treatments significantly altered these hormones. The ratio of testosterone to LH, a measure for possible compensated Leydig cell failure, also remained unaffected (Figure 2C). Figure 2. View largeDownload slide Serum testosterone and gonadotropin levels in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Serum testosterone (A), LH (B) and FSH (D) were assessed in 7–11 males from independent litters. The ratio of testosterone to LH (C), which allows detection of potential compensated Leydig cell failure, was calculated as well. No significant difference was detected using one-way ANOVA corrected by Dunnett’s multiple comparison test. Figure 2. View largeDownload slide Serum testosterone and gonadotropin levels in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Serum testosterone (A), LH (B) and FSH (D) were assessed in 7–11 males from independent litters. The ratio of testosterone to LH (C), which allows detection of potential compensated Leydig cell failure, was calculated as well. No significant difference was detected using one-way ANOVA corrected by Dunnett’s multiple comparison test. Sperm Count and Motility Testicular and epididymal sperm heads were counted to further investigate the effects of in utero and lactational exposure to DEHP, DINCH, and candidate replacement plasticizers. There were no significant effects on sperm counts in the testis, combined caput and corpus epididymides, or cauda epididymidis of exposed animals (Figs. 3A–C). Daily sperm production was unaltered by the treatments (Figure 3D). Immediately following necropsy, cauda sperm motility was also assessed using CASA. None of the treatments significantly altered sperm motility (Table 1). Table 1. CASA of Adult Cauda Sperm After In Utero and Lactational Exposure to Corn Oil (Vehicle), DEHP, DINCH, BDB, or DOS Dose (mg/kg/day) CORN OIL DEHP DINCH BDB DOS 30 300 30 300 30 300 30 300 Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Motile sperm (%) 73.1 1.6 72.1 0.8 73.0 2.1 72.4 2.7 72.8 3.2 74.6 2.6 72.5 2.2 71.7 2.3 74.1 1.3 Progressively motile (%) 32.9 1.9 30.1 1.9 31.5 2.3 32.3 2.5 28.0 3.4 32.8 2.1 33.1 2.5 35.1 2.9 33.5 2.1 Rapid velocity (%) 43.0 2.5 40.0 2.4 41.5 3.7 44.5 2.3 37.9 4.3 44.0 2.3 44.1 3.1 46.7 3.7 44.7 2.7 Medium velocity (%) 0.8 0.3 1.0 0.3 1.8 1.0 0.9 0.3 1.7 0.6 2.2 0.8 1.1 0.5 1.1 0.4 1.2 0.5 Slow velocity (%) 29.3 2.2 31.1 2.1 29.8 3.5 27.0 3.4 33.3 3.4 28.3 2.4 27.2 1.9 24.1 3.0 28.2 2.6 Static (%) 26.9 1.6 27.9 0.8 27.1 2.1 27.7 2.7 27.2 3.2 25.4 2.6 27.5 2.2 28.4 2.3 25.9 1.3 VAP (µm/s) 153.5 3.3 152.8 4.6 146.9 8.1 157.3 5.6 140.8 6.7 149.3 5.4 151.5 3.0 149.4 4.4 151.3 4.5 VSL (µm/s) 81.2 1.8 79.2 2.4 77.6 2.3 80.8 2.5 73.0 2.3 77.2 2.4 80.3 1.8 79.7 3.0 78.8 1.9 VCL (µm/s) 337.5 8.6 338.9 12.2 326.7 20.1 347.9 18.6 316.5 15.3 326.9 13.4 344.2 11.1 338.8 14.5 329.8 9.3 ALH (µm/s) 23.1 0.4 23.5 0.6 22.0 0.9 23.6 0.9 22.0 0.6 22.5 0.5 23.0 0.4 22.2 0.7 22.5 0.6 BCF (Hz) 33.0 0.5 32.9 0.6 33.6 0.6 33.4 0.9 35.0 0.7 34.1 0.8 35.5 0.8 33.9 0.6 34.0 0.7 STR (%) 55.4 0.8 54.5 0.8 56.2 2.0 54.0 1.2 55.5 1.5 55.0 1.0 55.7 0.9 56.1 1.2 55.1 0.9 LIN (%) 26.0 0.4 25.5 0.5 26.3 1.1 25.5 0.8 26.2 0.8 26.2 0.7 25.7 0.7 26.0 0.5 26.7 0.6 Dose (mg/kg/day) CORN OIL DEHP DINCH BDB DOS 30 300 30 300 30 300 30 300 Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Motile sperm (%) 73.1 1.6 72.1 0.8 73.0 2.1 72.4 2.7 72.8 3.2 74.6 2.6 72.5 2.2 71.7 2.3 74.1 1.3 Progressively motile (%) 32.9 1.9 30.1 1.9 31.5 2.3 32.3 2.5 28.0 3.4 32.8 2.1 33.1 2.5 35.1 2.9 33.5 2.1 Rapid velocity (%) 43.0 2.5 40.0 2.4 41.5 3.7 44.5 2.3 37.9 4.3 44.0 2.3 44.1 3.1 46.7 3.7 44.7 2.7 Medium velocity (%) 0.8 0.3 1.0 0.3 1.8 1.0 0.9 0.3 1.7 0.6 2.2 0.8 1.1 0.5 1.1 0.4 1.2 0.5 Slow velocity (%) 29.3 2.2 31.1 2.1 29.8 3.5 27.0 3.4 33.3 3.4 28.3 2.4 27.2 1.9 24.1 3.0 28.2 2.6 Static (%) 26.9 1.6 27.9 0.8 27.1 2.1 27.7 2.7 27.2 3.2 25.4 2.6 27.5 2.2 28.4 2.3 25.9 1.3 VAP (µm/s) 153.5 3.3 152.8 4.6 146.9 8.1 157.3 5.6 140.8 6.7 149.3 5.4 151.5 3.0 149.4 4.4 151.3 4.5 VSL (µm/s) 81.2 1.8 79.2 2.4 77.6 2.3 80.8 2.5 73.0 2.3 77.2 2.4 80.3 1.8 79.7 3.0 78.8 1.9 VCL (µm/s) 337.5 8.6 338.9 12.2 326.7 20.1 347.9 18.6 316.5 15.3 326.9 13.4 344.2 11.1 338.8 14.5 329.8 9.3 ALH (µm/s) 23.1 0.4 23.5 0.6 22.0 0.9 23.6 0.9 22.0 0.6 22.5 0.5 23.0 0.4 22.2 0.7 22.5 0.6 BCF (Hz) 33.0 0.5 32.9 0.6 33.6 0.6 33.4 0.9 35.0 0.7 34.1 0.8 35.5 0.8 33.9 0.6 34.0 0.7 STR (%) 55.4 0.8 54.5 0.8 56.2 2.0 54.0 1.2 55.5 1.5 55.0 1.0 55.7 0.9 56.1 1.2 55.1 0.9 LIN (%) 26.0 0.4 25.5 0.5 26.3 1.1 25.5 0.8 26.2 0.8 26.2 0.7 25.7 0.7 26.0 0.5 26.7 0.6 Mean and standard errors of the mean (SEM) are given for each measured parameter. VAP, average path velocity; VSL, progressive velocity; VCL, track speed; ALH, average lateral amplitude; BCF, beat frequency; STR, straightness; LIN, linearity Table 1. CASA of Adult Cauda Sperm After In Utero and Lactational Exposure to Corn Oil (Vehicle), DEHP, DINCH, BDB, or DOS Dose (mg/kg/day) CORN OIL DEHP DINCH BDB DOS 30 300 30 300 30 300 30 300 Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Motile sperm (%) 73.1 1.6 72.1 0.8 73.0 2.1 72.4 2.7 72.8 3.2 74.6 2.6 72.5 2.2 71.7 2.3 74.1 1.3 Progressively motile (%) 32.9 1.9 30.1 1.9 31.5 2.3 32.3 2.5 28.0 3.4 32.8 2.1 33.1 2.5 35.1 2.9 33.5 2.1 Rapid velocity (%) 43.0 2.5 40.0 2.4 41.5 3.7 44.5 2.3 37.9 4.3 44.0 2.3 44.1 3.1 46.7 3.7 44.7 2.7 Medium velocity (%) 0.8 0.3 1.0 0.3 1.8 1.0 0.9 0.3 1.7 0.6 2.2 0.8 1.1 0.5 1.1 0.4 1.2 0.5 Slow velocity (%) 29.3 2.2 31.1 2.1 29.8 3.5 27.0 3.4 33.3 3.4 28.3 2.4 27.2 1.9 24.1 3.0 28.2 2.6 Static (%) 26.9 1.6 27.9 0.8 27.1 2.1 27.7 2.7 27.2 3.2 25.4 2.6 27.5 2.2 28.4 2.3 25.9 1.3 VAP (µm/s) 153.5 3.3 152.8 4.6 146.9 8.1 157.3 5.6 140.8 6.7 149.3 5.4 151.5 3.0 149.4 4.4 151.3 4.5 VSL (µm/s) 81.2 1.8 79.2 2.4 77.6 2.3 80.8 2.5 73.0 2.3 77.2 2.4 80.3 1.8 79.7 3.0 78.8 1.9 VCL (µm/s) 337.5 8.6 338.9 12.2 326.7 20.1 347.9 18.6 316.5 15.3 326.9 13.4 344.2 11.1 338.8 14.5 329.8 9.3 ALH (µm/s) 23.1 0.4 23.5 0.6 22.0 0.9 23.6 0.9 22.0 0.6 22.5 0.5 23.0 0.4 22.2 0.7 22.5 0.6 BCF (Hz) 33.0 0.5 32.9 0.6 33.6 0.6 33.4 0.9 35.0 0.7 34.1 0.8 35.5 0.8 33.9 0.6 34.0 0.7 STR (%) 55.4 0.8 54.5 0.8 56.2 2.0 54.0 1.2 55.5 1.5 55.0 1.0 55.7 0.9 56.1 1.2 55.1 0.9 LIN (%) 26.0 0.4 25.5 0.5 26.3 1.1 25.5 0.8 26.2 0.8 26.2 0.7 25.7 0.7 26.0 0.5 26.7 0.6 Dose (mg/kg/day) CORN OIL DEHP DINCH BDB DOS 30 300 30 300 30 300 30 300 Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Motile sperm (%) 73.1 1.6 72.1 0.8 73.0 2.1 72.4 2.7 72.8 3.2 74.6 2.6 72.5 2.2 71.7 2.3 74.1 1.3 Progressively motile (%) 32.9 1.9 30.1 1.9 31.5 2.3 32.3 2.5 28.0 3.4 32.8 2.1 33.1 2.5 35.1 2.9 33.5 2.1 Rapid velocity (%) 43.0 2.5 40.0 2.4 41.5 3.7 44.5 2.3 37.9 4.3 44.0 2.3 44.1 3.1 46.7 3.7 44.7 2.7 Medium velocity (%) 0.8 0.3 1.0 0.3 1.8 1.0 0.9 0.3 1.7 0.6 2.2 0.8 1.1 0.5 1.1 0.4 1.2 0.5 Slow velocity (%) 29.3 2.2 31.1 2.1 29.8 3.5 27.0 3.4 33.3 3.4 28.3 2.4 27.2 1.9 24.1 3.0 28.2 2.6 Static (%) 26.9 1.6 27.9 0.8 27.1 2.1 27.7 2.7 27.2 3.2 25.4 2.6 27.5 2.2 28.4 2.3 25.9 1.3 VAP (µm/s) 153.5 3.3 152.8 4.6 146.9 8.1 157.3 5.6 140.8 6.7 149.3 5.4 151.5 3.0 149.4 4.4 151.3 4.5 VSL (µm/s) 81.2 1.8 79.2 2.4 77.6 2.3 80.8 2.5 73.0 2.3 77.2 2.4 80.3 1.8 79.7 3.0 78.8 1.9 VCL (µm/s) 337.5 8.6 338.9 12.2 326.7 20.1 347.9 18.6 316.5 15.3 326.9 13.4 344.2 11.1 338.8 14.5 329.8 9.3 ALH (µm/s) 23.1 0.4 23.5 0.6 22.0 0.9 23.6 0.9 22.0 0.6 22.5 0.5 23.0 0.4 22.2 0.7 22.5 0.6 BCF (Hz) 33.0 0.5 32.9 0.6 33.6 0.6 33.4 0.9 35.0 0.7 34.1 0.8 35.5 0.8 33.9 0.6 34.0 0.7 STR (%) 55.4 0.8 54.5 0.8 56.2 2.0 54.0 1.2 55.5 1.5 55.0 1.0 55.7 0.9 56.1 1.2 55.1 0.9 LIN (%) 26.0 0.4 25.5 0.5 26.3 1.1 25.5 0.8 26.2 0.8 26.2 0.7 25.7 0.7 26.0 0.5 26.7 0.6 Mean and standard errors of the mean (SEM) are given for each measured parameter. VAP, average path velocity; VSL, progressive velocity; VCL, track speed; ALH, average lateral amplitude; BCF, beat frequency; STR, straightness; LIN, linearity Figure 3. View largeDownload slide Sperm head counts in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Sperm heads were counted in the testis (A), caput and corpus epididymides (B) or cauda epididymidis (C) and reported as 108 per gram of each. Daily sperm production is reported for each treatment (D). n = 4–7 males from independent litters. No significant difference was detected using one-way ANOVA corrected by Dunnett’s multiple comparison test. Figure 3. View largeDownload slide Sperm head counts in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Sperm heads were counted in the testis (A), caput and corpus epididymides (B) or cauda epididymidis (C) and reported as 108 per gram of each. Daily sperm production is reported for each treatment (D). n = 4–7 males from independent litters. No significant difference was detected using one-way ANOVA corrected by Dunnett’s multiple comparison test. Testicular Gene Expression Analysis The consequences of perinatal exposure to our candidate compounds was assessed on global testicular gene expression to provide additional insights into their possible biological effects and to identify relevant mechanisms of action that could contribute to an increase in epididymal weight observed after exposure to DEHP. Using single-color microarrays, gene expression was analyzed in 6 independent PND 90 adult males per treatment group. Figure 4A depicts the overall relationship between transcript responses to all treatments using principal component analysis (PCA). This exploratory multidimensional data mining procedure converts a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components. Treatments producing similar gene expression responses are plotted in close proximity. Here, PCA analysis described three major components representing 23.66 (x-axis), 17.07 (y-axis), and 13.45% (z-axis) of the variance among all samples. Exposure to 30 mg/kg/day BDB or DOS produced expression profiles that clustered closely to that of corn oil (grey circle, Figure 4A); at the same dose, both DEHP and DINCH displayed profiles that were isolated and distant from this grouping. At 300 mg/kg/day, DEHP, DINCH, and BDB clustered together and further away from corn oil along the third component, while DOS presented a more isolated profile along the first and second components. Figure 4. View largeDownload slide Testicular gene expression in PND 90 rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. (A) PCA of single-color microarray data; (B) Number of uniquely mapped transcripts significantly up or downregulated by more than 1.5-fold determined by moderated t test and Benjamini–Hochberg FDR correction (p > .05); (C) Venn diagrams representing commonalities in differential gene expression between treatments at 30 and 300 mg/kg/day, respectively. All data were generated using n = 5–6 male rats from independent litters. Figure 4. View largeDownload slide Testicular gene expression in PND 90 rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. (A) PCA of single-color microarray data; (B) Number of uniquely mapped transcripts significantly up or downregulated by more than 1.5-fold determined by moderated t test and Benjamini–Hochberg FDR correction (p > .05); (C) Venn diagrams representing commonalities in differential gene expression between treatments at 30 and 300 mg/kg/day, respectively. All data were generated using n = 5–6 male rats from independent litters. Independent statistical comparisons were used to identify the numbers of transcripts that were significantly differentially expressed, with a fold-change threshold of 1.5, between corn oil and each individual treatment (Figure 4B). In the 30 mg/kg/day treatment groups, DEHP, DINCH, BDB, and DOS significantly altered the expression of 12, 19, 3, and 15 transcripts, respectively. Exposure to 300 mg/kg/day DEHP produced the greatest effect, with 55 affected transcripts (26 upregulated and 29 downregulated). Comparatively, at an equivalent dose, DINCH significantly altered 14 transcripts, BDB altered 27 transcripts, and treatment with DOS produced no significant effect. Venn diagrams in Figure 4C represent the overlap between significantly altered transcript sets for all treatments at a given dose. At both 30 and 300 mg/kg/day, BDB and DOS shared no differentially expressed transcript with DEHP or with each other. At 30 mg/kg/day, DINCH and DOS both significantly upregulated the expression of Upk3bl (uroplakin 3b-like protein), at fold-changes of 1.9 and 2.0, respectively. At 300 mg/kg/day, DEHP and DINCH both downregulated the expression of Hpgds by fold-changes of 1.7 and 1.6, respectively, and Ly6g6f (lymphocyte antigen 6 family member G6F) by fold-changes of 1.9 and 1.7, respectively. Finally, at 300 mg/kg/day, both DINCH and BDB downregulated the expression of HS3ST3A1 (heparan sulfate-glucosamine 3-sulfotransferase 3A1) by 2.0 and 2.1, respectively, as well as the expression of the LOC100910837 loci (olfactory receptor 2AK2-like) by fold-changes of 1.7 and 1.5, respectively. Commonalities between doses for each treatment were quite limited. Exposure to 30 and 300 mg/kg/day DEHP significantly upregulated the expression of Xkr9 (XK, Kell Blood Group Complex Subunit-Related Family, Member 9) by 3.3- and 3.1-fold, respectively, and downregulated the expression of mitogen activated protein kinase 4 (Mapk4) by 1.7- and 1.5-fold, respectively. Overall, the limited overlap between treatments suggests that our candidate compounds do not share key mechanisms of action with DEHP and DINCH. For each treatment, we further investigated the transcripts that were the most significantly altered based on absolute fold change (Table 2). To understand the biological relationships underlying such gene expression changes, the transcripts that were significantly altered by 1.5-fold or more were imported into Ingenuity Pathway Analysis. Pathways that were predicted to be significantly affected by the treatment and related transcripts are listed in Supplementary Table 3. While these pathways were not specifically predicted to be activated or inhibited (null z-score) due to the limited number of transcripts on these lists, they may identify gene enrichment for important physiological pathways within our datasets. Based on the latter information, and considering the transcripts with highest absolute fold changes and their role relative to testicular function, we selected 6 targets of interest to be validated by RT-qPCR (Figure 5). Table 2. Testicular Transcripts Significantly Affected With a >1.5-Fold Change After In Utero and Lactational Exposure to DEHP, DINCH, BDB, or DOS ID Symbol Expression p-value Expression fold change DEHP 30 A_64_P009798 Nr5a2* 3.79E−02 −7.306 A_64_P103179 Xkr9 3.79E−02 3.288 A_44_P260348 LOC689316 3.79E−02 −3.143 A_64_P159163 Gm1527/Gm6558 3.79E−02 −2.102 A_64_P048106 Foxp2 3.79E−02 1.91 A_44_P271529 Olfr380 3.79E−02 −1.808 A_64_P051307 Runx2* 3.79E−02 −1.768 A_43_P16031 Mapk4* 4.63E−02 −1.706 A_42_P794562 Cyp7a1 3.79E−02 1.701 A_64_P051039 Spon2 3.79E−02 −1.644 A_64_P072739 Flrt1 3.79E−02 −1.588 A_44_P161470 Defa6 (includes others) 4.48E−02 1.515 DEHP 300 A_44_P636808 9230104L09Rik 4.68E−02 3.585 A_64_P103179 Xkr9 1.88E−02 3.127 A_64_P023008 LOC100911534 4.48E−02 2.453 A_44_P419034 Olr181/Olr183 4.37E−02 −2.37 A_64_P232281 Vom2r1 (includes others) 5.59E−03 2.143 A_64_P064556 Slc36a4 1.28E−02 2.063 A_64_P047621 Ildr1 1.41E−02 −2.044 A_44_P405374 LOC290071 1.88E−02 2.027 A_64_P132182 Zeb2os 4.37E−02 1.956 A_43_P11449 Cpb1 1.41E−02 1.944 A_44_P351263 Ly6g6f 1.41E−02 −1.935 A_64_P087625 LOC102552143/LOC102552337 4.57E−02 1.927 A_44_P689645 Enox2 1.41E−02 −1.894 A_64_P038812 Erbb2 4.37E−02 1.87 A_64_P004007 Rd3l 4.37E−02 1.861 A_44_P140275 Alpk1 1.43E−02 1.856 A_64_P075532 Gm8882 (includes others) 4.02E−02 −1.827 A_64_P090866 Sstr1 4.37E−02 −1.808 A_44_P558411 Scrg1 4.81E−02 1.804 A_64_P063443 LOC103690069 2.71E−02 1.788 A_64_P131538 Olfr649 2.59E−03 −1.768 A_64_P080530 Nrxn3 4.93E−02 1.757 A_64_P060018 Pilrb 4.37E−02 −1.746 A_64_P108698 Cryge/Crygf 4.37E−02 1.711 A_43_P11278 Hpgd 2.71E−02 −1.707 A_44_P439292 Hpgds* 4.86E−02 −1.699 A_64_P111725 LOC103690800 2.66E−02 1.675 A_64_P160550 Gpr171 4.48E−02 1.67 A_64_P055137 Gldn 1.00E−02 −1.665 A_64_P150603 Ptger3 3.36E−02 −1.653 A_42_P736315 Rtp4 5.59E−03 −1.643 A_44_P515494 Ddr1 3.76E−02 −1.64 A_64_P033121 Rasa4 4.57E−02 1.635 A_44_P496572 Kmo 3.49E−02 −1.625 A_64_P156001 Olr396 4.67E−02 −1.616 A_64_P106933 Foxh1 5.59E−03 1.614 A_44_P426079 Kcnc1 3.49E−02 −1.604 A_64_P063558 LOC499764 4.02E−02 −1.593 A_44_P1013314 Iisg15 3.71E−02 −1.586 A_64_P032777 Ly49i2 (includes others) 5.59E−03 1.583 A_64_P095136 Fam163b 4.37E−02 −1.583 A_64_P056955 Tmprss2 4.57E−02 −1.579 A_44_P440207 Cd244 4.57E−02 1.577 A_44_P427596 Fabp1 4.57E−02 −1.573 A_64_P002502 Helb 4.37E−02 1.571 A_64_P290615 Vmn2r116 (includes others) 4.93E−02 −1.567 A_44_P1054324 Csta 1.43E−02 1.531 A_44_P438272 Crisp1/Crisp3 3.43E−02 −1.526 A_64_P100356 Or5k2 4.71E−02 −1.525 A_44_P282164 Pdia2 4.02E−02 −1.521 A_64_P092299 Olfr371 1.88E−02 −1.518 A_64_P131908 LOC103695256 4.37E−02 1.516 A_64_P249426 LOC285423 3.55E−02 1.506 A_43_P16031 Mapk4* 4.67E−02 −1.506 A_64_P036484 Chrm5 1.44E−02 −1.502 DINCH 30 A_44_P281300 Nxnl1 4.68E−02 3.054 A_44_P543128 Olfr742/Olfr743 4.68E−02 2.161 A_64_P103000 Scn3A 2.10E−02 2.11 A_64_P007789 LOC688970/RGD1561231 2.20E−03 2.073 A_44_P882995 Znf286a 2.63E−02 2.017 A_44_P324340 Mfsd6 4.47E−02 −1.985 A_64_P131908 LOC103695256 2.63E−02 1.963 A_43_P20178 Nid2 2.63E−02 1.943 A_64_P031806 Upk3bl 4.47E−02 1.892 A_64_P128687 Olfr67 4.68E−02 1.753 A_64_P317473 Tgm7 3.99E−02 1.662 A_42_P817417 Pvr 4.70E−02 −1.607 A_64_P017886 Robo2 4.68E−02 1.596 A_64_P052225 Slc22a8 2.63E−02 1.586 A_64_P013802 Dcp2 4.68E−02 −1.574 A_64_P017329 Syk* 2.63E−02 1.54 A_64_P063443 LOC103690069 4.68E−02 1.528 A_64_P033121 Rasa4 4.68E−02 1.526 A_64_P024454 Thsd7b 2.63E−02 1.505 DINCH 300 A_64_P037666 Iqsec3 4.54E−02 5.025 A_64_P048486 Hs3st3a1 2.99E−02 −2.033 A_64_P098882 Fcrl6 2.99E−02 1.97 A_44_P490784 Krt77 3.04E−02 1.967 A_44_P556319 Fgf18 2.99E−02 1.963 A_44_P538496 LOC100910837 (includes others) 6.83E−03 −1.667 A_44_P351263 Ly6g6f 4.54E−02 −1.654 A_44_P234013 Vn1r1 6.75E−03 −1.609 A_64_P031335 Stk32b 4.54E−02 −1.563 A_64_P062373 Vps37d 1.55E−02 −1.558 A_44_P439292 Hpgds* 4.11E−02 −1.551 A_64_P052380 Hibch 4.54E−02 1.544 A_64_P108160 Pou4f3 4.54E−02 −1.536 A_43_P23237 Sim2 4.54E−02 1.529 BDB 30 A_64_P120085 Sowahb 4.97E−02 −2.217 A_64_P031460 Vom2r1 (includes others) 2.81E−02 −2.216 A_64_P103610 Lmod3 2.81E−02 2.027 BDB 300 A_44_P292730 Rnase1 2.14E−02 3.377 A_64_P022561 Nlrp2 4.66E−02 2.308 A_64_P130627 2310002L09Rik 4.71E−02 2.227 A_64_P048486 Hs3st3a1 1.61E−02 −2.104 A_64_P030841 Ltf* 3.03E−02 −2.07 A_64_P083883 Samd11 2.14E−02 −1.997 A_64_P078624 Vtcn1 4.58E−02 1.888 A_64_P090851 Znf521 4.58E−02 1.884 A_64_P116670 Mta3 2.14E−02 −1.837 A_44_P1023538 C3 4.66E−02 −1.754 A_64_P128687 Olfr67 3.03E−02 1.7 A_64_P118323 Miat 4.45E−02 1.682 A_64_P059208 Trb 2.14E−02 −1.677 A_64_P120230 4921509C19Rik (includes others) 4.58E−02 −1.654 A_64_P092394 Dsg2 4.87E−02 1.645 A_64_P048506 S1pr5 4.45E−02 1.635 A_44_P384531 Rap1gds1 4.58E−02 1.616 A_64_P017329 Syk* 2.14E−02 1.597 A_64_P072219 Rtp3 4.71E−02 −1.568 A_64_P140886 Lrrc71 4.58E−02 −1.566 A_44_P299641 Olr1572 1.61E−02 −1.562 A_64_P161216 Kif5c 3.03E−02 1.543 A_44_P466189 Angptl2 3.03E−02 1.536 A_44_P538496 LOC100910837 (includes others) 1.61E−02 −1.527 A_44_P414421 Or1l1 2.14E−02 −1.526 A_64_P142640 Gjc3 3.03E−02 1.514 A_64_P101291 Hoxc12 3.03E−02 1.501 DOS 30 A_64_P014642 Mogat3 1.82E−02 3.72 A_43_P16740 Ogn 3.72E−02 −2.377 A_64_P036115 Ppp3ca 2.89E−02 2.308 A_64_P114465 Rab27b 4.27E−02 2.147 A_64_P031806 Upk3bl 3.92E−02 2.02 A_44_P990998 Klk8 4.85E−02 1.978 A_44_P700270 Fam64a 4.85E−02 1.911 A_44_P746666 Znf667 1.82E−02 −1.883 A_64_P069386 Gm378 4.98E−02 −1.654 A_64_P007372 Tmprss15 4.85E−02 −1.635 A_64_P120210 Galr1 2.27E−02 −1.591 A_64_P005972 Nhlrc3 4.27E−02 1.522 A_64_P031407 Mars2 3.92E−02 −1.516 A_42_P736530 Samd9 4.85E−02 −1.505 A_44_P468239 Sgtb 4.85E−02 −1.504 ID Symbol Expression p-value Expression fold change DEHP 30 A_64_P009798 Nr5a2* 3.79E−02 −7.306 A_64_P103179 Xkr9 3.79E−02 3.288 A_44_P260348 LOC689316 3.79E−02 −3.143 A_64_P159163 Gm1527/Gm6558 3.79E−02 −2.102 A_64_P048106 Foxp2 3.79E−02 1.91 A_44_P271529 Olfr380 3.79E−02 −1.808 A_64_P051307 Runx2* 3.79E−02 −1.768 A_43_P16031 Mapk4* 4.63E−02 −1.706 A_42_P794562 Cyp7a1 3.79E−02 1.701 A_64_P051039 Spon2 3.79E−02 −1.644 A_64_P072739 Flrt1 3.79E−02 −1.588 A_44_P161470 Defa6 (includes others) 4.48E−02 1.515 DEHP 300 A_44_P636808 9230104L09Rik 4.68E−02 3.585 A_64_P103179 Xkr9 1.88E−02 3.127 A_64_P023008 LOC100911534 4.48E−02 2.453 A_44_P419034 Olr181/Olr183 4.37E−02 −2.37 A_64_P232281 Vom2r1 (includes others) 5.59E−03 2.143 A_64_P064556 Slc36a4 1.28E−02 2.063 A_64_P047621 Ildr1 1.41E−02 −2.044 A_44_P405374 LOC290071 1.88E−02 2.027 A_64_P132182 Zeb2os 4.37E−02 1.956 A_43_P11449 Cpb1 1.41E−02 1.944 A_44_P351263 Ly6g6f 1.41E−02 −1.935 A_64_P087625 LOC102552143/LOC102552337 4.57E−02 1.927 A_44_P689645 Enox2 1.41E−02 −1.894 A_64_P038812 Erbb2 4.37E−02 1.87 A_64_P004007 Rd3l 4.37E−02 1.861 A_44_P140275 Alpk1 1.43E−02 1.856 A_64_P075532 Gm8882 (includes others) 4.02E−02 −1.827 A_64_P090866 Sstr1 4.37E−02 −1.808 A_44_P558411 Scrg1 4.81E−02 1.804 A_64_P063443 LOC103690069 2.71E−02 1.788 A_64_P131538 Olfr649 2.59E−03 −1.768 A_64_P080530 Nrxn3 4.93E−02 1.757 A_64_P060018 Pilrb 4.37E−02 −1.746 A_64_P108698 Cryge/Crygf 4.37E−02 1.711 A_43_P11278 Hpgd 2.71E−02 −1.707 A_44_P439292 Hpgds* 4.86E−02 −1.699 A_64_P111725 LOC103690800 2.66E−02 1.675 A_64_P160550 Gpr171 4.48E−02 1.67 A_64_P055137 Gldn 1.00E−02 −1.665 A_64_P150603 Ptger3 3.36E−02 −1.653 A_42_P736315 Rtp4 5.59E−03 −1.643 A_44_P515494 Ddr1 3.76E−02 −1.64 A_64_P033121 Rasa4 4.57E−02 1.635 A_44_P496572 Kmo 3.49E−02 −1.625 A_64_P156001 Olr396 4.67E−02 −1.616 A_64_P106933 Foxh1 5.59E−03 1.614 A_44_P426079 Kcnc1 3.49E−02 −1.604 A_64_P063558 LOC499764 4.02E−02 −1.593 A_44_P1013314 Iisg15 3.71E−02 −1.586 A_64_P032777 Ly49i2 (includes others) 5.59E−03 1.583 A_64_P095136 Fam163b 4.37E−02 −1.583 A_64_P056955 Tmprss2 4.57E−02 −1.579 A_44_P440207 Cd244 4.57E−02 1.577 A_44_P427596 Fabp1 4.57E−02 −1.573 A_64_P002502 Helb 4.37E−02 1.571 A_64_P290615 Vmn2r116 (includes others) 4.93E−02 −1.567 A_44_P1054324 Csta 1.43E−02 1.531 A_44_P438272 Crisp1/Crisp3 3.43E−02 −1.526 A_64_P100356 Or5k2 4.71E−02 −1.525 A_44_P282164 Pdia2 4.02E−02 −1.521 A_64_P092299 Olfr371 1.88E−02 −1.518 A_64_P131908 LOC103695256 4.37E−02 1.516 A_64_P249426 LOC285423 3.55E−02 1.506 A_43_P16031 Mapk4* 4.67E−02 −1.506 A_64_P036484 Chrm5 1.44E−02 −1.502 DINCH 30 A_44_P281300 Nxnl1 4.68E−02 3.054 A_44_P543128 Olfr742/Olfr743 4.68E−02 2.161 A_64_P103000 Scn3A 2.10E−02 2.11 A_64_P007789 LOC688970/RGD1561231 2.20E−03 2.073 A_44_P882995 Znf286a 2.63E−02 2.017 A_44_P324340 Mfsd6 4.47E−02 −1.985 A_64_P131908 LOC103695256 2.63E−02 1.963 A_43_P20178 Nid2 2.63E−02 1.943 A_64_P031806 Upk3bl 4.47E−02 1.892 A_64_P128687 Olfr67 4.68E−02 1.753 A_64_P317473 Tgm7 3.99E−02 1.662 A_42_P817417 Pvr 4.70E−02 −1.607 A_64_P017886 Robo2 4.68E−02 1.596 A_64_P052225 Slc22a8 2.63E−02 1.586 A_64_P013802 Dcp2 4.68E−02 −1.574 A_64_P017329 Syk* 2.63E−02 1.54 A_64_P063443 LOC103690069 4.68E−02 1.528 A_64_P033121 Rasa4 4.68E−02 1.526 A_64_P024454 Thsd7b 2.63E−02 1.505 DINCH 300 A_64_P037666 Iqsec3 4.54E−02 5.025 A_64_P048486 Hs3st3a1 2.99E−02 −2.033 A_64_P098882 Fcrl6 2.99E−02 1.97 A_44_P490784 Krt77 3.04E−02 1.967 A_44_P556319 Fgf18 2.99E−02 1.963 A_44_P538496 LOC100910837 (includes others) 6.83E−03 −1.667 A_44_P351263 Ly6g6f 4.54E−02 −1.654 A_44_P234013 Vn1r1 6.75E−03 −1.609 A_64_P031335 Stk32b 4.54E−02 −1.563 A_64_P062373 Vps37d 1.55E−02 −1.558 A_44_P439292 Hpgds* 4.11E−02 −1.551 A_64_P052380 Hibch 4.54E−02 1.544 A_64_P108160 Pou4f3 4.54E−02 −1.536 A_43_P23237 Sim2 4.54E−02 1.529 BDB 30 A_64_P120085 Sowahb 4.97E−02 −2.217 A_64_P031460 Vom2r1 (includes others) 2.81E−02 −2.216 A_64_P103610 Lmod3 2.81E−02 2.027 BDB 300 A_44_P292730 Rnase1 2.14E−02 3.377 A_64_P022561 Nlrp2 4.66E−02 2.308 A_64_P130627 2310002L09Rik 4.71E−02 2.227 A_64_P048486 Hs3st3a1 1.61E−02 −2.104 A_64_P030841 Ltf* 3.03E−02 −2.07 A_64_P083883 Samd11 2.14E−02 −1.997 A_64_P078624 Vtcn1 4.58E−02 1.888 A_64_P090851 Znf521 4.58E−02 1.884 A_64_P116670 Mta3 2.14E−02 −1.837 A_44_P1023538 C3 4.66E−02 −1.754 A_64_P128687 Olfr67 3.03E−02 1.7 A_64_P118323 Miat 4.45E−02 1.682 A_64_P059208 Trb 2.14E−02 −1.677 A_64_P120230 4921509C19Rik (includes others) 4.58E−02 −1.654 A_64_P092394 Dsg2 4.87E−02 1.645 A_64_P048506 S1pr5 4.45E−02 1.635 A_44_P384531 Rap1gds1 4.58E−02 1.616 A_64_P017329 Syk* 2.14E−02 1.597 A_64_P072219 Rtp3 4.71E−02 −1.568 A_64_P140886 Lrrc71 4.58E−02 −1.566 A_44_P299641 Olr1572 1.61E−02 −1.562 A_64_P161216 Kif5c 3.03E−02 1.543 A_44_P466189 Angptl2 3.03E−02 1.536 A_44_P538496 LOC100910837 (includes others) 1.61E−02 −1.527 A_44_P414421 Or1l1 2.14E−02 −1.526 A_64_P142640 Gjc3 3.03E−02 1.514 A_64_P101291 Hoxc12 3.03E−02 1.501 DOS 30 A_64_P014642 Mogat3 1.82E−02 3.72 A_43_P16740 Ogn 3.72E−02 −2.377 A_64_P036115 Ppp3ca 2.89E−02 2.308 A_64_P114465 Rab27b 4.27E−02 2.147 A_64_P031806 Upk3bl 3.92E−02 2.02 A_44_P990998 Klk8 4.85E−02 1.978 A_44_P700270 Fam64a 4.85E−02 1.911 A_44_P746666 Znf667 1.82E−02 −1.883 A_64_P069386 Gm378 4.98E−02 −1.654 A_64_P007372 Tmprss15 4.85E−02 −1.635 A_64_P120210 Galr1 2.27E−02 −1.591 A_64_P005972 Nhlrc3 4.27E−02 1.522 A_64_P031407 Mars2 3.92E−02 −1.516 A_42_P736530 Samd9 4.85E−02 −1.505 A_44_P468239 Sgtb 4.85E−02 −1.504 Transcripts selected for further RT-qPCR validation are indicated with an asterisk. Table 2. Testicular Transcripts Significantly Affected With a >1.5-Fold Change After In Utero and Lactational Exposure to DEHP, DINCH, BDB, or DOS ID Symbol Expression p-value Expression fold change DEHP 30 A_64_P009798 Nr5a2* 3.79E−02 −7.306 A_64_P103179 Xkr9 3.79E−02 3.288 A_44_P260348 LOC689316 3.79E−02 −3.143 A_64_P159163 Gm1527/Gm6558 3.79E−02 −2.102 A_64_P048106 Foxp2 3.79E−02 1.91 A_44_P271529 Olfr380 3.79E−02 −1.808 A_64_P051307 Runx2* 3.79E−02 −1.768 A_43_P16031 Mapk4* 4.63E−02 −1.706 A_42_P794562 Cyp7a1 3.79E−02 1.701 A_64_P051039 Spon2 3.79E−02 −1.644 A_64_P072739 Flrt1 3.79E−02 −1.588 A_44_P161470 Defa6 (includes others) 4.48E−02 1.515 DEHP 300 A_44_P636808 9230104L09Rik 4.68E−02 3.585 A_64_P103179 Xkr9 1.88E−02 3.127 A_64_P023008 LOC100911534 4.48E−02 2.453 A_44_P419034 Olr181/Olr183 4.37E−02 −2.37 A_64_P232281 Vom2r1 (includes others) 5.59E−03 2.143 A_64_P064556 Slc36a4 1.28E−02 2.063 A_64_P047621 Ildr1 1.41E−02 −2.044 A_44_P405374 LOC290071 1.88E−02 2.027 A_64_P132182 Zeb2os 4.37E−02 1.956 A_43_P11449 Cpb1 1.41E−02 1.944 A_44_P351263 Ly6g6f 1.41E−02 −1.935 A_64_P087625 LOC102552143/LOC102552337 4.57E−02 1.927 A_44_P689645 Enox2 1.41E−02 −1.894 A_64_P038812 Erbb2 4.37E−02 1.87 A_64_P004007 Rd3l 4.37E−02 1.861 A_44_P140275 Alpk1 1.43E−02 1.856 A_64_P075532 Gm8882 (includes others) 4.02E−02 −1.827 A_64_P090866 Sstr1 4.37E−02 −1.808 A_44_P558411 Scrg1 4.81E−02 1.804 A_64_P063443 LOC103690069 2.71E−02 1.788 A_64_P131538 Olfr649 2.59E−03 −1.768 A_64_P080530 Nrxn3 4.93E−02 1.757 A_64_P060018 Pilrb 4.37E−02 −1.746 A_64_P108698 Cryge/Crygf 4.37E−02 1.711 A_43_P11278 Hpgd 2.71E−02 −1.707 A_44_P439292 Hpgds* 4.86E−02 −1.699 A_64_P111725 LOC103690800 2.66E−02 1.675 A_64_P160550 Gpr171 4.48E−02 1.67 A_64_P055137 Gldn 1.00E−02 −1.665 A_64_P150603 Ptger3 3.36E−02 −1.653 A_42_P736315 Rtp4 5.59E−03 −1.643 A_44_P515494 Ddr1 3.76E−02 −1.64 A_64_P033121 Rasa4 4.57E−02 1.635 A_44_P496572 Kmo 3.49E−02 −1.625 A_64_P156001 Olr396 4.67E−02 −1.616 A_64_P106933 Foxh1 5.59E−03 1.614 A_44_P426079 Kcnc1 3.49E−02 −1.604 A_64_P063558 LOC499764 4.02E−02 −1.593 A_44_P1013314 Iisg15 3.71E−02 −1.586 A_64_P032777 Ly49i2 (includes others) 5.59E−03 1.583 A_64_P095136 Fam163b 4.37E−02 −1.583 A_64_P056955 Tmprss2 4.57E−02 −1.579 A_44_P440207 Cd244 4.57E−02 1.577 A_44_P427596 Fabp1 4.57E−02 −1.573 A_64_P002502 Helb 4.37E−02 1.571 A_64_P290615 Vmn2r116 (includes others) 4.93E−02 −1.567 A_44_P1054324 Csta 1.43E−02 1.531 A_44_P438272 Crisp1/Crisp3 3.43E−02 −1.526 A_64_P100356 Or5k2 4.71E−02 −1.525 A_44_P282164 Pdia2 4.02E−02 −1.521 A_64_P092299 Olfr371 1.88E−02 −1.518 A_64_P131908 LOC103695256 4.37E−02 1.516 A_64_P249426 LOC285423 3.55E−02 1.506 A_43_P16031 Mapk4* 4.67E−02 −1.506 A_64_P036484 Chrm5 1.44E−02 −1.502 DINCH 30 A_44_P281300 Nxnl1 4.68E−02 3.054 A_44_P543128 Olfr742/Olfr743 4.68E−02 2.161 A_64_P103000 Scn3A 2.10E−02 2.11 A_64_P007789 LOC688970/RGD1561231 2.20E−03 2.073 A_44_P882995 Znf286a 2.63E−02 2.017 A_44_P324340 Mfsd6 4.47E−02 −1.985 A_64_P131908 LOC103695256 2.63E−02 1.963 A_43_P20178 Nid2 2.63E−02 1.943 A_64_P031806 Upk3bl 4.47E−02 1.892 A_64_P128687 Olfr67 4.68E−02 1.753 A_64_P317473 Tgm7 3.99E−02 1.662 A_42_P817417 Pvr 4.70E−02 −1.607 A_64_P017886 Robo2 4.68E−02 1.596 A_64_P052225 Slc22a8 2.63E−02 1.586 A_64_P013802 Dcp2 4.68E−02 −1.574 A_64_P017329 Syk* 2.63E−02 1.54 A_64_P063443 LOC103690069 4.68E−02 1.528 A_64_P033121 Rasa4 4.68E−02 1.526 A_64_P024454 Thsd7b 2.63E−02 1.505 DINCH 300 A_64_P037666 Iqsec3 4.54E−02 5.025 A_64_P048486 Hs3st3a1 2.99E−02 −2.033 A_64_P098882 Fcrl6 2.99E−02 1.97 A_44_P490784 Krt77 3.04E−02 1.967 A_44_P556319 Fgf18 2.99E−02 1.963 A_44_P538496 LOC100910837 (includes others) 6.83E−03 −1.667 A_44_P351263 Ly6g6f 4.54E−02 −1.654 A_44_P234013 Vn1r1 6.75E−03 −1.609 A_64_P031335 Stk32b 4.54E−02 −1.563 A_64_P062373 Vps37d 1.55E−02 −1.558 A_44_P439292 Hpgds* 4.11E−02 −1.551 A_64_P052380 Hibch 4.54E−02 1.544 A_64_P108160 Pou4f3 4.54E−02 −1.536 A_43_P23237 Sim2 4.54E−02 1.529 BDB 30 A_64_P120085 Sowahb 4.97E−02 −2.217 A_64_P031460 Vom2r1 (includes others) 2.81E−02 −2.216 A_64_P103610 Lmod3 2.81E−02 2.027 BDB 300 A_44_P292730 Rnase1 2.14E−02 3.377 A_64_P022561 Nlrp2 4.66E−02 2.308 A_64_P130627 2310002L09Rik 4.71E−02 2.227 A_64_P048486 Hs3st3a1 1.61E−02 −2.104 A_64_P030841 Ltf* 3.03E−02 −2.07 A_64_P083883 Samd11 2.14E−02 −1.997 A_64_P078624 Vtcn1 4.58E−02 1.888 A_64_P090851 Znf521 4.58E−02 1.884 A_64_P116670 Mta3 2.14E−02 −1.837 A_44_P1023538 C3 4.66E−02 −1.754 A_64_P128687 Olfr67 3.03E−02 1.7 A_64_P118323 Miat 4.45E−02 1.682 A_64_P059208 Trb 2.14E−02 −1.677 A_64_P120230 4921509C19Rik (includes others) 4.58E−02 −1.654 A_64_P092394 Dsg2 4.87E−02 1.645 A_64_P048506 S1pr5 4.45E−02 1.635 A_44_P384531 Rap1gds1 4.58E−02 1.616 A_64_P017329 Syk* 2.14E−02 1.597 A_64_P072219 Rtp3 4.71E−02 −1.568 A_64_P140886 Lrrc71 4.58E−02 −1.566 A_44_P299641 Olr1572 1.61E−02 −1.562 A_64_P161216 Kif5c 3.03E−02 1.543 A_44_P466189 Angptl2 3.03E−02 1.536 A_44_P538496 LOC100910837 (includes others) 1.61E−02 −1.527 A_44_P414421 Or1l1 2.14E−02 −1.526 A_64_P142640 Gjc3 3.03E−02 1.514 A_64_P101291 Hoxc12 3.03E−02 1.501 DOS 30 A_64_P014642 Mogat3 1.82E−02 3.72 A_43_P16740 Ogn 3.72E−02 −2.377 A_64_P036115 Ppp3ca 2.89E−02 2.308 A_64_P114465 Rab27b 4.27E−02 2.147 A_64_P031806 Upk3bl 3.92E−02 2.02 A_44_P990998 Klk8 4.85E−02 1.978 A_44_P700270 Fam64a 4.85E−02 1.911 A_44_P746666 Znf667 1.82E−02 −1.883 A_64_P069386 Gm378 4.98E−02 −1.654 A_64_P007372 Tmprss15 4.85E−02 −1.635 A_64_P120210 Galr1 2.27E−02 −1.591 A_64_P005972 Nhlrc3 4.27E−02 1.522 A_64_P031407 Mars2 3.92E−02 −1.516 A_42_P736530 Samd9 4.85E−02 −1.505 A_44_P468239 Sgtb 4.85E−02 −1.504 ID Symbol Expression p-value Expression fold change DEHP 30 A_64_P009798 Nr5a2* 3.79E−02 −7.306 A_64_P103179 Xkr9 3.79E−02 3.288 A_44_P260348 LOC689316 3.79E−02 −3.143 A_64_P159163 Gm1527/Gm6558 3.79E−02 −2.102 A_64_P048106 Foxp2 3.79E−02 1.91 A_44_P271529 Olfr380 3.79E−02 −1.808 A_64_P051307 Runx2* 3.79E−02 −1.768 A_43_P16031 Mapk4* 4.63E−02 −1.706 A_42_P794562 Cyp7a1 3.79E−02 1.701 A_64_P051039 Spon2 3.79E−02 −1.644 A_64_P072739 Flrt1 3.79E−02 −1.588 A_44_P161470 Defa6 (includes others) 4.48E−02 1.515 DEHP 300 A_44_P636808 9230104L09Rik 4.68E−02 3.585 A_64_P103179 Xkr9 1.88E−02 3.127 A_64_P023008 LOC100911534 4.48E−02 2.453 A_44_P419034 Olr181/Olr183 4.37E−02 −2.37 A_64_P232281 Vom2r1 (includes others) 5.59E−03 2.143 A_64_P064556 Slc36a4 1.28E−02 2.063 A_64_P047621 Ildr1 1.41E−02 −2.044 A_44_P405374 LOC290071 1.88E−02 2.027 A_64_P132182 Zeb2os 4.37E−02 1.956 A_43_P11449 Cpb1 1.41E−02 1.944 A_44_P351263 Ly6g6f 1.41E−02 −1.935 A_64_P087625 LOC102552143/LOC102552337 4.57E−02 1.927 A_44_P689645 Enox2 1.41E−02 −1.894 A_64_P038812 Erbb2 4.37E−02 1.87 A_64_P004007 Rd3l 4.37E−02 1.861 A_44_P140275 Alpk1 1.43E−02 1.856 A_64_P075532 Gm8882 (includes others) 4.02E−02 −1.827 A_64_P090866 Sstr1 4.37E−02 −1.808 A_44_P558411 Scrg1 4.81E−02 1.804 A_64_P063443 LOC103690069 2.71E−02 1.788 A_64_P131538 Olfr649 2.59E−03 −1.768 A_64_P080530 Nrxn3 4.93E−02 1.757 A_64_P060018 Pilrb 4.37E−02 −1.746 A_64_P108698 Cryge/Crygf 4.37E−02 1.711 A_43_P11278 Hpgd 2.71E−02 −1.707 A_44_P439292 Hpgds* 4.86E−02 −1.699 A_64_P111725 LOC103690800 2.66E−02 1.675 A_64_P160550 Gpr171 4.48E−02 1.67 A_64_P055137 Gldn 1.00E−02 −1.665 A_64_P150603 Ptger3 3.36E−02 −1.653 A_42_P736315 Rtp4 5.59E−03 −1.643 A_44_P515494 Ddr1 3.76E−02 −1.64 A_64_P033121 Rasa4 4.57E−02 1.635 A_44_P496572 Kmo 3.49E−02 −1.625 A_64_P156001 Olr396 4.67E−02 −1.616 A_64_P106933 Foxh1 5.59E−03 1.614 A_44_P426079 Kcnc1 3.49E−02 −1.604 A_64_P063558 LOC499764 4.02E−02 −1.593 A_44_P1013314 Iisg15 3.71E−02 −1.586 A_64_P032777 Ly49i2 (includes others) 5.59E−03 1.583 A_64_P095136 Fam163b 4.37E−02 −1.583 A_64_P056955 Tmprss2 4.57E−02 −1.579 A_44_P440207 Cd244 4.57E−02 1.577 A_44_P427596 Fabp1 4.57E−02 −1.573 A_64_P002502 Helb 4.37E−02 1.571 A_64_P290615 Vmn2r116 (includes others) 4.93E−02 −1.567 A_44_P1054324 Csta 1.43E−02 1.531 A_44_P438272 Crisp1/Crisp3 3.43E−02 −1.526 A_64_P100356 Or5k2 4.71E−02 −1.525 A_44_P282164 Pdia2 4.02E−02 −1.521 A_64_P092299 Olfr371 1.88E−02 −1.518 A_64_P131908 LOC103695256 4.37E−02 1.516 A_64_P249426 LOC285423 3.55E−02 1.506 A_43_P16031 Mapk4* 4.67E−02 −1.506 A_64_P036484 Chrm5 1.44E−02 −1.502 DINCH 30 A_44_P281300 Nxnl1 4.68E−02 3.054 A_44_P543128 Olfr742/Olfr743 4.68E−02 2.161 A_64_P103000 Scn3A 2.10E−02 2.11 A_64_P007789 LOC688970/RGD1561231 2.20E−03 2.073 A_44_P882995 Znf286a 2.63E−02 2.017 A_44_P324340 Mfsd6 4.47E−02 −1.985 A_64_P131908 LOC103695256 2.63E−02 1.963 A_43_P20178 Nid2 2.63E−02 1.943 A_64_P031806 Upk3bl 4.47E−02 1.892 A_64_P128687 Olfr67 4.68E−02 1.753 A_64_P317473 Tgm7 3.99E−02 1.662 A_42_P817417 Pvr 4.70E−02 −1.607 A_64_P017886 Robo2 4.68E−02 1.596 A_64_P052225 Slc22a8 2.63E−02 1.586 A_64_P013802 Dcp2 4.68E−02 −1.574 A_64_P017329 Syk* 2.63E−02 1.54 A_64_P063443 LOC103690069 4.68E−02 1.528 A_64_P033121 Rasa4 4.68E−02 1.526 A_64_P024454 Thsd7b 2.63E−02 1.505 DINCH 300 A_64_P037666 Iqsec3 4.54E−02 5.025 A_64_P048486 Hs3st3a1 2.99E−02 −2.033 A_64_P098882 Fcrl6 2.99E−02 1.97 A_44_P490784 Krt77 3.04E−02 1.967 A_44_P556319 Fgf18 2.99E−02 1.963 A_44_P538496 LOC100910837 (includes others) 6.83E−03 −1.667 A_44_P351263 Ly6g6f 4.54E−02 −1.654 A_44_P234013 Vn1r1 6.75E−03 −1.609 A_64_P031335 Stk32b 4.54E−02 −1.563 A_64_P062373 Vps37d 1.55E−02 −1.558 A_44_P439292 Hpgds* 4.11E−02 −1.551 A_64_P052380 Hibch 4.54E−02 1.544 A_64_P108160 Pou4f3 4.54E−02 −1.536 A_43_P23237 Sim2 4.54E−02 1.529 BDB 30 A_64_P120085 Sowahb 4.97E−02 −2.217 A_64_P031460 Vom2r1 (includes others) 2.81E−02 −2.216 A_64_P103610 Lmod3 2.81E−02 2.027 BDB 300 A_44_P292730 Rnase1 2.14E−02 3.377 A_64_P022561 Nlrp2 4.66E−02 2.308 A_64_P130627 2310002L09Rik 4.71E−02 2.227 A_64_P048486 Hs3st3a1 1.61E−02 −2.104 A_64_P030841 Ltf* 3.03E−02 −2.07 A_64_P083883 Samd11 2.14E−02 −1.997 A_64_P078624 Vtcn1 4.58E−02 1.888 A_64_P090851 Znf521 4.58E−02 1.884 A_64_P116670 Mta3 2.14E−02 −1.837 A_44_P1023538 C3 4.66E−02 −1.754 A_64_P128687 Olfr67 3.03E−02 1.7 A_64_P118323 Miat 4.45E−02 1.682 A_64_P059208 Trb 2.14E−02 −1.677 A_64_P120230 4921509C19Rik (includes others) 4.58E−02 −1.654 A_64_P092394 Dsg2 4.87E−02 1.645 A_64_P048506 S1pr5 4.45E−02 1.635 A_44_P384531 Rap1gds1 4.58E−02 1.616 A_64_P017329 Syk* 2.14E−02 1.597 A_64_P072219 Rtp3 4.71E−02 −1.568 A_64_P140886 Lrrc71 4.58E−02 −1.566 A_44_P299641 Olr1572 1.61E−02 −1.562 A_64_P161216 Kif5c 3.03E−02 1.543 A_44_P466189 Angptl2 3.03E−02 1.536 A_44_P538496 LOC100910837 (includes others) 1.61E−02 −1.527 A_44_P414421 Or1l1 2.14E−02 −1.526 A_64_P142640 Gjc3 3.03E−02 1.514 A_64_P101291 Hoxc12 3.03E−02 1.501 DOS 30 A_64_P014642 Mogat3 1.82E−02 3.72 A_43_P16740 Ogn 3.72E−02 −2.377 A_64_P036115 Ppp3ca 2.89E−02 2.308 A_64_P114465 Rab27b 4.27E−02 2.147 A_64_P031806 Upk3bl 3.92E−02 2.02 A_44_P990998 Klk8 4.85E−02 1.978 A_44_P700270 Fam64a 4.85E−02 1.911 A_44_P746666 Znf667 1.82E−02 −1.883 A_64_P069386 Gm378 4.98E−02 −1.654 A_64_P007372 Tmprss15 4.85E−02 −1.635 A_64_P120210 Galr1 2.27E−02 −1.591 A_64_P005972 Nhlrc3 4.27E−02 1.522 A_64_P031407 Mars2 3.92E−02 −1.516 A_42_P736530 Samd9 4.85E−02 −1.505 A_44_P468239 Sgtb 4.85E−02 −1.504 Transcripts selected for further RT-qPCR validation are indicated with an asterisk. Figure 5. View largeDownload slide Gene expression quantification by RT-qPCR for selected transcripts following in utero and lactational exposure to DEHP, DINCH, BDB or DOS. Relative levels of gene amplification compared with the control group and housekeeping gene Ppia were calculated following the ΔΔCt method. Significance was determined by one-way ANOVA corrected by Dunnett’s multiple comparison test; n = 6 biological replicates plated in triplicate; **p < .01; ****p < .0001. Figure 5. View largeDownload slide Gene expression quantification by RT-qPCR for selected transcripts following in utero and lactational exposure to DEHP, DINCH, BDB or DOS. Relative levels of gene amplification compared with the control group and housekeeping gene Ppia were calculated following the ΔΔCt method. Significance was determined by one-way ANOVA corrected by Dunnett’s multiple comparison test; n = 6 biological replicates plated in triplicate; **p < .01; ****p < .0001. Interestingly, among all transcripts displaying modified expression, several were related to estrogen function and signaling. Hence, the nuclear receptor NR5A2, which plays a predominant role in reverse cholesterol transport and steroidogenesis (Pezzi et al., 2004; Sirianni et al., 2002), was downregulated by 7.3-fold after exposure to 30 mg/kg/day DEHP (Table 2). Validation with RT-qPCR confirmed this downregulation and revealed that Nr5a2 expression was also targeted by exposure to 300 mg/kg/day DEHP and by 30 mg/kg/day DINCH and BDB (Figure 5A). Lactotransferrin, the expression of which is dependent on estrogen (Teng, 2006), was also of interest in this context. Microarray data revealed that exposure to 300 mg/kg/day BDB downregulated the latter by 2.1-fold (Table 2). Validation by RT-qPCR not only confirmed this effect, but also revealed a stronger downregulation of Ltf by 300 mg/kg/day DEHP (Figure 5B). Finally, exposure to 30 mg/kg/day DEHP led to a significant 1.8-fold downregulation of Runx2 (runt-related factor 2) expression (Table 2). The latter regulates the expression of many steroid hormone-responsive genes in osteoblasts, and is regulated itself by estrogens at the transcriptional level (Teplyuk et al., 2009). qRT-PCR analysis did not reveal a statistically significant effect on the expression of this gene (Figure 5C). Other targets of interest included the hematopoietic prostaglandin D-synthase HPGDS, which is involved in inflammatory responses, and was downregulated by 1.7 and 1.6-fold after exposure to 300 mg/kg/day DEHP and DINCH, respectively (Table 2). Because many putative endocrine disruptors have been shown to disrupt prostaglandin synthesis, we further assessed Hpgds expression by RT-qPCR but found no significant effect by any of the treatments (Figure 5D). Mapk4 was downregulated by 1.7 and 1.5-fold after exposure to 30 and 300 mg/kg/day DEHP, respectively (Table 2). The tyrosine-protein kinase SYK, also involved in the MAPK signaling cascade, was downregulated by 1.5 and 1.6-fold, respectively, after exposure to 30 mg/kg/day DINCH or 300 mg/kg/day BDB (Table 2). However, RT-qPCR did not reveal statistically significant differences in the expression of these genes (Figs. 5D and 5E). DISCUSSION In a previous study, we demonstrated that in utero and lactational exposure to DEHP produced classically described endocrine-disruptive phenotypes such as a decreased anogenital index and increased multi-nucleated gonocytes at PND 3, as well as hemorrhagic testes at PND 8 (Nardelli et al., 2017). In comparison, exposure to BDB and DOS did not produce any significant effect on these endpoints. Here, we explored the impact of in utero and lactational exposure to DEHP, DINCH, BDB, and DOS in adult offspring. This study provides evidence that perinatal exposure to DINCH, BDB, and DOS produces no significant effects on organ weights, serum gonadotropin, and testosterone levels or sperm quality in the adult. In addition, we demonstrate that exposure to one green plasticizer, DOS, has fewer effects on testicular gene expression than DEHP, and identify estrogen signaling in the testis to be a potential long-term target of DEHP. Several studies have reported significant decreases in testicular or seminal vesicle weights in the adult male resulting from perinatal DEHP exposure. These effects were observed solely after exposure to relatively high doses of DEHP. Exposure to 938 or 1250 mg/kg/day DEHP from GD 14 to PND 0 produced a higher incidence of testicular atrophy (Culty et al., 2008), while exposure to 500 mg/kg/day DEHP from GD3 to PND 21 produced a significant decrease in testicular weight (Dorostghoal et al., 2012). In other studies, exposures to 405 mg/kg/day DEHP from GD 6 to PND 21 (Andrade et al., 2006) or to 500 mg/kg/day DEHP from GD 0 to PND 21 (Dalsenter et al., 2006) produced significant decreases in seminal vesicle weights; these effects were not associated with changes in testicular weight. To the best of our knowledge, there are no studies showing an alteration of adult reproductive organ weights after in utero and lactational exposure to DEHP at doses below 405 mg/kg/day. Consistent with these findings, we did not observe a significant effect of DEHP exposures at 30 and 300 mg/kg/day on testicular or seminal vesicle weights. However, exposure to 300 mg/kg/day DEHP did produce a significant increase in paired epididymal weight. Importantly, we did not find any significant effect of exposure to DINCH, BDB, or DOS on androgen-dependent organ weights. We measured serum steroids and gonadotropins to investigate whether exposure to our candidate compounds would affect steroidogenesis. Testosterone, LH and FSH levels remained unaffected by all treatments. The ratio of testosterone to LH a measure for possible compensated Leydig cell failure, remained unaffected as well; this ratio was decreased in adult rats following perinatal exposure to dibutyl phthalate, a potent antiandrogen (Kilcoyne et al., 2014). The literature on the long-term consequences of perinatal exposure to DEHP on steroidogenesis in males remains quite scarce. Exposure of Long Evans dams to 100 mg/kg/day DEHP from GD 12 to GD 21 produced inhibitory effects on testosterone and LH levels in the male offspring that were no longer apparent at PND 90 (Akingbemi et al., 2001). Two other studies report significant decreases in circulating testosterone levels at PND 60 following in utero exposure of Sprague Dawley dams to doses of DEHP starting at 100 mg/kg/day (Culty et al., 2008; Martinez-Arguelles et al., 2009); here, the treatment windows, GD 14 to PND0 or GD 14 to GD 19, respectively, were limited to fetal life. In contrast, the male offspring of Wistar dams showed increased testosterone levels at PND 90 after exposure to 0.045, 0.405, or 405 mg/kg/day DEHP from GD 6 to PND 21(Andrade et al., 2006). These divergences seem to be highly dependent on the rat strain, the dose and the window of exposure used, and call for a deeper exploration of the long-term consequences of exposure to DEHP. DINCH and our candidate compounds, BDB and DOS, did not affect testosterone, LH or FSH concentrations in serum. We assessed sperm production at PND 90 to investigate whether exposure to our candidate compounds affects spermatogenesis in the adult. None of the treatments significantly affected testicular or epididymal sperm head counts or cauda sperm motility. In a previous study, in utero and lactational exposure to DEHP has been reported to reduce daily sperm production 19%–25% in relation to control in animals exposed to 15, 45, 135, or 405 mg/kg/day (Andrade et al., 2006). Reduced sperm counts were also observed in the male offspring of dams exposed to 100 or 500 mg/kg/day DEHP from GD 3 to PND 21 (Dorostghoal et al., 2012). Similarly, exposure to 500 mg/kg/day DEHP from GD 0 to PND 21 significantly reduced sperm production in adult males (Dalsenter et al., 2006); however, these effects were not observed at lower doses. The latter 3 studies have in common the use of Wistar rats. In our study, we used Sprague Dawley rats, which have been shown to be more resistant to some endocrine disrupting compounds in different experimental settings (Abuelhija et al., 2013; Kacew and Festing, 1996), including to diethylstilbestrol (Shellabarger et al., 1978). Such strain differences in susceptibility may contribute to the absence of effects on sperm counts in our experiments. Using toxicogenomics, we determined whether our candidate compounds would produce detrimental effects on testicular gene expression in the adult in comparison to DEHP. We observed that in utero and lactational exposure to 300 mg/kg/day DEHP produced the most substantial effect on adult testicular gene expression, while the overall gene expression profile after exposure to BDB and DOS was closer to that of corn oil. We also identified several related genes presenting altered expression following exposure to DEHP, DINCH, or BDB. Based on significance, fold changes and pathway analysis, we narrowed down our list of targets to 6 transcripts, and validated our data using RT-qPCR. Interestingly, we found 3 of these transcripts to be associated with estrogens. We identified the expression of Nr5a2 to be significantly altered after exposure to both doses of DEHP, and 30 mg/kg/day DINCH and BDB. Also called liver receptor homologue-1 (LRH-1), NR5A2 is an orphan nuclear hormone receptor closely related to steroidogenic factor 1 (SF1). It is expressed in several steroidogenic tissues in many species, and has been hypothesized to play a critical role in development and function of the endocrine and reproductive systems (Sirianni et al., 2002). NR5A2 has been demonstrated to regulate the transcription of genes encoding steroidogenic enzymes, more specifically aromatase (Pezzi et al., 2004). In the adult male, aromatase activity is higher than at any other age (Tsai-Morris et al., 1985). Furthermore, exposure to 300 mg/kg/day DEHP and BDB produced a significant decrease in Ltf expression. The secretion in reproductive tissues of lactotransferrin, a multifunctional glycoprotein, is estrogen-dependent (Teng, 2006). While its role in the male reproductive tract remains largely unknown, it is found in the testis, epididymis, vas deferens, and prostate (Yu and Chen, 1993). Lactotransferrin is also abundant in seminal fluid and is believed to be directly correlated to gonadal function and sperm concentration in several species (Kikuchi et al., 2003a, b). Finally, the significant downregulation of Runx2 by DEHP is also in agreement with a dysregulation of estrogen signaling in the testis. There are indeed several examples of gene regulatory interplay between Runx2 and estrogens (Teplyuk et al., 2009), notably its role in the regulation of steroidogenic enzyme Cyp11a1. Estrogens have profound implications in testicular and epididymal development and function, and therefore on male fertility (Cooke et al., 2017; O'Donnell et al., 2001). In adult males, estrogen administration or deficiency may affect the maintenance of the hypothalamo-pituitary-testis axis (O’Donnell et al., 2001; Robaire et al., 1979). Testicular estrogens have also been shown to play a role in the regulation of luminal fluid reabsorption by the epithelial cells lining the efferent ductules (Hess et al., 2011). Finally, many studies provide indirect evidence for a role of estrogen in spermatogenesis involving germ cell proliferation, differentiation, and maturation of spermatids, as well as germ cell survival and apoptosis (reviewed in Carreau and Hess, 2010). Our gene expression data point toward a subtle but significant dysregulation of estrogen function in the testis after exposure to DEHP, and to a lesser extent to DINCH and BDB. Using high performance liquid chromatography/mass spectrometry, serum estradiols were below levels of detection in this study (data not shown). However, the increased epididymal weight we observed after exposure to 300 mg/kg/day DEHP is consistent with a potential impairment of fluid reabsorption by the efferent ductules that might be related to estrogen signaling. This study provides evidence that in utero and lactational exposures to our candidate compounds, BDB and DOS, as well as exposure to DINCH, do not produce significant alterations in adult male reproductive function. However, our data indicate that exposure to DEHP and, to a lesser extent, exposure to DINCH and BDB could produce subtle but significant alterations of estrogen signaling in the adult testis. These data need to be placed in the context of exposure to multiple endocrine disruptive compounds: gene expression modifications that are observed more than 2 months after exposure has ceased may suggest that exposure to multiple compounds throughout life may have long-lasting effects on adult male testicular function. Importantly, exposure to DOS did not produce significant changes in the expression of the estrogen signaling targets we studied, confirming its potential as a substitute for DEHP. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. ACKNOWLEDGMENTS The authors should like to thank Hanno Erythropel, Milan Maric, and Richard Leask from the McGill University Department of Chemical Engineering for designing and/or providing raw materials, Sheila Ernest and Elise Kolasa for their technical help, and Elise Boivin-Ford for her help with data entry. FUNDING Canadian Institutes of Health Research (CIHR) Institute of Human Development, Child and Youth Health [RHF100626]; post-doctoral fellowships from the CIHR Training Program in Reproduction, Early Development, and the Impact on Health (REDIH) and the Fonds de Recherche du Québec en Santé (FRQS) [to O.A.]; studentships from the Réseau Québécois en Reproduction NSERC-CREATE and the CIHR REDIH Program [to T.C.N.]. B.R. and B.F.H. are James McGill Professors. REFERENCES Abuelhija M. , Weng C. C. , Shetty G. , Meistrich M. L. ( 2013 ). Rat models of post-irradiation recovery of spermatogenesis: interstrain differences . Andrology 1 , 206 – 215 . Google Scholar CrossRef Search ADS PubMed Akingbemi B. T. , Youker R. T. , Sottas C. M. , Ge R. , Katz E. , Klinefelter G. R. , Zirkin B. R. , Hardy M. P. ( 2001 ). Modulation of rat Leydig cell steroidogenic function by di(2-ethylhexyl)phthalate . Biol. Reprod . 65 , 1252 – 1259 . Google Scholar CrossRef Search ADS PubMed Albert O. , Jégou B. ( 2014 ). A critical assessment of the endocrine susceptibility of the human testis to phthalates from fetal life to adulthood . Hum. Reprod. Update 20 , 231 – 249 . Google Scholar CrossRef Search ADS PubMed Albert O. , Nardelli T. C. , Hales B. F. , Robaire B. ( 2018 ). Identifying Greener and Safer Plasticizers: A 4-Step Approach . Toxicol. Sci . 161 , 266 – 275 . Google Scholar CrossRef Search ADS PubMed Andrade A. J. M. , Grande S. W. , Talsness C. E. , Gericke C. , Grote K. , Golombiewski A. , Sterner-Kock A. , Chahoud I. ( 2006 ). A dose response study following in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP): Reproductive effects on adult male offspring rats . Toxicology 228 , 85 – 97 . Google Scholar CrossRef Search ADS PubMed Boisvert A. , Jones S. , Issop L. , Erythropel H. C. , Papadopoulos V. , Culty M. ( 2016 ). In vitro functional screening as a means to identify new plasticizers devoid of reproductive toxicity . Environ. Res . 150 , 496 – 512 . Google Scholar CrossRef Search ADS PubMed Calafat A. M. , Slakman A. R. , Silva M. J. , Herbert A. R. , Needham L. L. ( 2004 ). Automated solid phase extraction and quantitative analysis of human milk for 13 phthalate metabolites . J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci . 805 , 49 – 56 . Google Scholar CrossRef Search ADS PubMed Campioli E. , Duong T. B. , Deschamps F. , Papadopoulos V. ( 2015 ). Cyclohexane-1, 2-dicarboxylic acid diisononyl ester and metabolite effects on rat epididymal stromal vascular fraction differentiation of adipose tissue . Environ. Res . 140 , 145 – 156 . Google Scholar CrossRef Search ADS PubMed Campioli E. , Lee S. , Lau M. , Marques L. , Papadopoulos V. ( 2017 ). Effect of prenatal DINCH plasticizer exposure on rat offspring testicular function and metabolism . Sci. Rep . 7 , 11072. Google Scholar CrossRef Search ADS PubMed Carreau S. , Hess R. A. ( 2010 ). Oestrogens and spermatogenesis . Philos. Trans. R. Soc. Lond. B. Biol. Sci . 365 , 1517 – 1535 . Google Scholar CrossRef Search ADS PubMed Cooke P. S. , Nanjappa M. K. , Ko C. , Prins G. S. , Hess R. A. ( 2017 ). Estrogens in male physiology . Physiol. Rev . 97 , 995 – 1043 . Google Scholar CrossRef Search ADS PubMed Culty M. , Thuillier R. , Li W. , Wang Y. , Martinez-Arguelles D. B. , Benjamin C. G. , Triantafilou K. M. , Zirkin B. R. , Papadopoulos V. ( 2008 ). In utero exposure to di-(2-ethylhexyl) phthalate exerts both short-term and long-lasting suppressive effects on testosterone production in the rat . Biol. Reprod . 78 , 1018 – 1028 . Google Scholar CrossRef Search ADS PubMed Dalsenter P. R. , Santana G. M. , Grande S. W. , Andrade A. J. M. , Araújo S. L. ( 2006 ). Phthalate affect the reproductive function and sexual behavior of male Wistar rats . Hum. Exp. Toxicol . 25 , 297 – 303 . Google Scholar CrossRef Search ADS PubMed Dorostghoal M. , Moazedi A. A. , Zardkaf A. ( 2012 ). Long-term effects of maternal exposure to Di (2-ethylhexyl) Phthalate on sperm and testicular parameters in Wistar rats offspring . Iran. J. Reprod. Med . 10 , 7 – 14 . Google Scholar PubMed Duty S. M. , Silva M. J. , Barr D. B. , Brock J. W. , Ryan L. , Chen Z. , Herrick R. F. , Christiani D. C. , Hauser R. ( 2003 ). Phthalate exposure and human semen parameters . Epidemiology 14 , 269 – 277 . Google Scholar PubMed Erythropel H. C. , Dodd P. , Leask R. L. , Maric M. , Cooper D. G. ( 2013 ). Designing green plasticizers: Influence of alkyl chain length on biodegradation and plasticization properties of succinate based plasticizers . Chemosphere 91 , 358 – 365 . Google Scholar CrossRef Search ADS PubMed Erythropel H. C. , Maric M. , Nicell J. A. , Leask R. L. , Yargeau V. ( 2014 ). Leaching of the plasticizer di(2-ethylhexyl)phthalate (DEHP) from plastic containers and the question of human exposure . Appl. Microbiol. Biotechnol . 98 , 9967 – 9981 . Google Scholar CrossRef Search ADS PubMed Firlotte N. , Cooper D. G. , Maric M. , Nicell J. A. ( 2009 ). Characterization of 1,5‐pentanediol dibenzoate as a potential “green” plasticizer for poly(vinyl chloride) . J. Vinyl Addit. Technol . 15 , 99 – 107 . Giovanoulis G. , Alves A. , Papadopoulou E. , Cousins A. P. , Schütze A. , Koch H. M. , Haug L. S. , Covaci A. , Magnér J. , Voorspoels S. ( 2016 ). Evaluation of exposure to phthalate esters and DINCH in urine and nails from a Norwegian study population . Environ. Res . 151 , 80 – 90 . Google Scholar CrossRef Search ADS PubMed Gray L. E. , Barlow N. J. , Howdeshell K. L. , Ostby J. S. , Furr J. R. , Gray C. L. ( 2009 ). Transgenerational effects of di (2-ethylhexyl) phthalate in the male CRL: CD(SD) rat: added value of assessing multiple offspring per litter . Toxicol. Sci . 110 , 411 – 425 . Google Scholar CrossRef Search ADS PubMed Gray L. E. , Ostby J. , Furr J. , Price M. , Veeramachaneni D. N. , Parks L. ( 2000 ). Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat . Toxicol. Sci . 58 , 350 – 365 . Google Scholar CrossRef Search ADS PubMed Hannon P. R. , Flaws J. A. ( 2015 ). The effects of phthalates on the ovary . Front. Endocrinol . 2 , 8 . Hauser R. , Calafat A. M. ( 2005 ). Phthalates and human health . Occup. Environ. Med . 62 , 806 – 818 . Google Scholar CrossRef Search ADS PubMed Hess R. A. , Fernandes S. A. F. , Gomes G. R. O. , Oliveira C. A. , Lazari M. F. M. , Porto C. S. ( 2011 ). Estrogen and its receptors in efferent ductules and epididymis . J. Androl . 32 , 600 – 613 . Google Scholar CrossRef Search ADS PubMed Heudorf U. , Mersch-Sundermann V. , Angerer J. ( 2007 ). Phthalates: toxicology and exposure . Int. J. Hyg. Environ. Health 210 , 623 – 634 . Google Scholar CrossRef Search ADS PubMed Kacew S. , Festing M. F. ( 1996 ). Role of rat strain in the differential sensitivity to pharmaceutical agents and naturally occurring substances . J. Toxicol. Environ. Health 47 , 1 – 30 . Google Scholar PubMed Kermanshahi-pour A. , Cooper D. G. , Mamer O. A. , Maric M. , Nicell J. A. ( 2009 ). Mechanisms of biodegradation of dibenzoate plasticizers . Chemosphere 77 , 258 – 263 . Google Scholar CrossRef Search ADS PubMed Kikuchi M. , Mizoroki S. , Kubo T. , Ohiwa Y. , Kubota M. , Yamada N. , Orino K. , Ohnami Y. , Watanabe K. ( 2003a ). Seminal plasma lactoferrin but not transferrin reflects gonadal function in dogs . J. Vet. Med. Sci . 65 , 679 – 684 . Google Scholar CrossRef Search ADS Kikuchi M. , Takao Y. , Tokuda N. , Ohnami Y. , Orino K. , Watanabe K. ( 2003b ). Relationship between seminal plasma lactoferrin and gonadal function in horses . J. Vet. Med. Sci . 65 , 1273 – 1274 . Google Scholar CrossRef Search ADS Kilcoyne K. R. , Smith L. B. , Atanassova N. , Macpherson S. , McKinnell C. , van den Driesche S. , Jobling M. S. , Chambers T. J. G. , De Gendt K. , Verhoeven G. et al. , . ( 2014 ). Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells . Proc. Natl Acad. Sci. USA 111 , E1924 – E1932 . Google Scholar CrossRef Search ADS Koch H. M. , Drexler H. , Angerer J. ( 2003 ). An estimation of the daily intake of di(2-ethylhexyl)phthalate (DEHP) and other phthalates in the general population . Int. J. Hyg. Environ. Health 206 , 77 – 83 . Google Scholar CrossRef Search ADS PubMed Latini G. , De Felice C. , Presta G. , Del Vecchio A. , Paris I. , Ruggieri F. , Mazzeo P. ( 2003 ). Exposure to Di(2-ethylhexyl)phthalate in humans during pregnancy. A preliminary report . Biol. Neonate 83 , 22 – 24 . Google Scholar CrossRef Search ADS PubMed Martinez-Arguelles D. B. , Culty M. , Zirkin B. R. , Papadopoulos V. ( 2009 ). In utero exposure to di-(2-ethylhexyl) phthalate decreases mineralocorticoid receptor expression in the adult testis . Endocrinology 150 , 5575 – 5585 . Google Scholar CrossRef Search ADS PubMed Nair A. B. , Jacob S. ( 2016 ). A simple practice guide for dose conversion between animals and human . J. Basic Clin. Pharm . 7 , 27 – 31 . Google Scholar CrossRef Search ADS PubMed Nardelli T. C. , Albert O. , Lalancette C. , Culty M. , Hales B. F. , Robaire B. ( 2017 ). In utero and lactational exposure study in rats to identify replacements for Di(2-ethylhexyl) phthalate . Sci. Rep . 7 , 623. Google Scholar CrossRef Search ADS PubMed Nardelli T. C. , Erythropel H. C. , Robaire B. ( 2015 ). Toxicogenomic screening of replacements for di(2-ethylhexyl) phthalate (DEHP) using the immortalized TM4 sertoli cell line . PLoS ONE 10 , e0138421. Google Scholar CrossRef Search ADS PubMed O'Donnell L. , Robertson K. M. , Jones M. E. , Simpson E. R. ( 2001 ). Estrogen and spermatogenesis . Endocr. Rev . 22 , 289 – 318 . Google Scholar CrossRef Search ADS PubMed Parks L. G. , Ostby J. S. , Lambright C. R. , Abbott B. D. , Klinefelter G. R. , Barlow N. J. , Gray L. E. ( 2000 ). The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat . Toxicol. Sci . 58 , 339 – 349 . Google Scholar CrossRef Search ADS PubMed Pezzi V. , Sirianni R. , Chimento A. , Maggiolini M. , Bourguiba S. , Delalande C. , Carreau S. , Andò S. , Simpson E. R. , Clyne C. D. ( 2004 ). Differential expression of steroidogenic factor-1/adrenal 4 binding protein and liver receptor homolog-1 (LRH-1)/fetoprotein transcription factor in the rat testis: LRH-1 as a potential regulator of testicular aromatase expression . Endocrinology 145 , 2186 – 2196 . Google Scholar CrossRef Search ADS PubMed Robaire B. , Ewing L. L. , Irby D. C. , Desjardins C. ( 1979 ). Interactions of testosterone and estradiol-17 beta on the reproductive tract of the male rat . Biol. Reprod . 21 , 455 – 463 . Google Scholar CrossRef Search ADS PubMed Robb G. W. , Amann R. P. , Killian G. J. ( 1978 ). Daily sperm production and epididymal sperm reserves of pubertal and adult rats . J. Reprod. Fertil . 54 , 103 – 107 . Google Scholar CrossRef Search ADS PubMed Schettler T. ( 2006 ). Human exposure to phthalates via consumer products . Int. J. Androl . 29 , 134 – 139 . discussion181–5. Google Scholar CrossRef Search ADS PubMed Shellabarger C. J. , Stone J. P. , Holtzman S. ( 1978 ). Rat differences in mammary tumor induction with estrogen and neutron radiation . J. Natl Cancer Inst . 61 , 1505 – 1508 . Google Scholar PubMed Silva M. J. , Barr D. B. , Reidy J. A. , Malek N. A. , Hodge C. C. , Caudill S. P. , Brock J. W. , Needham L. L. , Calafat A. M. ( 2004a ). Urinary levels of seven phthalate metabolites in the U.S. population from the National Health and Nutrition Examination Survey (NHANES) 1999–2000 . Environ. Health Perspect . 112 , 331 – 338 . Google Scholar CrossRef Search ADS Silva M. J. , Jia T. , Samandar E. , Preau J. L. , Calafat A. M. ( 2013 ). Environmental exposure to the plasticizer 1, 2-cyclohexane dicarboxylic acid, diisononyl ester (DINCH) in U.S. adults (2000–2012) . Environ. Res . 126 , 159 – 163 . Google Scholar CrossRef Search ADS PubMed Silva M. J. , Reidy J. A. , Herbert A. R. , Preau J. L. , Needham L. L. , Calafat A. M. ( 2004b ). Detection of phthalate metabolites in human amniotic fluid . Bull. Environ. Contam. Toxicol . 72 , 1226 – 1231 . Google Scholar CrossRef Search ADS Sirianni R. , Seely J. B. , Attia G. , Stocco D. M. , Carr B. R. , Pezzi V. , Rainey W. E. ( 2002 ). Liver receptor homologue-1 is expressed in human steroidogenic tissues and activates transcription of genes encoding steroidogenic enzymes . J. Endocrinol . 174 , R13 – R17 . Google Scholar CrossRef Search ADS PubMed Skakkebaek N. E. , Rajpert-De Meyts E. , Main K. M. ( 2001 ). Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects . Hum. Reprod . 16 , 972 – 978 . Google Scholar CrossRef Search ADS PubMed Teng C. T. ( 2006 ). Factors regulating lactoferrin gene expression . Biochem. Cell Biol . 84 , 263 – 267 . Google Scholar CrossRef Search ADS PubMed Teplyuk N. M. , Zhang Y. , Lou Y. , Hawse J. R. , Hassan M. Q. , Teplyuk V. I. , Pratap J. , Galindo M. , Stein J. L. , Stein G. S. et al. , . ( 2009 ). The osteogenic transcription factor runx2 controls genes involved in sterol/steroid metabolism, including CYP11A1 in osteoblasts . Mol. Endocrinol . 23 , 849 – 861 . Google Scholar CrossRef Search ADS PubMed Thomas J. A. , Thomas M. J. ( 1984 ). Biological effects of di-(2-ethylhexyl) phthalate and other phthalic acid esters . Crit. Rev. Toxicol . 13 , 283 – 317 . Google Scholar CrossRef Search ADS PubMed Tsai-Morris C. H. , Aquilano D. R. , Dufau M. L. ( 1985 ). Cellular localization of rat testicular aromatase activity during development . Endocrinology 116 , 38 – 46 . Google Scholar CrossRef Search ADS PubMed U.S. Food and Drug Administration ( 2002 ). Safety assessment of di(2-ethylhexyl)phthalate (DEHP) released from PVC medical devices. Available at: https://www.fda.gov/downloads/MedicalDevices/…/UCM080457.pdf. Accessed March 8, 2018. Wittassek M. , Koch H. M. , Angerer J. , Brüning T. ( 2011 ). Assessing exposure to phthalates—the human biomonitoring approach . Mol. Nutr. Food Res . 55 , 7 – 31 . Google Scholar CrossRef Search ADS PubMed Yu L. C. , Chen Y. H. ( 1993 ). The developmental profile of lactoferrin in mouse epididymis . Biochem. J . 296 , 107 – 111 . Google Scholar CrossRef Search ADS PubMed Zubkova E. V. , Robaire B. ( 2004 ). Effect of glutathione depletion on antioxidant enzymes in the epididymis, seminal vesicles, and liver and on spermatozoa motility in the aging brown Norway rat . Biol. Reprod . 71 , 1002 – 1008 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Toxicological Sciences Oxford University Press

Effects of In Utero and Lactational Exposure to New Generation Green Plasticizers on Adult Male Rats: A Comparative Study With Di(2-Ethylhexyl) Phthalate

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© The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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10.1093/toxsci/kfy072
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Abstract

Abstract Di(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, is a ubiquitous environmental contaminant and may act as an endocrine disruptor. Early life exposures to DEHP may result in anti-androgenic effects, impairing the development of the male reproductive tract. However, data on the long-lasting consequences of such DEHP exposures on adult male reproductive function are still rare and discrepant. Previously, we identified 2 novel plasticizers, 1,4-butanediol dibenzoate (BDB) and dioctyl succinate (DOS), as potential substitutes for DEHP that did not reproduce classically described endocrine disrupting phenotypes in prepubertal male offspring after maternal exposure. Here, we investigated the consequences of in utero and lactational exposure to BDB and DOS on adult male rat reproductive function in a comparative study with DEHP and a commercially available alternative plasticizer, 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH). Timed pregnant Sprague Dawley rats were gavaged with vehicle or a test chemical (30 or 300 mg/kg/day) from gestation day 8 to postnatal day 21. While DEHP exposure (300 mg/kg/day) significantly increased epididymal weight in the adult, exposure to DINCH, BDB, or DOS did not affect reproductive organ weights, steroid levels, or sperm quality. Using a toxicogenomic microarray approach, we found that adult testicular gene expression was affected by exposure to the higher dose of DEHP; transcripts such as Nr5a2, Ltf, or Runx2 were significantly downregulated, suggesting that DEHP was targeting estrogen signaling. Lesser effects were observed after treatment with either DINCH or BDB. DOS exposure did not produce such effects, confirming its potential as a responsible substitute for DEHP. endocrine disruptors, testis, epididymis, androgens, estrogens, sperm Phthalates are plasticizing agents used as emollients, matrices, solvents, and excipients in numerous industrial applications (Heudorf et al., 2007). Among them, di(2-ethylhexyl) phthalate (DEHP) is the most commonly used additive to provide flexibility to polyvinyl chloride (PVC) and is found in products such as construction materials, household products, toys, cosmetics, food packaging, and medical tubing (Schettler, 2006). Since phthalates are not covalently bound to their supports, they leach into the environment over time (Erythropel et al., 2014; Thomas and Thomas, 1984), resulting in widespread human exposure (Hauser and Calafat, 2005; Koch et al., 2003; Wittassek et al., 2011). Biomonitoring studies reveal their presence in several body fluids, including blood, urine (Silva et al., 2004a), semen (Duty et al., 2003), amniotic fluid (Silva et al., 2004b), umbilical cord blood (Latini et al., 2003), and breast milk (Calafat et al., 2004). Over the past few decades, phthalates, including DEHP, have been established to act as endocrine disrupting compounds, producing adverse effects in various in vitro and in vivo models in both males (Albert and Jégou, 2014) and females (Hannon and Flaws, 2015). Numerous studies have demonstrated the effects of in utero and lactational exposure to these agents on the developing fetus, a sensitive target for chemical insults (Skakkebaek et al., 2001). Pre- and perinatal exposure to DEHP in rodents results in decreased testosterone production (Parks et al., 2000) and anogenital index (Gray et al., 2000; Parks et al., 2000), increased nipple retention (Gray et al., 2000), hemorrhagic testes (Gray et al., 2000; Nardelli et al., 2017), and multinucleated gonocytes in the seminiferous chords (Gray et al., 2000; Nardelli et al., 2017; Parks et al., 2000) in male pups. Information on the impact of early life exposure to DEHP in the adult remains limited. In utero and lactational exposure to DEHP in rat models has been reported to result in either unchanged (Akingbemi et al., 2001), increased (Andrade et al., 2006) or decreased (Culty et al., 2008; Martinez-Arguelles et al., 2009) testosterone levels in the adult male. Such changes are sometimes associated with decreased sperm production (Andrade et al., 2006; Dorostghoal et al., 2012) and testicular histological changes and lesions (Andrade et al., 2006; Culty et al., 2008; Dorostghoal et al., 2012), but the underlying mechanisms are still unclear. The growing body of evidence in support of the deleterious effects of phthalates has prompted public health authorities to regulate their use in specific applications, triggering a search for safer replacements. For example, 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) was first introduced to the European market in 2002, received final approval from the European Food Safety Authority in 2006, and has been marketed as a safe replacement for DEHP ever since. Exposure to DINCH is now widespread, as assessed by increased urinary levels of DINCH metabolites (Giovanoulis et al., 2016; Silva et al., 2013). However, recent reports identify DINCH as a bioactive compound (Nardelli et al., 2015), a potent metabolic disruptor (Campioli et al., 2015), and a potential endocrine disruptor (Boisvert et al., 2016; Campioli et al., 2017; Nardelli et al., 2017), raising concerns about its possible impact on public health. In previous studies, we used a 4-step approach to identify greener replacements for DEHP with desirable plasticizing properties, biodegradability, minimal leaching, and fewer deleterious effects. We identified 2 candidate compounds, 1,4-butanediol dibenzoate (BDB) and dioctyl succinate (DOS), that did not cause significant physiological perturbations in several immortalized cell lines or after acute exposure in vivo (Albert et al., 2018; Boisvert et al., 2016; Nardelli et al., 2015). Recently, we demonstrated that in utero and lactational exposure to BDB and DOS did not produce the phenotypes described after exposure to DEHP from gestational day (GD) 8 to post-natal day (PND) 3, 8, or 21 (Nardelli et al., 2017). Here, we sought to investigate the consequences of in utero and lactational exposure to BDB and DOS on the adult male rat reproductive function, in a comparative study with DEHP and DINCH. MATERIALS AND METHODS Chemicals and reagents DEHP was purchased from Sigma-Aldrich Corporation (CAS #117-81-7; Cat #80030, St. Louis, MO). DINCH (CAS #474919-59-0 and 166412-78-8) was purchased from BASF Canada (Mississauga, ON). BDB (CAS #19924-27-2) and DOS (CAS #14491-66-8) were synthesized as previously described (Erythropel et al., 2013; Firlotte et al., 2009; Kermanshahi-pour et al., 2009), and their purity was determined to be 99% by NMR analysis. Chemicals were stored in a vacuum chamber with desiccant at room temperature until mixed with corn oil on each day of treatment (Catalogue #C8267; Lot#MKBN5383V, Sigma-Aldrich). Animals All manipulations and terminal procedures were approved by the McGill University Animal Care Committee (protocol #7317). Mating and treatments were done as described in Nardelli et al. (2017). In brief, virgin female and proven-breeder male Sprague Dawley rats were purchased from Charles River Laboratories (St-Constant, Quebec, Canada) and mated on the morning of proestrus. The next morning, sperm-positive females were placed in individual cages; this was considered GD 0. On GD 8, pregnant dams were randomly assigned to a treatment group, weighed and administered doses of 30 or 300 mg/kg of DEHP, DINCH, BDB, or DOS by gavage; control animals were administered 1 ml of vehicle (corn oil). The lower dose (30 mg/kg) is representative of high human exposure to DEHP (U.S. Food and Drug Administration, 2002) with an adjustment for interspecies metabolism (Nair and Jacob, 2016), and the higher dose (300 mg/kg) was selected based on previous literature that reported deleterious reproductive outcomes following exposure to DEHP during gestation and lactation (Gray et al., 2009). 15–19 dams per group were treated daily across 3 cohorts, except on the day of delivery, until pups were weaned at PND 21. Animals were maintained on a 12-h light/dark cycle and provided food and water ad libitum. At PND 90, 1 male per litter was euthanized by CO2 asphyxiation followed by cardiac puncture using a 10 ml syringe with 21 G 1-1/2-in. needle. Whole blood was collected in a BD Vacutainer SST tube (Becton, Dickinson and Company, Mississauga, ON) by negative pressure. The tubes were inverted several times and allowed to clot at room temperature for 30 min. To isolate serum, tubes were spun at 1000 × g in an Allegra-X 15R benchtop centrifuge (Beckman Coulter, Pasadena CA) at 4°C with a SX4750 swinging bucket rotor. Serum was aliquoted and kept at −80°C until further use. Organs were collected, weighed, flash frozen in liquid nitrogen, and stored at −80°C for further RNA extraction and sperm counts. Sperm head counts 5–10 mm3 portions of frozen testes were thawed on ice and weighed. Frozen epididymides were separated into 2 parts (caput-corpus and cauda) and weighed. Samples were homogenized in 5 ml of 0.9% sodium chloride, 0.1% thimerosal, and 0.05% Triton X-100 using the Polytron PT10-35GT (Kinematica Inc, Bohemia, NY) at 20 000 rpm for 2 intervals of 15 s separated by a 30-s interval. Sperm heads were counted using a hemocytometer (Hausser Scientific, Horsham, PA). Daily sperm production was calculated as described in Robb et al. (1978). Computer-assisted sperm analysis (CASA) The motility of spermatozoa was measured by CASA, as previously described (Zubkova and Robaire, 2004). In brief, immediately following euthanasia, the left cauda epididymidis was clamped, excised and minced in 3 ml of motility buffer [Hanks Balanced Salt Solution (Thermo Fisher Scientific, Waltham, MA), 0.35 g/l sodium bicarbonate (ACP Chemicals, Montreal, QC, CA), 4.2 g/l HEPES (Sigma-Aldrich), 0.9 g/l d-glucose (Sigma-Aldrich), 0.1 g/l sodium pyruvate (Thermo Fisher Scientific), 2 g/l bovine serum albumin (Sigma-Aldrich), 0.025 g/l soybean trypsin inhibitor (Sigma-Aldrich), pH 7.4]. Spermatozoa were allowed to disperse for 3 min and diluted 1:30 in motility buffer. 20 µl of this suspension were loaded onto 80 µm 2X-Cel Dual Sided Sperm Analysis Chambers and covered with 2X-Cel Cover Glass (Hamilton-Thorne Research, Beverly, MA). Movement characteristics were analyzed using the Hamilton-Thorne TOX Integrated Visual Optical System for sperm analysis (version 12). Settings were as follows: stage temperature: 37°C; frames acquired: 30; frame rate: 60 Hz; minimum contrast: 80; minimum cell size: 4 pixels; minimum static contrast: 15; cell intensity: 80: magnification: 0.82; static size limits: 0.29–8.82; static intensity: 0.18–1.8. A minimum of 350 spermatozoa and 5 fields were analyzed per sample. For each animal, data are expressed as the mean of the 5 fields with the highest percentage of motile sperm. Hormone assays Serum testosterone levels were assessed using enzyme-linked immunosorbent assay kits (IB79106; IBL America, Minneapolis, MN) according to the manufacturer’s instructions (dynamic range: 0.083–16 ng/ml; intra-assay variability: 3.2%–4.2%; inter-assay variability: 4.7%–9.9%). Luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations were assessed by the Ligand Assay & Analysis Core of the University of Virginia School of Medicine Center for Research in Reproduction using Millipore Pituitary Panel Multiplex kits (EMD Millipore, Saint Charles, MO). RNA extraction RNA was extracted from entire testes using the Qiagen RNEasy Mini Kit (Qiagen, Toronto, ON). In brief, frozen testes were pulverized to a thin powder using a chilled mortar and pestle. Approximately 30 mg of powder were placed in 1 ml of RLT buffer supplemented with ß-mercaptoethanol and mechanically disrupted using a rotor–stator homogenizer (Polytron PT10-35GT, Kinematica Inc.) at 20 000 rpm for 20 s. Homogenates were further processed with a QIAshredder column (Qiagen), and RNA was isolated as per the manufacturer’s instructions. RNA quality was measured using a NanoDrop 2000 Spectrophotometer (Thermo Fisher), with 260/280 and 260/230 absorbance ratios comprised between 1.9–2.1 and 2.0–2.2, respectively. Microarrays RNA (100 ng) was labeled with Cy3 using the Agilent One Color Low Input Quick Amp kit and hybridized on arrays using the SurePrint G3 Rat Gene Expression 8x60K microarray kit (Agilent Technologies, Santa Clara, CA) as per manufacturer’s instructions. All arrays were confirmed by the manufacturer to be from the same batch. Arrays were done using RNA from the testes of 6 animals from independent litters for each treatment group and read on an Agilent SureScan Microarray Scanner G2600D. The resulting data were analyzed using GeneSpring version 14.9 (Agilent Technologies). Data were normalized using percentile shift normalization to the 75th percentile. Following quality control using principal component scores on all arrays, 1 sample was removed in the DINCH 30 group. Transcripts affected by more than 1.5-fold were determined using a moderated t test and Benjamini–Hochberg FDR correction for each treatment. The microarray data have been uploaded to Gene Expression Omnibus (accession number GSE110553). The biological relationships between transcripts were analyzed using Ingenuity Pathway Analysis, version 01.12 (42012434). RT-qPCR We identified 6 genes of interest to be validated (Supplementary Table 1) from the microarray data. RNA from whole testis was converted to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen), as per the manufacturer’s instructions. Gene expression was quantified using the QuantiTect SYBR Green PCR Kit (Qiagen) using the StepOne Plus Real Time PCR System (Applied Biosytems, Foster City, CA) with the following thermal conditions: initial heat activation for 15 min at 95°C, followed by 50 cycles of denaturation for 15 s at 94°C, annealing for 30 s at 55°C, and extension for 30 s at 72°C. Serial dilutions of whole testis RNA pooled from animals from all treatment groups were used as an internal reference and to create standard curves for primer efficiency and template concentration optimization. Each gene was amplified in triplicate from 6 independent samples for each treatment group. Relative levels of gene amplification compared with the control group and housekeeping gene, Ppia, were calculated using StepOnePlus Software (version 2.1). A melt curve was systematically generated to ensure the specificity of the PCR reaction. Statistical analysis Significance was determined by ANOVA followed by Dunnett’s post hoc test. Outliers due to biological differences have not been removed from any of the data. Statistical calculations were generated using GraphPad Prism 6.07 (GraphPad Software, La Jolla, CA). RESULTS Overall Health and Androgen-Sensitive Organ Weights Maternal and offspring overall health data were previously published in Nardelli et al. (2017). In brief, there was no significant effect of the treatments on maternal weight gain or general health except for a significant decrease in heart weight after exposure to 300 mg/kg/day DEHP. Litter sizes, offspring viability, and postnatal growth remained unchanged by treatment. Animals were weighed and necropsied at PND 90 to assess whether exposure to the treatments altered general health. In utero and lactational exposure to DEHP, DINCH, BDB, and DOS did not alter body weight in adult males. This was also the case for the weights of the heart, lungs, liver, spleen, and kidneys (Supplementary Table 2). Exposure to 300 mg/kg/day DEHP produced a significant increase in paired epididymides weight (p = .02), but other androgen-responsive and reproductive tract organs, including paired testes and seminal vesicles, were unaffected by the treatment (Figure 1). Figure 1. View largeDownload slide Androgen-sensitive organ weights in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Data are standardized to 100 g body weight (bw) except for testes, which are encapsulated organs. Significance was determined by one-way ANOVA corrected by Dunnett’s multiple comparison test; n = 8–11 animals from independent litters per group; *p < .05. Figure 1. View largeDownload slide Androgen-sensitive organ weights in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Data are standardized to 100 g body weight (bw) except for testes, which are encapsulated organs. Significance was determined by one-way ANOVA corrected by Dunnett’s multiple comparison test; n = 8–11 animals from independent litters per group; *p < .05. Hormonal Measurements Serum from males from 7 to 11 independent litters was assessed for testosterone, LH, and FSH concentrations to determine whether early life exposures disrupt hormonal function in adult males (Figure 2). None of the treatments significantly altered these hormones. The ratio of testosterone to LH, a measure for possible compensated Leydig cell failure, also remained unaffected (Figure 2C). Figure 2. View largeDownload slide Serum testosterone and gonadotropin levels in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Serum testosterone (A), LH (B) and FSH (D) were assessed in 7–11 males from independent litters. The ratio of testosterone to LH (C), which allows detection of potential compensated Leydig cell failure, was calculated as well. No significant difference was detected using one-way ANOVA corrected by Dunnett’s multiple comparison test. Figure 2. View largeDownload slide Serum testosterone and gonadotropin levels in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Serum testosterone (A), LH (B) and FSH (D) were assessed in 7–11 males from independent litters. The ratio of testosterone to LH (C), which allows detection of potential compensated Leydig cell failure, was calculated as well. No significant difference was detected using one-way ANOVA corrected by Dunnett’s multiple comparison test. Sperm Count and Motility Testicular and epididymal sperm heads were counted to further investigate the effects of in utero and lactational exposure to DEHP, DINCH, and candidate replacement plasticizers. There were no significant effects on sperm counts in the testis, combined caput and corpus epididymides, or cauda epididymidis of exposed animals (Figs. 3A–C). Daily sperm production was unaltered by the treatments (Figure 3D). Immediately following necropsy, cauda sperm motility was also assessed using CASA. None of the treatments significantly altered sperm motility (Table 1). Table 1. CASA of Adult Cauda Sperm After In Utero and Lactational Exposure to Corn Oil (Vehicle), DEHP, DINCH, BDB, or DOS Dose (mg/kg/day) CORN OIL DEHP DINCH BDB DOS 30 300 30 300 30 300 30 300 Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Motile sperm (%) 73.1 1.6 72.1 0.8 73.0 2.1 72.4 2.7 72.8 3.2 74.6 2.6 72.5 2.2 71.7 2.3 74.1 1.3 Progressively motile (%) 32.9 1.9 30.1 1.9 31.5 2.3 32.3 2.5 28.0 3.4 32.8 2.1 33.1 2.5 35.1 2.9 33.5 2.1 Rapid velocity (%) 43.0 2.5 40.0 2.4 41.5 3.7 44.5 2.3 37.9 4.3 44.0 2.3 44.1 3.1 46.7 3.7 44.7 2.7 Medium velocity (%) 0.8 0.3 1.0 0.3 1.8 1.0 0.9 0.3 1.7 0.6 2.2 0.8 1.1 0.5 1.1 0.4 1.2 0.5 Slow velocity (%) 29.3 2.2 31.1 2.1 29.8 3.5 27.0 3.4 33.3 3.4 28.3 2.4 27.2 1.9 24.1 3.0 28.2 2.6 Static (%) 26.9 1.6 27.9 0.8 27.1 2.1 27.7 2.7 27.2 3.2 25.4 2.6 27.5 2.2 28.4 2.3 25.9 1.3 VAP (µm/s) 153.5 3.3 152.8 4.6 146.9 8.1 157.3 5.6 140.8 6.7 149.3 5.4 151.5 3.0 149.4 4.4 151.3 4.5 VSL (µm/s) 81.2 1.8 79.2 2.4 77.6 2.3 80.8 2.5 73.0 2.3 77.2 2.4 80.3 1.8 79.7 3.0 78.8 1.9 VCL (µm/s) 337.5 8.6 338.9 12.2 326.7 20.1 347.9 18.6 316.5 15.3 326.9 13.4 344.2 11.1 338.8 14.5 329.8 9.3 ALH (µm/s) 23.1 0.4 23.5 0.6 22.0 0.9 23.6 0.9 22.0 0.6 22.5 0.5 23.0 0.4 22.2 0.7 22.5 0.6 BCF (Hz) 33.0 0.5 32.9 0.6 33.6 0.6 33.4 0.9 35.0 0.7 34.1 0.8 35.5 0.8 33.9 0.6 34.0 0.7 STR (%) 55.4 0.8 54.5 0.8 56.2 2.0 54.0 1.2 55.5 1.5 55.0 1.0 55.7 0.9 56.1 1.2 55.1 0.9 LIN (%) 26.0 0.4 25.5 0.5 26.3 1.1 25.5 0.8 26.2 0.8 26.2 0.7 25.7 0.7 26.0 0.5 26.7 0.6 Dose (mg/kg/day) CORN OIL DEHP DINCH BDB DOS 30 300 30 300 30 300 30 300 Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Motile sperm (%) 73.1 1.6 72.1 0.8 73.0 2.1 72.4 2.7 72.8 3.2 74.6 2.6 72.5 2.2 71.7 2.3 74.1 1.3 Progressively motile (%) 32.9 1.9 30.1 1.9 31.5 2.3 32.3 2.5 28.0 3.4 32.8 2.1 33.1 2.5 35.1 2.9 33.5 2.1 Rapid velocity (%) 43.0 2.5 40.0 2.4 41.5 3.7 44.5 2.3 37.9 4.3 44.0 2.3 44.1 3.1 46.7 3.7 44.7 2.7 Medium velocity (%) 0.8 0.3 1.0 0.3 1.8 1.0 0.9 0.3 1.7 0.6 2.2 0.8 1.1 0.5 1.1 0.4 1.2 0.5 Slow velocity (%) 29.3 2.2 31.1 2.1 29.8 3.5 27.0 3.4 33.3 3.4 28.3 2.4 27.2 1.9 24.1 3.0 28.2 2.6 Static (%) 26.9 1.6 27.9 0.8 27.1 2.1 27.7 2.7 27.2 3.2 25.4 2.6 27.5 2.2 28.4 2.3 25.9 1.3 VAP (µm/s) 153.5 3.3 152.8 4.6 146.9 8.1 157.3 5.6 140.8 6.7 149.3 5.4 151.5 3.0 149.4 4.4 151.3 4.5 VSL (µm/s) 81.2 1.8 79.2 2.4 77.6 2.3 80.8 2.5 73.0 2.3 77.2 2.4 80.3 1.8 79.7 3.0 78.8 1.9 VCL (µm/s) 337.5 8.6 338.9 12.2 326.7 20.1 347.9 18.6 316.5 15.3 326.9 13.4 344.2 11.1 338.8 14.5 329.8 9.3 ALH (µm/s) 23.1 0.4 23.5 0.6 22.0 0.9 23.6 0.9 22.0 0.6 22.5 0.5 23.0 0.4 22.2 0.7 22.5 0.6 BCF (Hz) 33.0 0.5 32.9 0.6 33.6 0.6 33.4 0.9 35.0 0.7 34.1 0.8 35.5 0.8 33.9 0.6 34.0 0.7 STR (%) 55.4 0.8 54.5 0.8 56.2 2.0 54.0 1.2 55.5 1.5 55.0 1.0 55.7 0.9 56.1 1.2 55.1 0.9 LIN (%) 26.0 0.4 25.5 0.5 26.3 1.1 25.5 0.8 26.2 0.8 26.2 0.7 25.7 0.7 26.0 0.5 26.7 0.6 Mean and standard errors of the mean (SEM) are given for each measured parameter. VAP, average path velocity; VSL, progressive velocity; VCL, track speed; ALH, average lateral amplitude; BCF, beat frequency; STR, straightness; LIN, linearity Table 1. CASA of Adult Cauda Sperm After In Utero and Lactational Exposure to Corn Oil (Vehicle), DEHP, DINCH, BDB, or DOS Dose (mg/kg/day) CORN OIL DEHP DINCH BDB DOS 30 300 30 300 30 300 30 300 Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Motile sperm (%) 73.1 1.6 72.1 0.8 73.0 2.1 72.4 2.7 72.8 3.2 74.6 2.6 72.5 2.2 71.7 2.3 74.1 1.3 Progressively motile (%) 32.9 1.9 30.1 1.9 31.5 2.3 32.3 2.5 28.0 3.4 32.8 2.1 33.1 2.5 35.1 2.9 33.5 2.1 Rapid velocity (%) 43.0 2.5 40.0 2.4 41.5 3.7 44.5 2.3 37.9 4.3 44.0 2.3 44.1 3.1 46.7 3.7 44.7 2.7 Medium velocity (%) 0.8 0.3 1.0 0.3 1.8 1.0 0.9 0.3 1.7 0.6 2.2 0.8 1.1 0.5 1.1 0.4 1.2 0.5 Slow velocity (%) 29.3 2.2 31.1 2.1 29.8 3.5 27.0 3.4 33.3 3.4 28.3 2.4 27.2 1.9 24.1 3.0 28.2 2.6 Static (%) 26.9 1.6 27.9 0.8 27.1 2.1 27.7 2.7 27.2 3.2 25.4 2.6 27.5 2.2 28.4 2.3 25.9 1.3 VAP (µm/s) 153.5 3.3 152.8 4.6 146.9 8.1 157.3 5.6 140.8 6.7 149.3 5.4 151.5 3.0 149.4 4.4 151.3 4.5 VSL (µm/s) 81.2 1.8 79.2 2.4 77.6 2.3 80.8 2.5 73.0 2.3 77.2 2.4 80.3 1.8 79.7 3.0 78.8 1.9 VCL (µm/s) 337.5 8.6 338.9 12.2 326.7 20.1 347.9 18.6 316.5 15.3 326.9 13.4 344.2 11.1 338.8 14.5 329.8 9.3 ALH (µm/s) 23.1 0.4 23.5 0.6 22.0 0.9 23.6 0.9 22.0 0.6 22.5 0.5 23.0 0.4 22.2 0.7 22.5 0.6 BCF (Hz) 33.0 0.5 32.9 0.6 33.6 0.6 33.4 0.9 35.0 0.7 34.1 0.8 35.5 0.8 33.9 0.6 34.0 0.7 STR (%) 55.4 0.8 54.5 0.8 56.2 2.0 54.0 1.2 55.5 1.5 55.0 1.0 55.7 0.9 56.1 1.2 55.1 0.9 LIN (%) 26.0 0.4 25.5 0.5 26.3 1.1 25.5 0.8 26.2 0.8 26.2 0.7 25.7 0.7 26.0 0.5 26.7 0.6 Dose (mg/kg/day) CORN OIL DEHP DINCH BDB DOS 30 300 30 300 30 300 30 300 Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Motile sperm (%) 73.1 1.6 72.1 0.8 73.0 2.1 72.4 2.7 72.8 3.2 74.6 2.6 72.5 2.2 71.7 2.3 74.1 1.3 Progressively motile (%) 32.9 1.9 30.1 1.9 31.5 2.3 32.3 2.5 28.0 3.4 32.8 2.1 33.1 2.5 35.1 2.9 33.5 2.1 Rapid velocity (%) 43.0 2.5 40.0 2.4 41.5 3.7 44.5 2.3 37.9 4.3 44.0 2.3 44.1 3.1 46.7 3.7 44.7 2.7 Medium velocity (%) 0.8 0.3 1.0 0.3 1.8 1.0 0.9 0.3 1.7 0.6 2.2 0.8 1.1 0.5 1.1 0.4 1.2 0.5 Slow velocity (%) 29.3 2.2 31.1 2.1 29.8 3.5 27.0 3.4 33.3 3.4 28.3 2.4 27.2 1.9 24.1 3.0 28.2 2.6 Static (%) 26.9 1.6 27.9 0.8 27.1 2.1 27.7 2.7 27.2 3.2 25.4 2.6 27.5 2.2 28.4 2.3 25.9 1.3 VAP (µm/s) 153.5 3.3 152.8 4.6 146.9 8.1 157.3 5.6 140.8 6.7 149.3 5.4 151.5 3.0 149.4 4.4 151.3 4.5 VSL (µm/s) 81.2 1.8 79.2 2.4 77.6 2.3 80.8 2.5 73.0 2.3 77.2 2.4 80.3 1.8 79.7 3.0 78.8 1.9 VCL (µm/s) 337.5 8.6 338.9 12.2 326.7 20.1 347.9 18.6 316.5 15.3 326.9 13.4 344.2 11.1 338.8 14.5 329.8 9.3 ALH (µm/s) 23.1 0.4 23.5 0.6 22.0 0.9 23.6 0.9 22.0 0.6 22.5 0.5 23.0 0.4 22.2 0.7 22.5 0.6 BCF (Hz) 33.0 0.5 32.9 0.6 33.6 0.6 33.4 0.9 35.0 0.7 34.1 0.8 35.5 0.8 33.9 0.6 34.0 0.7 STR (%) 55.4 0.8 54.5 0.8 56.2 2.0 54.0 1.2 55.5 1.5 55.0 1.0 55.7 0.9 56.1 1.2 55.1 0.9 LIN (%) 26.0 0.4 25.5 0.5 26.3 1.1 25.5 0.8 26.2 0.8 26.2 0.7 25.7 0.7 26.0 0.5 26.7 0.6 Mean and standard errors of the mean (SEM) are given for each measured parameter. VAP, average path velocity; VSL, progressive velocity; VCL, track speed; ALH, average lateral amplitude; BCF, beat frequency; STR, straightness; LIN, linearity Figure 3. View largeDownload slide Sperm head counts in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Sperm heads were counted in the testis (A), caput and corpus epididymides (B) or cauda epididymidis (C) and reported as 108 per gram of each. Daily sperm production is reported for each treatment (D). n = 4–7 males from independent litters. No significant difference was detected using one-way ANOVA corrected by Dunnett’s multiple comparison test. Figure 3. View largeDownload slide Sperm head counts in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Sperm heads were counted in the testis (A), caput and corpus epididymides (B) or cauda epididymidis (C) and reported as 108 per gram of each. Daily sperm production is reported for each treatment (D). n = 4–7 males from independent litters. No significant difference was detected using one-way ANOVA corrected by Dunnett’s multiple comparison test. Testicular Gene Expression Analysis The consequences of perinatal exposure to our candidate compounds was assessed on global testicular gene expression to provide additional insights into their possible biological effects and to identify relevant mechanisms of action that could contribute to an increase in epididymal weight observed after exposure to DEHP. Using single-color microarrays, gene expression was analyzed in 6 independent PND 90 adult males per treatment group. Figure 4A depicts the overall relationship between transcript responses to all treatments using principal component analysis (PCA). This exploratory multidimensional data mining procedure converts a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components. Treatments producing similar gene expression responses are plotted in close proximity. Here, PCA analysis described three major components representing 23.66 (x-axis), 17.07 (y-axis), and 13.45% (z-axis) of the variance among all samples. Exposure to 30 mg/kg/day BDB or DOS produced expression profiles that clustered closely to that of corn oil (grey circle, Figure 4A); at the same dose, both DEHP and DINCH displayed profiles that were isolated and distant from this grouping. At 300 mg/kg/day, DEHP, DINCH, and BDB clustered together and further away from corn oil along the third component, while DOS presented a more isolated profile along the first and second components. Figure 4. View largeDownload slide Testicular gene expression in PND 90 rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. (A) PCA of single-color microarray data; (B) Number of uniquely mapped transcripts significantly up or downregulated by more than 1.5-fold determined by moderated t test and Benjamini–Hochberg FDR correction (p > .05); (C) Venn diagrams representing commonalities in differential gene expression between treatments at 30 and 300 mg/kg/day, respectively. All data were generated using n = 5–6 male rats from independent litters. Figure 4. View largeDownload slide Testicular gene expression in PND 90 rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. (A) PCA of single-color microarray data; (B) Number of uniquely mapped transcripts significantly up or downregulated by more than 1.5-fold determined by moderated t test and Benjamini–Hochberg FDR correction (p > .05); (C) Venn diagrams representing commonalities in differential gene expression between treatments at 30 and 300 mg/kg/day, respectively. All data were generated using n = 5–6 male rats from independent litters. Independent statistical comparisons were used to identify the numbers of transcripts that were significantly differentially expressed, with a fold-change threshold of 1.5, between corn oil and each individual treatment (Figure 4B). In the 30 mg/kg/day treatment groups, DEHP, DINCH, BDB, and DOS significantly altered the expression of 12, 19, 3, and 15 transcripts, respectively. Exposure to 300 mg/kg/day DEHP produced the greatest effect, with 55 affected transcripts (26 upregulated and 29 downregulated). Comparatively, at an equivalent dose, DINCH significantly altered 14 transcripts, BDB altered 27 transcripts, and treatment with DOS produced no significant effect. Venn diagrams in Figure 4C represent the overlap between significantly altered transcript sets for all treatments at a given dose. At both 30 and 300 mg/kg/day, BDB and DOS shared no differentially expressed transcript with DEHP or with each other. At 30 mg/kg/day, DINCH and DOS both significantly upregulated the expression of Upk3bl (uroplakin 3b-like protein), at fold-changes of 1.9 and 2.0, respectively. At 300 mg/kg/day, DEHP and DINCH both downregulated the expression of Hpgds by fold-changes of 1.7 and 1.6, respectively, and Ly6g6f (lymphocyte antigen 6 family member G6F) by fold-changes of 1.9 and 1.7, respectively. Finally, at 300 mg/kg/day, both DINCH and BDB downregulated the expression of HS3ST3A1 (heparan sulfate-glucosamine 3-sulfotransferase 3A1) by 2.0 and 2.1, respectively, as well as the expression of the LOC100910837 loci (olfactory receptor 2AK2-like) by fold-changes of 1.7 and 1.5, respectively. Commonalities between doses for each treatment were quite limited. Exposure to 30 and 300 mg/kg/day DEHP significantly upregulated the expression of Xkr9 (XK, Kell Blood Group Complex Subunit-Related Family, Member 9) by 3.3- and 3.1-fold, respectively, and downregulated the expression of mitogen activated protein kinase 4 (Mapk4) by 1.7- and 1.5-fold, respectively. Overall, the limited overlap between treatments suggests that our candidate compounds do not share key mechanisms of action with DEHP and DINCH. For each treatment, we further investigated the transcripts that were the most significantly altered based on absolute fold change (Table 2). To understand the biological relationships underlying such gene expression changes, the transcripts that were significantly altered by 1.5-fold or more were imported into Ingenuity Pathway Analysis. Pathways that were predicted to be significantly affected by the treatment and related transcripts are listed in Supplementary Table 3. While these pathways were not specifically predicted to be activated or inhibited (null z-score) due to the limited number of transcripts on these lists, they may identify gene enrichment for important physiological pathways within our datasets. Based on the latter information, and considering the transcripts with highest absolute fold changes and their role relative to testicular function, we selected 6 targets of interest to be validated by RT-qPCR (Figure 5). Table 2. Testicular Transcripts Significantly Affected With a >1.5-Fold Change After In Utero and Lactational Exposure to DEHP, DINCH, BDB, or DOS ID Symbol Expression p-value Expression fold change DEHP 30 A_64_P009798 Nr5a2* 3.79E−02 −7.306 A_64_P103179 Xkr9 3.79E−02 3.288 A_44_P260348 LOC689316 3.79E−02 −3.143 A_64_P159163 Gm1527/Gm6558 3.79E−02 −2.102 A_64_P048106 Foxp2 3.79E−02 1.91 A_44_P271529 Olfr380 3.79E−02 −1.808 A_64_P051307 Runx2* 3.79E−02 −1.768 A_43_P16031 Mapk4* 4.63E−02 −1.706 A_42_P794562 Cyp7a1 3.79E−02 1.701 A_64_P051039 Spon2 3.79E−02 −1.644 A_64_P072739 Flrt1 3.79E−02 −1.588 A_44_P161470 Defa6 (includes others) 4.48E−02 1.515 DEHP 300 A_44_P636808 9230104L09Rik 4.68E−02 3.585 A_64_P103179 Xkr9 1.88E−02 3.127 A_64_P023008 LOC100911534 4.48E−02 2.453 A_44_P419034 Olr181/Olr183 4.37E−02 −2.37 A_64_P232281 Vom2r1 (includes others) 5.59E−03 2.143 A_64_P064556 Slc36a4 1.28E−02 2.063 A_64_P047621 Ildr1 1.41E−02 −2.044 A_44_P405374 LOC290071 1.88E−02 2.027 A_64_P132182 Zeb2os 4.37E−02 1.956 A_43_P11449 Cpb1 1.41E−02 1.944 A_44_P351263 Ly6g6f 1.41E−02 −1.935 A_64_P087625 LOC102552143/LOC102552337 4.57E−02 1.927 A_44_P689645 Enox2 1.41E−02 −1.894 A_64_P038812 Erbb2 4.37E−02 1.87 A_64_P004007 Rd3l 4.37E−02 1.861 A_44_P140275 Alpk1 1.43E−02 1.856 A_64_P075532 Gm8882 (includes others) 4.02E−02 −1.827 A_64_P090866 Sstr1 4.37E−02 −1.808 A_44_P558411 Scrg1 4.81E−02 1.804 A_64_P063443 LOC103690069 2.71E−02 1.788 A_64_P131538 Olfr649 2.59E−03 −1.768 A_64_P080530 Nrxn3 4.93E−02 1.757 A_64_P060018 Pilrb 4.37E−02 −1.746 A_64_P108698 Cryge/Crygf 4.37E−02 1.711 A_43_P11278 Hpgd 2.71E−02 −1.707 A_44_P439292 Hpgds* 4.86E−02 −1.699 A_64_P111725 LOC103690800 2.66E−02 1.675 A_64_P160550 Gpr171 4.48E−02 1.67 A_64_P055137 Gldn 1.00E−02 −1.665 A_64_P150603 Ptger3 3.36E−02 −1.653 A_42_P736315 Rtp4 5.59E−03 −1.643 A_44_P515494 Ddr1 3.76E−02 −1.64 A_64_P033121 Rasa4 4.57E−02 1.635 A_44_P496572 Kmo 3.49E−02 −1.625 A_64_P156001 Olr396 4.67E−02 −1.616 A_64_P106933 Foxh1 5.59E−03 1.614 A_44_P426079 Kcnc1 3.49E−02 −1.604 A_64_P063558 LOC499764 4.02E−02 −1.593 A_44_P1013314 Iisg15 3.71E−02 −1.586 A_64_P032777 Ly49i2 (includes others) 5.59E−03 1.583 A_64_P095136 Fam163b 4.37E−02 −1.583 A_64_P056955 Tmprss2 4.57E−02 −1.579 A_44_P440207 Cd244 4.57E−02 1.577 A_44_P427596 Fabp1 4.57E−02 −1.573 A_64_P002502 Helb 4.37E−02 1.571 A_64_P290615 Vmn2r116 (includes others) 4.93E−02 −1.567 A_44_P1054324 Csta 1.43E−02 1.531 A_44_P438272 Crisp1/Crisp3 3.43E−02 −1.526 A_64_P100356 Or5k2 4.71E−02 −1.525 A_44_P282164 Pdia2 4.02E−02 −1.521 A_64_P092299 Olfr371 1.88E−02 −1.518 A_64_P131908 LOC103695256 4.37E−02 1.516 A_64_P249426 LOC285423 3.55E−02 1.506 A_43_P16031 Mapk4* 4.67E−02 −1.506 A_64_P036484 Chrm5 1.44E−02 −1.502 DINCH 30 A_44_P281300 Nxnl1 4.68E−02 3.054 A_44_P543128 Olfr742/Olfr743 4.68E−02 2.161 A_64_P103000 Scn3A 2.10E−02 2.11 A_64_P007789 LOC688970/RGD1561231 2.20E−03 2.073 A_44_P882995 Znf286a 2.63E−02 2.017 A_44_P324340 Mfsd6 4.47E−02 −1.985 A_64_P131908 LOC103695256 2.63E−02 1.963 A_43_P20178 Nid2 2.63E−02 1.943 A_64_P031806 Upk3bl 4.47E−02 1.892 A_64_P128687 Olfr67 4.68E−02 1.753 A_64_P317473 Tgm7 3.99E−02 1.662 A_42_P817417 Pvr 4.70E−02 −1.607 A_64_P017886 Robo2 4.68E−02 1.596 A_64_P052225 Slc22a8 2.63E−02 1.586 A_64_P013802 Dcp2 4.68E−02 −1.574 A_64_P017329 Syk* 2.63E−02 1.54 A_64_P063443 LOC103690069 4.68E−02 1.528 A_64_P033121 Rasa4 4.68E−02 1.526 A_64_P024454 Thsd7b 2.63E−02 1.505 DINCH 300 A_64_P037666 Iqsec3 4.54E−02 5.025 A_64_P048486 Hs3st3a1 2.99E−02 −2.033 A_64_P098882 Fcrl6 2.99E−02 1.97 A_44_P490784 Krt77 3.04E−02 1.967 A_44_P556319 Fgf18 2.99E−02 1.963 A_44_P538496 LOC100910837 (includes others) 6.83E−03 −1.667 A_44_P351263 Ly6g6f 4.54E−02 −1.654 A_44_P234013 Vn1r1 6.75E−03 −1.609 A_64_P031335 Stk32b 4.54E−02 −1.563 A_64_P062373 Vps37d 1.55E−02 −1.558 A_44_P439292 Hpgds* 4.11E−02 −1.551 A_64_P052380 Hibch 4.54E−02 1.544 A_64_P108160 Pou4f3 4.54E−02 −1.536 A_43_P23237 Sim2 4.54E−02 1.529 BDB 30 A_64_P120085 Sowahb 4.97E−02 −2.217 A_64_P031460 Vom2r1 (includes others) 2.81E−02 −2.216 A_64_P103610 Lmod3 2.81E−02 2.027 BDB 300 A_44_P292730 Rnase1 2.14E−02 3.377 A_64_P022561 Nlrp2 4.66E−02 2.308 A_64_P130627 2310002L09Rik 4.71E−02 2.227 A_64_P048486 Hs3st3a1 1.61E−02 −2.104 A_64_P030841 Ltf* 3.03E−02 −2.07 A_64_P083883 Samd11 2.14E−02 −1.997 A_64_P078624 Vtcn1 4.58E−02 1.888 A_64_P090851 Znf521 4.58E−02 1.884 A_64_P116670 Mta3 2.14E−02 −1.837 A_44_P1023538 C3 4.66E−02 −1.754 A_64_P128687 Olfr67 3.03E−02 1.7 A_64_P118323 Miat 4.45E−02 1.682 A_64_P059208 Trb 2.14E−02 −1.677 A_64_P120230 4921509C19Rik (includes others) 4.58E−02 −1.654 A_64_P092394 Dsg2 4.87E−02 1.645 A_64_P048506 S1pr5 4.45E−02 1.635 A_44_P384531 Rap1gds1 4.58E−02 1.616 A_64_P017329 Syk* 2.14E−02 1.597 A_64_P072219 Rtp3 4.71E−02 −1.568 A_64_P140886 Lrrc71 4.58E−02 −1.566 A_44_P299641 Olr1572 1.61E−02 −1.562 A_64_P161216 Kif5c 3.03E−02 1.543 A_44_P466189 Angptl2 3.03E−02 1.536 A_44_P538496 LOC100910837 (includes others) 1.61E−02 −1.527 A_44_P414421 Or1l1 2.14E−02 −1.526 A_64_P142640 Gjc3 3.03E−02 1.514 A_64_P101291 Hoxc12 3.03E−02 1.501 DOS 30 A_64_P014642 Mogat3 1.82E−02 3.72 A_43_P16740 Ogn 3.72E−02 −2.377 A_64_P036115 Ppp3ca 2.89E−02 2.308 A_64_P114465 Rab27b 4.27E−02 2.147 A_64_P031806 Upk3bl 3.92E−02 2.02 A_44_P990998 Klk8 4.85E−02 1.978 A_44_P700270 Fam64a 4.85E−02 1.911 A_44_P746666 Znf667 1.82E−02 −1.883 A_64_P069386 Gm378 4.98E−02 −1.654 A_64_P007372 Tmprss15 4.85E−02 −1.635 A_64_P120210 Galr1 2.27E−02 −1.591 A_64_P005972 Nhlrc3 4.27E−02 1.522 A_64_P031407 Mars2 3.92E−02 −1.516 A_42_P736530 Samd9 4.85E−02 −1.505 A_44_P468239 Sgtb 4.85E−02 −1.504 ID Symbol Expression p-value Expression fold change DEHP 30 A_64_P009798 Nr5a2* 3.79E−02 −7.306 A_64_P103179 Xkr9 3.79E−02 3.288 A_44_P260348 LOC689316 3.79E−02 −3.143 A_64_P159163 Gm1527/Gm6558 3.79E−02 −2.102 A_64_P048106 Foxp2 3.79E−02 1.91 A_44_P271529 Olfr380 3.79E−02 −1.808 A_64_P051307 Runx2* 3.79E−02 −1.768 A_43_P16031 Mapk4* 4.63E−02 −1.706 A_42_P794562 Cyp7a1 3.79E−02 1.701 A_64_P051039 Spon2 3.79E−02 −1.644 A_64_P072739 Flrt1 3.79E−02 −1.588 A_44_P161470 Defa6 (includes others) 4.48E−02 1.515 DEHP 300 A_44_P636808 9230104L09Rik 4.68E−02 3.585 A_64_P103179 Xkr9 1.88E−02 3.127 A_64_P023008 LOC100911534 4.48E−02 2.453 A_44_P419034 Olr181/Olr183 4.37E−02 −2.37 A_64_P232281 Vom2r1 (includes others) 5.59E−03 2.143 A_64_P064556 Slc36a4 1.28E−02 2.063 A_64_P047621 Ildr1 1.41E−02 −2.044 A_44_P405374 LOC290071 1.88E−02 2.027 A_64_P132182 Zeb2os 4.37E−02 1.956 A_43_P11449 Cpb1 1.41E−02 1.944 A_44_P351263 Ly6g6f 1.41E−02 −1.935 A_64_P087625 LOC102552143/LOC102552337 4.57E−02 1.927 A_44_P689645 Enox2 1.41E−02 −1.894 A_64_P038812 Erbb2 4.37E−02 1.87 A_64_P004007 Rd3l 4.37E−02 1.861 A_44_P140275 Alpk1 1.43E−02 1.856 A_64_P075532 Gm8882 (includes others) 4.02E−02 −1.827 A_64_P090866 Sstr1 4.37E−02 −1.808 A_44_P558411 Scrg1 4.81E−02 1.804 A_64_P063443 LOC103690069 2.71E−02 1.788 A_64_P131538 Olfr649 2.59E−03 −1.768 A_64_P080530 Nrxn3 4.93E−02 1.757 A_64_P060018 Pilrb 4.37E−02 −1.746 A_64_P108698 Cryge/Crygf 4.37E−02 1.711 A_43_P11278 Hpgd 2.71E−02 −1.707 A_44_P439292 Hpgds* 4.86E−02 −1.699 A_64_P111725 LOC103690800 2.66E−02 1.675 A_64_P160550 Gpr171 4.48E−02 1.67 A_64_P055137 Gldn 1.00E−02 −1.665 A_64_P150603 Ptger3 3.36E−02 −1.653 A_42_P736315 Rtp4 5.59E−03 −1.643 A_44_P515494 Ddr1 3.76E−02 −1.64 A_64_P033121 Rasa4 4.57E−02 1.635 A_44_P496572 Kmo 3.49E−02 −1.625 A_64_P156001 Olr396 4.67E−02 −1.616 A_64_P106933 Foxh1 5.59E−03 1.614 A_44_P426079 Kcnc1 3.49E−02 −1.604 A_64_P063558 LOC499764 4.02E−02 −1.593 A_44_P1013314 Iisg15 3.71E−02 −1.586 A_64_P032777 Ly49i2 (includes others) 5.59E−03 1.583 A_64_P095136 Fam163b 4.37E−02 −1.583 A_64_P056955 Tmprss2 4.57E−02 −1.579 A_44_P440207 Cd244 4.57E−02 1.577 A_44_P427596 Fabp1 4.57E−02 −1.573 A_64_P002502 Helb 4.37E−02 1.571 A_64_P290615 Vmn2r116 (includes others) 4.93E−02 −1.567 A_44_P1054324 Csta 1.43E−02 1.531 A_44_P438272 Crisp1/Crisp3 3.43E−02 −1.526 A_64_P100356 Or5k2 4.71E−02 −1.525 A_44_P282164 Pdia2 4.02E−02 −1.521 A_64_P092299 Olfr371 1.88E−02 −1.518 A_64_P131908 LOC103695256 4.37E−02 1.516 A_64_P249426 LOC285423 3.55E−02 1.506 A_43_P16031 Mapk4* 4.67E−02 −1.506 A_64_P036484 Chrm5 1.44E−02 −1.502 DINCH 30 A_44_P281300 Nxnl1 4.68E−02 3.054 A_44_P543128 Olfr742/Olfr743 4.68E−02 2.161 A_64_P103000 Scn3A 2.10E−02 2.11 A_64_P007789 LOC688970/RGD1561231 2.20E−03 2.073 A_44_P882995 Znf286a 2.63E−02 2.017 A_44_P324340 Mfsd6 4.47E−02 −1.985 A_64_P131908 LOC103695256 2.63E−02 1.963 A_43_P20178 Nid2 2.63E−02 1.943 A_64_P031806 Upk3bl 4.47E−02 1.892 A_64_P128687 Olfr67 4.68E−02 1.753 A_64_P317473 Tgm7 3.99E−02 1.662 A_42_P817417 Pvr 4.70E−02 −1.607 A_64_P017886 Robo2 4.68E−02 1.596 A_64_P052225 Slc22a8 2.63E−02 1.586 A_64_P013802 Dcp2 4.68E−02 −1.574 A_64_P017329 Syk* 2.63E−02 1.54 A_64_P063443 LOC103690069 4.68E−02 1.528 A_64_P033121 Rasa4 4.68E−02 1.526 A_64_P024454 Thsd7b 2.63E−02 1.505 DINCH 300 A_64_P037666 Iqsec3 4.54E−02 5.025 A_64_P048486 Hs3st3a1 2.99E−02 −2.033 A_64_P098882 Fcrl6 2.99E−02 1.97 A_44_P490784 Krt77 3.04E−02 1.967 A_44_P556319 Fgf18 2.99E−02 1.963 A_44_P538496 LOC100910837 (includes others) 6.83E−03 −1.667 A_44_P351263 Ly6g6f 4.54E−02 −1.654 A_44_P234013 Vn1r1 6.75E−03 −1.609 A_64_P031335 Stk32b 4.54E−02 −1.563 A_64_P062373 Vps37d 1.55E−02 −1.558 A_44_P439292 Hpgds* 4.11E−02 −1.551 A_64_P052380 Hibch 4.54E−02 1.544 A_64_P108160 Pou4f3 4.54E−02 −1.536 A_43_P23237 Sim2 4.54E−02 1.529 BDB 30 A_64_P120085 Sowahb 4.97E−02 −2.217 A_64_P031460 Vom2r1 (includes others) 2.81E−02 −2.216 A_64_P103610 Lmod3 2.81E−02 2.027 BDB 300 A_44_P292730 Rnase1 2.14E−02 3.377 A_64_P022561 Nlrp2 4.66E−02 2.308 A_64_P130627 2310002L09Rik 4.71E−02 2.227 A_64_P048486 Hs3st3a1 1.61E−02 −2.104 A_64_P030841 Ltf* 3.03E−02 −2.07 A_64_P083883 Samd11 2.14E−02 −1.997 A_64_P078624 Vtcn1 4.58E−02 1.888 A_64_P090851 Znf521 4.58E−02 1.884 A_64_P116670 Mta3 2.14E−02 −1.837 A_44_P1023538 C3 4.66E−02 −1.754 A_64_P128687 Olfr67 3.03E−02 1.7 A_64_P118323 Miat 4.45E−02 1.682 A_64_P059208 Trb 2.14E−02 −1.677 A_64_P120230 4921509C19Rik (includes others) 4.58E−02 −1.654 A_64_P092394 Dsg2 4.87E−02 1.645 A_64_P048506 S1pr5 4.45E−02 1.635 A_44_P384531 Rap1gds1 4.58E−02 1.616 A_64_P017329 Syk* 2.14E−02 1.597 A_64_P072219 Rtp3 4.71E−02 −1.568 A_64_P140886 Lrrc71 4.58E−02 −1.566 A_44_P299641 Olr1572 1.61E−02 −1.562 A_64_P161216 Kif5c 3.03E−02 1.543 A_44_P466189 Angptl2 3.03E−02 1.536 A_44_P538496 LOC100910837 (includes others) 1.61E−02 −1.527 A_44_P414421 Or1l1 2.14E−02 −1.526 A_64_P142640 Gjc3 3.03E−02 1.514 A_64_P101291 Hoxc12 3.03E−02 1.501 DOS 30 A_64_P014642 Mogat3 1.82E−02 3.72 A_43_P16740 Ogn 3.72E−02 −2.377 A_64_P036115 Ppp3ca 2.89E−02 2.308 A_64_P114465 Rab27b 4.27E−02 2.147 A_64_P031806 Upk3bl 3.92E−02 2.02 A_44_P990998 Klk8 4.85E−02 1.978 A_44_P700270 Fam64a 4.85E−02 1.911 A_44_P746666 Znf667 1.82E−02 −1.883 A_64_P069386 Gm378 4.98E−02 −1.654 A_64_P007372 Tmprss15 4.85E−02 −1.635 A_64_P120210 Galr1 2.27E−02 −1.591 A_64_P005972 Nhlrc3 4.27E−02 1.522 A_64_P031407 Mars2 3.92E−02 −1.516 A_42_P736530 Samd9 4.85E−02 −1.505 A_44_P468239 Sgtb 4.85E−02 −1.504 Transcripts selected for further RT-qPCR validation are indicated with an asterisk. Table 2. Testicular Transcripts Significantly Affected With a >1.5-Fold Change After In Utero and Lactational Exposure to DEHP, DINCH, BDB, or DOS ID Symbol Expression p-value Expression fold change DEHP 30 A_64_P009798 Nr5a2* 3.79E−02 −7.306 A_64_P103179 Xkr9 3.79E−02 3.288 A_44_P260348 LOC689316 3.79E−02 −3.143 A_64_P159163 Gm1527/Gm6558 3.79E−02 −2.102 A_64_P048106 Foxp2 3.79E−02 1.91 A_44_P271529 Olfr380 3.79E−02 −1.808 A_64_P051307 Runx2* 3.79E−02 −1.768 A_43_P16031 Mapk4* 4.63E−02 −1.706 A_42_P794562 Cyp7a1 3.79E−02 1.701 A_64_P051039 Spon2 3.79E−02 −1.644 A_64_P072739 Flrt1 3.79E−02 −1.588 A_44_P161470 Defa6 (includes others) 4.48E−02 1.515 DEHP 300 A_44_P636808 9230104L09Rik 4.68E−02 3.585 A_64_P103179 Xkr9 1.88E−02 3.127 A_64_P023008 LOC100911534 4.48E−02 2.453 A_44_P419034 Olr181/Olr183 4.37E−02 −2.37 A_64_P232281 Vom2r1 (includes others) 5.59E−03 2.143 A_64_P064556 Slc36a4 1.28E−02 2.063 A_64_P047621 Ildr1 1.41E−02 −2.044 A_44_P405374 LOC290071 1.88E−02 2.027 A_64_P132182 Zeb2os 4.37E−02 1.956 A_43_P11449 Cpb1 1.41E−02 1.944 A_44_P351263 Ly6g6f 1.41E−02 −1.935 A_64_P087625 LOC102552143/LOC102552337 4.57E−02 1.927 A_44_P689645 Enox2 1.41E−02 −1.894 A_64_P038812 Erbb2 4.37E−02 1.87 A_64_P004007 Rd3l 4.37E−02 1.861 A_44_P140275 Alpk1 1.43E−02 1.856 A_64_P075532 Gm8882 (includes others) 4.02E−02 −1.827 A_64_P090866 Sstr1 4.37E−02 −1.808 A_44_P558411 Scrg1 4.81E−02 1.804 A_64_P063443 LOC103690069 2.71E−02 1.788 A_64_P131538 Olfr649 2.59E−03 −1.768 A_64_P080530 Nrxn3 4.93E−02 1.757 A_64_P060018 Pilrb 4.37E−02 −1.746 A_64_P108698 Cryge/Crygf 4.37E−02 1.711 A_43_P11278 Hpgd 2.71E−02 −1.707 A_44_P439292 Hpgds* 4.86E−02 −1.699 A_64_P111725 LOC103690800 2.66E−02 1.675 A_64_P160550 Gpr171 4.48E−02 1.67 A_64_P055137 Gldn 1.00E−02 −1.665 A_64_P150603 Ptger3 3.36E−02 −1.653 A_42_P736315 Rtp4 5.59E−03 −1.643 A_44_P515494 Ddr1 3.76E−02 −1.64 A_64_P033121 Rasa4 4.57E−02 1.635 A_44_P496572 Kmo 3.49E−02 −1.625 A_64_P156001 Olr396 4.67E−02 −1.616 A_64_P106933 Foxh1 5.59E−03 1.614 A_44_P426079 Kcnc1 3.49E−02 −1.604 A_64_P063558 LOC499764 4.02E−02 −1.593 A_44_P1013314 Iisg15 3.71E−02 −1.586 A_64_P032777 Ly49i2 (includes others) 5.59E−03 1.583 A_64_P095136 Fam163b 4.37E−02 −1.583 A_64_P056955 Tmprss2 4.57E−02 −1.579 A_44_P440207 Cd244 4.57E−02 1.577 A_44_P427596 Fabp1 4.57E−02 −1.573 A_64_P002502 Helb 4.37E−02 1.571 A_64_P290615 Vmn2r116 (includes others) 4.93E−02 −1.567 A_44_P1054324 Csta 1.43E−02 1.531 A_44_P438272 Crisp1/Crisp3 3.43E−02 −1.526 A_64_P100356 Or5k2 4.71E−02 −1.525 A_44_P282164 Pdia2 4.02E−02 −1.521 A_64_P092299 Olfr371 1.88E−02 −1.518 A_64_P131908 LOC103695256 4.37E−02 1.516 A_64_P249426 LOC285423 3.55E−02 1.506 A_43_P16031 Mapk4* 4.67E−02 −1.506 A_64_P036484 Chrm5 1.44E−02 −1.502 DINCH 30 A_44_P281300 Nxnl1 4.68E−02 3.054 A_44_P543128 Olfr742/Olfr743 4.68E−02 2.161 A_64_P103000 Scn3A 2.10E−02 2.11 A_64_P007789 LOC688970/RGD1561231 2.20E−03 2.073 A_44_P882995 Znf286a 2.63E−02 2.017 A_44_P324340 Mfsd6 4.47E−02 −1.985 A_64_P131908 LOC103695256 2.63E−02 1.963 A_43_P20178 Nid2 2.63E−02 1.943 A_64_P031806 Upk3bl 4.47E−02 1.892 A_64_P128687 Olfr67 4.68E−02 1.753 A_64_P317473 Tgm7 3.99E−02 1.662 A_42_P817417 Pvr 4.70E−02 −1.607 A_64_P017886 Robo2 4.68E−02 1.596 A_64_P052225 Slc22a8 2.63E−02 1.586 A_64_P013802 Dcp2 4.68E−02 −1.574 A_64_P017329 Syk* 2.63E−02 1.54 A_64_P063443 LOC103690069 4.68E−02 1.528 A_64_P033121 Rasa4 4.68E−02 1.526 A_64_P024454 Thsd7b 2.63E−02 1.505 DINCH 300 A_64_P037666 Iqsec3 4.54E−02 5.025 A_64_P048486 Hs3st3a1 2.99E−02 −2.033 A_64_P098882 Fcrl6 2.99E−02 1.97 A_44_P490784 Krt77 3.04E−02 1.967 A_44_P556319 Fgf18 2.99E−02 1.963 A_44_P538496 LOC100910837 (includes others) 6.83E−03 −1.667 A_44_P351263 Ly6g6f 4.54E−02 −1.654 A_44_P234013 Vn1r1 6.75E−03 −1.609 A_64_P031335 Stk32b 4.54E−02 −1.563 A_64_P062373 Vps37d 1.55E−02 −1.558 A_44_P439292 Hpgds* 4.11E−02 −1.551 A_64_P052380 Hibch 4.54E−02 1.544 A_64_P108160 Pou4f3 4.54E−02 −1.536 A_43_P23237 Sim2 4.54E−02 1.529 BDB 30 A_64_P120085 Sowahb 4.97E−02 −2.217 A_64_P031460 Vom2r1 (includes others) 2.81E−02 −2.216 A_64_P103610 Lmod3 2.81E−02 2.027 BDB 300 A_44_P292730 Rnase1 2.14E−02 3.377 A_64_P022561 Nlrp2 4.66E−02 2.308 A_64_P130627 2310002L09Rik 4.71E−02 2.227 A_64_P048486 Hs3st3a1 1.61E−02 −2.104 A_64_P030841 Ltf* 3.03E−02 −2.07 A_64_P083883 Samd11 2.14E−02 −1.997 A_64_P078624 Vtcn1 4.58E−02 1.888 A_64_P090851 Znf521 4.58E−02 1.884 A_64_P116670 Mta3 2.14E−02 −1.837 A_44_P1023538 C3 4.66E−02 −1.754 A_64_P128687 Olfr67 3.03E−02 1.7 A_64_P118323 Miat 4.45E−02 1.682 A_64_P059208 Trb 2.14E−02 −1.677 A_64_P120230 4921509C19Rik (includes others) 4.58E−02 −1.654 A_64_P092394 Dsg2 4.87E−02 1.645 A_64_P048506 S1pr5 4.45E−02 1.635 A_44_P384531 Rap1gds1 4.58E−02 1.616 A_64_P017329 Syk* 2.14E−02 1.597 A_64_P072219 Rtp3 4.71E−02 −1.568 A_64_P140886 Lrrc71 4.58E−02 −1.566 A_44_P299641 Olr1572 1.61E−02 −1.562 A_64_P161216 Kif5c 3.03E−02 1.543 A_44_P466189 Angptl2 3.03E−02 1.536 A_44_P538496 LOC100910837 (includes others) 1.61E−02 −1.527 A_44_P414421 Or1l1 2.14E−02 −1.526 A_64_P142640 Gjc3 3.03E−02 1.514 A_64_P101291 Hoxc12 3.03E−02 1.501 DOS 30 A_64_P014642 Mogat3 1.82E−02 3.72 A_43_P16740 Ogn 3.72E−02 −2.377 A_64_P036115 Ppp3ca 2.89E−02 2.308 A_64_P114465 Rab27b 4.27E−02 2.147 A_64_P031806 Upk3bl 3.92E−02 2.02 A_44_P990998 Klk8 4.85E−02 1.978 A_44_P700270 Fam64a 4.85E−02 1.911 A_44_P746666 Znf667 1.82E−02 −1.883 A_64_P069386 Gm378 4.98E−02 −1.654 A_64_P007372 Tmprss15 4.85E−02 −1.635 A_64_P120210 Galr1 2.27E−02 −1.591 A_64_P005972 Nhlrc3 4.27E−02 1.522 A_64_P031407 Mars2 3.92E−02 −1.516 A_42_P736530 Samd9 4.85E−02 −1.505 A_44_P468239 Sgtb 4.85E−02 −1.504 ID Symbol Expression p-value Expression fold change DEHP 30 A_64_P009798 Nr5a2* 3.79E−02 −7.306 A_64_P103179 Xkr9 3.79E−02 3.288 A_44_P260348 LOC689316 3.79E−02 −3.143 A_64_P159163 Gm1527/Gm6558 3.79E−02 −2.102 A_64_P048106 Foxp2 3.79E−02 1.91 A_44_P271529 Olfr380 3.79E−02 −1.808 A_64_P051307 Runx2* 3.79E−02 −1.768 A_43_P16031 Mapk4* 4.63E−02 −1.706 A_42_P794562 Cyp7a1 3.79E−02 1.701 A_64_P051039 Spon2 3.79E−02 −1.644 A_64_P072739 Flrt1 3.79E−02 −1.588 A_44_P161470 Defa6 (includes others) 4.48E−02 1.515 DEHP 300 A_44_P636808 9230104L09Rik 4.68E−02 3.585 A_64_P103179 Xkr9 1.88E−02 3.127 A_64_P023008 LOC100911534 4.48E−02 2.453 A_44_P419034 Olr181/Olr183 4.37E−02 −2.37 A_64_P232281 Vom2r1 (includes others) 5.59E−03 2.143 A_64_P064556 Slc36a4 1.28E−02 2.063 A_64_P047621 Ildr1 1.41E−02 −2.044 A_44_P405374 LOC290071 1.88E−02 2.027 A_64_P132182 Zeb2os 4.37E−02 1.956 A_43_P11449 Cpb1 1.41E−02 1.944 A_44_P351263 Ly6g6f 1.41E−02 −1.935 A_64_P087625 LOC102552143/LOC102552337 4.57E−02 1.927 A_44_P689645 Enox2 1.41E−02 −1.894 A_64_P038812 Erbb2 4.37E−02 1.87 A_64_P004007 Rd3l 4.37E−02 1.861 A_44_P140275 Alpk1 1.43E−02 1.856 A_64_P075532 Gm8882 (includes others) 4.02E−02 −1.827 A_64_P090866 Sstr1 4.37E−02 −1.808 A_44_P558411 Scrg1 4.81E−02 1.804 A_64_P063443 LOC103690069 2.71E−02 1.788 A_64_P131538 Olfr649 2.59E−03 −1.768 A_64_P080530 Nrxn3 4.93E−02 1.757 A_64_P060018 Pilrb 4.37E−02 −1.746 A_64_P108698 Cryge/Crygf 4.37E−02 1.711 A_43_P11278 Hpgd 2.71E−02 −1.707 A_44_P439292 Hpgds* 4.86E−02 −1.699 A_64_P111725 LOC103690800 2.66E−02 1.675 A_64_P160550 Gpr171 4.48E−02 1.67 A_64_P055137 Gldn 1.00E−02 −1.665 A_64_P150603 Ptger3 3.36E−02 −1.653 A_42_P736315 Rtp4 5.59E−03 −1.643 A_44_P515494 Ddr1 3.76E−02 −1.64 A_64_P033121 Rasa4 4.57E−02 1.635 A_44_P496572 Kmo 3.49E−02 −1.625 A_64_P156001 Olr396 4.67E−02 −1.616 A_64_P106933 Foxh1 5.59E−03 1.614 A_44_P426079 Kcnc1 3.49E−02 −1.604 A_64_P063558 LOC499764 4.02E−02 −1.593 A_44_P1013314 Iisg15 3.71E−02 −1.586 A_64_P032777 Ly49i2 (includes others) 5.59E−03 1.583 A_64_P095136 Fam163b 4.37E−02 −1.583 A_64_P056955 Tmprss2 4.57E−02 −1.579 A_44_P440207 Cd244 4.57E−02 1.577 A_44_P427596 Fabp1 4.57E−02 −1.573 A_64_P002502 Helb 4.37E−02 1.571 A_64_P290615 Vmn2r116 (includes others) 4.93E−02 −1.567 A_44_P1054324 Csta 1.43E−02 1.531 A_44_P438272 Crisp1/Crisp3 3.43E−02 −1.526 A_64_P100356 Or5k2 4.71E−02 −1.525 A_44_P282164 Pdia2 4.02E−02 −1.521 A_64_P092299 Olfr371 1.88E−02 −1.518 A_64_P131908 LOC103695256 4.37E−02 1.516 A_64_P249426 LOC285423 3.55E−02 1.506 A_43_P16031 Mapk4* 4.67E−02 −1.506 A_64_P036484 Chrm5 1.44E−02 −1.502 DINCH 30 A_44_P281300 Nxnl1 4.68E−02 3.054 A_44_P543128 Olfr742/Olfr743 4.68E−02 2.161 A_64_P103000 Scn3A 2.10E−02 2.11 A_64_P007789 LOC688970/RGD1561231 2.20E−03 2.073 A_44_P882995 Znf286a 2.63E−02 2.017 A_44_P324340 Mfsd6 4.47E−02 −1.985 A_64_P131908 LOC103695256 2.63E−02 1.963 A_43_P20178 Nid2 2.63E−02 1.943 A_64_P031806 Upk3bl 4.47E−02 1.892 A_64_P128687 Olfr67 4.68E−02 1.753 A_64_P317473 Tgm7 3.99E−02 1.662 A_42_P817417 Pvr 4.70E−02 −1.607 A_64_P017886 Robo2 4.68E−02 1.596 A_64_P052225 Slc22a8 2.63E−02 1.586 A_64_P013802 Dcp2 4.68E−02 −1.574 A_64_P017329 Syk* 2.63E−02 1.54 A_64_P063443 LOC103690069 4.68E−02 1.528 A_64_P033121 Rasa4 4.68E−02 1.526 A_64_P024454 Thsd7b 2.63E−02 1.505 DINCH 300 A_64_P037666 Iqsec3 4.54E−02 5.025 A_64_P048486 Hs3st3a1 2.99E−02 −2.033 A_64_P098882 Fcrl6 2.99E−02 1.97 A_44_P490784 Krt77 3.04E−02 1.967 A_44_P556319 Fgf18 2.99E−02 1.963 A_44_P538496 LOC100910837 (includes others) 6.83E−03 −1.667 A_44_P351263 Ly6g6f 4.54E−02 −1.654 A_44_P234013 Vn1r1 6.75E−03 −1.609 A_64_P031335 Stk32b 4.54E−02 −1.563 A_64_P062373 Vps37d 1.55E−02 −1.558 A_44_P439292 Hpgds* 4.11E−02 −1.551 A_64_P052380 Hibch 4.54E−02 1.544 A_64_P108160 Pou4f3 4.54E−02 −1.536 A_43_P23237 Sim2 4.54E−02 1.529 BDB 30 A_64_P120085 Sowahb 4.97E−02 −2.217 A_64_P031460 Vom2r1 (includes others) 2.81E−02 −2.216 A_64_P103610 Lmod3 2.81E−02 2.027 BDB 300 A_44_P292730 Rnase1 2.14E−02 3.377 A_64_P022561 Nlrp2 4.66E−02 2.308 A_64_P130627 2310002L09Rik 4.71E−02 2.227 A_64_P048486 Hs3st3a1 1.61E−02 −2.104 A_64_P030841 Ltf* 3.03E−02 −2.07 A_64_P083883 Samd11 2.14E−02 −1.997 A_64_P078624 Vtcn1 4.58E−02 1.888 A_64_P090851 Znf521 4.58E−02 1.884 A_64_P116670 Mta3 2.14E−02 −1.837 A_44_P1023538 C3 4.66E−02 −1.754 A_64_P128687 Olfr67 3.03E−02 1.7 A_64_P118323 Miat 4.45E−02 1.682 A_64_P059208 Trb 2.14E−02 −1.677 A_64_P120230 4921509C19Rik (includes others) 4.58E−02 −1.654 A_64_P092394 Dsg2 4.87E−02 1.645 A_64_P048506 S1pr5 4.45E−02 1.635 A_44_P384531 Rap1gds1 4.58E−02 1.616 A_64_P017329 Syk* 2.14E−02 1.597 A_64_P072219 Rtp3 4.71E−02 −1.568 A_64_P140886 Lrrc71 4.58E−02 −1.566 A_44_P299641 Olr1572 1.61E−02 −1.562 A_64_P161216 Kif5c 3.03E−02 1.543 A_44_P466189 Angptl2 3.03E−02 1.536 A_44_P538496 LOC100910837 (includes others) 1.61E−02 −1.527 A_44_P414421 Or1l1 2.14E−02 −1.526 A_64_P142640 Gjc3 3.03E−02 1.514 A_64_P101291 Hoxc12 3.03E−02 1.501 DOS 30 A_64_P014642 Mogat3 1.82E−02 3.72 A_43_P16740 Ogn 3.72E−02 −2.377 A_64_P036115 Ppp3ca 2.89E−02 2.308 A_64_P114465 Rab27b 4.27E−02 2.147 A_64_P031806 Upk3bl 3.92E−02 2.02 A_44_P990998 Klk8 4.85E−02 1.978 A_44_P700270 Fam64a 4.85E−02 1.911 A_44_P746666 Znf667 1.82E−02 −1.883 A_64_P069386 Gm378 4.98E−02 −1.654 A_64_P007372 Tmprss15 4.85E−02 −1.635 A_64_P120210 Galr1 2.27E−02 −1.591 A_64_P005972 Nhlrc3 4.27E−02 1.522 A_64_P031407 Mars2 3.92E−02 −1.516 A_42_P736530 Samd9 4.85E−02 −1.505 A_44_P468239 Sgtb 4.85E−02 −1.504 Transcripts selected for further RT-qPCR validation are indicated with an asterisk. Figure 5. View largeDownload slide Gene expression quantification by RT-qPCR for selected transcripts following in utero and lactational exposure to DEHP, DINCH, BDB or DOS. Relative levels of gene amplification compared with the control group and housekeeping gene Ppia were calculated following the ΔΔCt method. Significance was determined by one-way ANOVA corrected by Dunnett’s multiple comparison test; n = 6 biological replicates plated in triplicate; **p < .01; ****p < .0001. Figure 5. View largeDownload slide Gene expression quantification by RT-qPCR for selected transcripts following in utero and lactational exposure to DEHP, DINCH, BDB or DOS. Relative levels of gene amplification compared with the control group and housekeeping gene Ppia were calculated following the ΔΔCt method. Significance was determined by one-way ANOVA corrected by Dunnett’s multiple comparison test; n = 6 biological replicates plated in triplicate; **p < .01; ****p < .0001. Interestingly, among all transcripts displaying modified expression, several were related to estrogen function and signaling. Hence, the nuclear receptor NR5A2, which plays a predominant role in reverse cholesterol transport and steroidogenesis (Pezzi et al., 2004; Sirianni et al., 2002), was downregulated by 7.3-fold after exposure to 30 mg/kg/day DEHP (Table 2). Validation with RT-qPCR confirmed this downregulation and revealed that Nr5a2 expression was also targeted by exposure to 300 mg/kg/day DEHP and by 30 mg/kg/day DINCH and BDB (Figure 5A). Lactotransferrin, the expression of which is dependent on estrogen (Teng, 2006), was also of interest in this context. Microarray data revealed that exposure to 300 mg/kg/day BDB downregulated the latter by 2.1-fold (Table 2). Validation by RT-qPCR not only confirmed this effect, but also revealed a stronger downregulation of Ltf by 300 mg/kg/day DEHP (Figure 5B). Finally, exposure to 30 mg/kg/day DEHP led to a significant 1.8-fold downregulation of Runx2 (runt-related factor 2) expression (Table 2). The latter regulates the expression of many steroid hormone-responsive genes in osteoblasts, and is regulated itself by estrogens at the transcriptional level (Teplyuk et al., 2009). qRT-PCR analysis did not reveal a statistically significant effect on the expression of this gene (Figure 5C). Other targets of interest included the hematopoietic prostaglandin D-synthase HPGDS, which is involved in inflammatory responses, and was downregulated by 1.7 and 1.6-fold after exposure to 300 mg/kg/day DEHP and DINCH, respectively (Table 2). Because many putative endocrine disruptors have been shown to disrupt prostaglandin synthesis, we further assessed Hpgds expression by RT-qPCR but found no significant effect by any of the treatments (Figure 5D). Mapk4 was downregulated by 1.7 and 1.5-fold after exposure to 30 and 300 mg/kg/day DEHP, respectively (Table 2). The tyrosine-protein kinase SYK, also involved in the MAPK signaling cascade, was downregulated by 1.5 and 1.6-fold, respectively, after exposure to 30 mg/kg/day DINCH or 300 mg/kg/day BDB (Table 2). However, RT-qPCR did not reveal statistically significant differences in the expression of these genes (Figs. 5D and 5E). DISCUSSION In a previous study, we demonstrated that in utero and lactational exposure to DEHP produced classically described endocrine-disruptive phenotypes such as a decreased anogenital index and increased multi-nucleated gonocytes at PND 3, as well as hemorrhagic testes at PND 8 (Nardelli et al., 2017). In comparison, exposure to BDB and DOS did not produce any significant effect on these endpoints. Here, we explored the impact of in utero and lactational exposure to DEHP, DINCH, BDB, and DOS in adult offspring. This study provides evidence that perinatal exposure to DINCH, BDB, and DOS produces no significant effects on organ weights, serum gonadotropin, and testosterone levels or sperm quality in the adult. In addition, we demonstrate that exposure to one green plasticizer, DOS, has fewer effects on testicular gene expression than DEHP, and identify estrogen signaling in the testis to be a potential long-term target of DEHP. Several studies have reported significant decreases in testicular or seminal vesicle weights in the adult male resulting from perinatal DEHP exposure. These effects were observed solely after exposure to relatively high doses of DEHP. Exposure to 938 or 1250 mg/kg/day DEHP from GD 14 to PND 0 produced a higher incidence of testicular atrophy (Culty et al., 2008), while exposure to 500 mg/kg/day DEHP from GD3 to PND 21 produced a significant decrease in testicular weight (Dorostghoal et al., 2012). In other studies, exposures to 405 mg/kg/day DEHP from GD 6 to PND 21 (Andrade et al., 2006) or to 500 mg/kg/day DEHP from GD 0 to PND 21 (Dalsenter et al., 2006) produced significant decreases in seminal vesicle weights; these effects were not associated with changes in testicular weight. To the best of our knowledge, there are no studies showing an alteration of adult reproductive organ weights after in utero and lactational exposure to DEHP at doses below 405 mg/kg/day. Consistent with these findings, we did not observe a significant effect of DEHP exposures at 30 and 300 mg/kg/day on testicular or seminal vesicle weights. However, exposure to 300 mg/kg/day DEHP did produce a significant increase in paired epididymal weight. Importantly, we did not find any significant effect of exposure to DINCH, BDB, or DOS on androgen-dependent organ weights. We measured serum steroids and gonadotropins to investigate whether exposure to our candidate compounds would affect steroidogenesis. Testosterone, LH and FSH levels remained unaffected by all treatments. The ratio of testosterone to LH a measure for possible compensated Leydig cell failure, remained unaffected as well; this ratio was decreased in adult rats following perinatal exposure to dibutyl phthalate, a potent antiandrogen (Kilcoyne et al., 2014). The literature on the long-term consequences of perinatal exposure to DEHP on steroidogenesis in males remains quite scarce. Exposure of Long Evans dams to 100 mg/kg/day DEHP from GD 12 to GD 21 produced inhibitory effects on testosterone and LH levels in the male offspring that were no longer apparent at PND 90 (Akingbemi et al., 2001). Two other studies report significant decreases in circulating testosterone levels at PND 60 following in utero exposure of Sprague Dawley dams to doses of DEHP starting at 100 mg/kg/day (Culty et al., 2008; Martinez-Arguelles et al., 2009); here, the treatment windows, GD 14 to PND0 or GD 14 to GD 19, respectively, were limited to fetal life. In contrast, the male offspring of Wistar dams showed increased testosterone levels at PND 90 after exposure to 0.045, 0.405, or 405 mg/kg/day DEHP from GD 6 to PND 21(Andrade et al., 2006). These divergences seem to be highly dependent on the rat strain, the dose and the window of exposure used, and call for a deeper exploration of the long-term consequences of exposure to DEHP. DINCH and our candidate compounds, BDB and DOS, did not affect testosterone, LH or FSH concentrations in serum. We assessed sperm production at PND 90 to investigate whether exposure to our candidate compounds affects spermatogenesis in the adult. None of the treatments significantly affected testicular or epididymal sperm head counts or cauda sperm motility. In a previous study, in utero and lactational exposure to DEHP has been reported to reduce daily sperm production 19%–25% in relation to control in animals exposed to 15, 45, 135, or 405 mg/kg/day (Andrade et al., 2006). Reduced sperm counts were also observed in the male offspring of dams exposed to 100 or 500 mg/kg/day DEHP from GD 3 to PND 21 (Dorostghoal et al., 2012). Similarly, exposure to 500 mg/kg/day DEHP from GD 0 to PND 21 significantly reduced sperm production in adult males (Dalsenter et al., 2006); however, these effects were not observed at lower doses. The latter 3 studies have in common the use of Wistar rats. In our study, we used Sprague Dawley rats, which have been shown to be more resistant to some endocrine disrupting compounds in different experimental settings (Abuelhija et al., 2013; Kacew and Festing, 1996), including to diethylstilbestrol (Shellabarger et al., 1978). Such strain differences in susceptibility may contribute to the absence of effects on sperm counts in our experiments. Using toxicogenomics, we determined whether our candidate compounds would produce detrimental effects on testicular gene expression in the adult in comparison to DEHP. We observed that in utero and lactational exposure to 300 mg/kg/day DEHP produced the most substantial effect on adult testicular gene expression, while the overall gene expression profile after exposure to BDB and DOS was closer to that of corn oil. We also identified several related genes presenting altered expression following exposure to DEHP, DINCH, or BDB. Based on significance, fold changes and pathway analysis, we narrowed down our list of targets to 6 transcripts, and validated our data using RT-qPCR. Interestingly, we found 3 of these transcripts to be associated with estrogens. We identified the expression of Nr5a2 to be significantly altered after exposure to both doses of DEHP, and 30 mg/kg/day DINCH and BDB. Also called liver receptor homologue-1 (LRH-1), NR5A2 is an orphan nuclear hormone receptor closely related to steroidogenic factor 1 (SF1). It is expressed in several steroidogenic tissues in many species, and has been hypothesized to play a critical role in development and function of the endocrine and reproductive systems (Sirianni et al., 2002). NR5A2 has been demonstrated to regulate the transcription of genes encoding steroidogenic enzymes, more specifically aromatase (Pezzi et al., 2004). In the adult male, aromatase activity is higher than at any other age (Tsai-Morris et al., 1985). Furthermore, exposure to 300 mg/kg/day DEHP and BDB produced a significant decrease in Ltf expression. The secretion in reproductive tissues of lactotransferrin, a multifunctional glycoprotein, is estrogen-dependent (Teng, 2006). While its role in the male reproductive tract remains largely unknown, it is found in the testis, epididymis, vas deferens, and prostate (Yu and Chen, 1993). Lactotransferrin is also abundant in seminal fluid and is believed to be directly correlated to gonadal function and sperm concentration in several species (Kikuchi et al., 2003a, b). Finally, the significant downregulation of Runx2 by DEHP is also in agreement with a dysregulation of estrogen signaling in the testis. There are indeed several examples of gene regulatory interplay between Runx2 and estrogens (Teplyuk et al., 2009), notably its role in the regulation of steroidogenic enzyme Cyp11a1. Estrogens have profound implications in testicular and epididymal development and function, and therefore on male fertility (Cooke et al., 2017; O'Donnell et al., 2001). In adult males, estrogen administration or deficiency may affect the maintenance of the hypothalamo-pituitary-testis axis (O’Donnell et al., 2001; Robaire et al., 1979). Testicular estrogens have also been shown to play a role in the regulation of luminal fluid reabsorption by the epithelial cells lining the efferent ductules (Hess et al., 2011). Finally, many studies provide indirect evidence for a role of estrogen in spermatogenesis involving germ cell proliferation, differentiation, and maturation of spermatids, as well as germ cell survival and apoptosis (reviewed in Carreau and Hess, 2010). Our gene expression data point toward a subtle but significant dysregulation of estrogen function in the testis after exposure to DEHP, and to a lesser extent to DINCH and BDB. Using high performance liquid chromatography/mass spectrometry, serum estradiols were below levels of detection in this study (data not shown). However, the increased epididymal weight we observed after exposure to 300 mg/kg/day DEHP is consistent with a potential impairment of fluid reabsorption by the efferent ductules that might be related to estrogen signaling. This study provides evidence that in utero and lactational exposures to our candidate compounds, BDB and DOS, as well as exposure to DINCH, do not produce significant alterations in adult male reproductive function. However, our data indicate that exposure to DEHP and, to a lesser extent, exposure to DINCH and BDB could produce subtle but significant alterations of estrogen signaling in the adult testis. These data need to be placed in the context of exposure to multiple endocrine disruptive compounds: gene expression modifications that are observed more than 2 months after exposure has ceased may suggest that exposure to multiple compounds throughout life may have long-lasting effects on adult male testicular function. Importantly, exposure to DOS did not produce significant changes in the expression of the estrogen signaling targets we studied, confirming its potential as a substitute for DEHP. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. ACKNOWLEDGMENTS The authors should like to thank Hanno Erythropel, Milan Maric, and Richard Leask from the McGill University Department of Chemical Engineering for designing and/or providing raw materials, Sheila Ernest and Elise Kolasa for their technical help, and Elise Boivin-Ford for her help with data entry. FUNDING Canadian Institutes of Health Research (CIHR) Institute of Human Development, Child and Youth Health [RHF100626]; post-doctoral fellowships from the CIHR Training Program in Reproduction, Early Development, and the Impact on Health (REDIH) and the Fonds de Recherche du Québec en Santé (FRQS) [to O.A.]; studentships from the Réseau Québécois en Reproduction NSERC-CREATE and the CIHR REDIH Program [to T.C.N.]. B.R. and B.F.H. are James McGill Professors. REFERENCES Abuelhija M. , Weng C. C. , Shetty G. , Meistrich M. L. ( 2013 ). Rat models of post-irradiation recovery of spermatogenesis: interstrain differences . Andrology 1 , 206 – 215 . Google Scholar CrossRef Search ADS PubMed Akingbemi B. T. , Youker R. T. , Sottas C. M. , Ge R. , Katz E. , Klinefelter G. R. , Zirkin B. R. , Hardy M. P. ( 2001 ). Modulation of rat Leydig cell steroidogenic function by di(2-ethylhexyl)phthalate . Biol. Reprod . 65 , 1252 – 1259 . Google Scholar CrossRef Search ADS PubMed Albert O. , Jégou B. ( 2014 ). A critical assessment of the endocrine susceptibility of the human testis to phthalates from fetal life to adulthood . Hum. Reprod. Update 20 , 231 – 249 . Google Scholar CrossRef Search ADS PubMed Albert O. , Nardelli T. C. , Hales B. F. , Robaire B. ( 2018 ). Identifying Greener and Safer Plasticizers: A 4-Step Approach . Toxicol. Sci . 161 , 266 – 275 . Google Scholar CrossRef Search ADS PubMed Andrade A. J. M. , Grande S. W. , Talsness C. E. , Gericke C. , Grote K. , Golombiewski A. , Sterner-Kock A. , Chahoud I. ( 2006 ). A dose response study following in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP): Reproductive effects on adult male offspring rats . Toxicology 228 , 85 – 97 . Google Scholar CrossRef Search ADS PubMed Boisvert A. , Jones S. , Issop L. , Erythropel H. C. , Papadopoulos V. , Culty M. ( 2016 ). In vitro functional screening as a means to identify new plasticizers devoid of reproductive toxicity . Environ. Res . 150 , 496 – 512 . Google Scholar CrossRef Search ADS PubMed Calafat A. M. , Slakman A. R. , Silva M. J. , Herbert A. R. , Needham L. L. ( 2004 ). Automated solid phase extraction and quantitative analysis of human milk for 13 phthalate metabolites . J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci . 805 , 49 – 56 . Google Scholar CrossRef Search ADS PubMed Campioli E. , Duong T. B. , Deschamps F. , Papadopoulos V. ( 2015 ). Cyclohexane-1, 2-dicarboxylic acid diisononyl ester and metabolite effects on rat epididymal stromal vascular fraction differentiation of adipose tissue . Environ. Res . 140 , 145 – 156 . Google Scholar CrossRef Search ADS PubMed Campioli E. , Lee S. , Lau M. , Marques L. , Papadopoulos V. ( 2017 ). Effect of prenatal DINCH plasticizer exposure on rat offspring testicular function and metabolism . Sci. Rep . 7 , 11072. Google Scholar CrossRef Search ADS PubMed Carreau S. , Hess R. A. ( 2010 ). Oestrogens and spermatogenesis . Philos. Trans. R. Soc. Lond. B. Biol. Sci . 365 , 1517 – 1535 . Google Scholar CrossRef Search ADS PubMed Cooke P. S. , Nanjappa M. K. , Ko C. , Prins G. S. , Hess R. A. ( 2017 ). Estrogens in male physiology . Physiol. Rev . 97 , 995 – 1043 . Google Scholar CrossRef Search ADS PubMed Culty M. , Thuillier R. , Li W. , Wang Y. , Martinez-Arguelles D. B. , Benjamin C. G. , Triantafilou K. M. , Zirkin B. R. , Papadopoulos V. ( 2008 ). In utero exposure to di-(2-ethylhexyl) phthalate exerts both short-term and long-lasting suppressive effects on testosterone production in the rat . Biol. Reprod . 78 , 1018 – 1028 . Google Scholar CrossRef Search ADS PubMed Dalsenter P. R. , Santana G. M. , Grande S. W. , Andrade A. J. M. , Araújo S. L. ( 2006 ). Phthalate affect the reproductive function and sexual behavior of male Wistar rats . Hum. Exp. Toxicol . 25 , 297 – 303 . Google Scholar CrossRef Search ADS PubMed Dorostghoal M. , Moazedi A. A. , Zardkaf A. ( 2012 ). Long-term effects of maternal exposure to Di (2-ethylhexyl) Phthalate on sperm and testicular parameters in Wistar rats offspring . Iran. J. Reprod. Med . 10 , 7 – 14 . Google Scholar PubMed Duty S. M. , Silva M. J. , Barr D. B. , Brock J. W. , Ryan L. , Chen Z. , Herrick R. F. , Christiani D. C. , Hauser R. ( 2003 ). Phthalate exposure and human semen parameters . Epidemiology 14 , 269 – 277 . Google Scholar PubMed Erythropel H. C. , Dodd P. , Leask R. L. , Maric M. , Cooper D. G. ( 2013 ). Designing green plasticizers: Influence of alkyl chain length on biodegradation and plasticization properties of succinate based plasticizers . Chemosphere 91 , 358 – 365 . Google Scholar CrossRef Search ADS PubMed Erythropel H. C. , Maric M. , Nicell J. A. , Leask R. L. , Yargeau V. ( 2014 ). Leaching of the plasticizer di(2-ethylhexyl)phthalate (DEHP) from plastic containers and the question of human exposure . Appl. Microbiol. Biotechnol . 98 , 9967 – 9981 . Google Scholar CrossRef Search ADS PubMed Firlotte N. , Cooper D. G. , Maric M. , Nicell J. A. ( 2009 ). Characterization of 1,5‐pentanediol dibenzoate as a potential “green” plasticizer for poly(vinyl chloride) . J. Vinyl Addit. Technol . 15 , 99 – 107 . Giovanoulis G. , Alves A. , Papadopoulou E. , Cousins A. P. , Schütze A. , Koch H. M. , Haug L. S. , Covaci A. , Magnér J. , Voorspoels S. ( 2016 ). Evaluation of exposure to phthalate esters and DINCH in urine and nails from a Norwegian study population . Environ. Res . 151 , 80 – 90 . Google Scholar CrossRef Search ADS PubMed Gray L. E. , Barlow N. J. , Howdeshell K. L. , Ostby J. S. , Furr J. R. , Gray C. L. ( 2009 ). Transgenerational effects of di (2-ethylhexyl) phthalate in the male CRL: CD(SD) rat: added value of assessing multiple offspring per litter . Toxicol. Sci . 110 , 411 – 425 . Google Scholar CrossRef Search ADS PubMed Gray L. E. , Ostby J. , Furr J. , Price M. , Veeramachaneni D. N. , Parks L. ( 2000 ). Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat . Toxicol. Sci . 58 , 350 – 365 . Google Scholar CrossRef Search ADS PubMed Hannon P. R. , Flaws J. A. ( 2015 ). The effects of phthalates on the ovary . Front. Endocrinol . 2 , 8 . Hauser R. , Calafat A. M. ( 2005 ). Phthalates and human health . Occup. Environ. Med . 62 , 806 – 818 . Google Scholar CrossRef Search ADS PubMed Hess R. A. , Fernandes S. A. F. , Gomes G. R. O. , Oliveira C. A. , Lazari M. F. M. , Porto C. S. ( 2011 ). Estrogen and its receptors in efferent ductules and epididymis . J. Androl . 32 , 600 – 613 . Google Scholar CrossRef Search ADS PubMed Heudorf U. , Mersch-Sundermann V. , Angerer J. ( 2007 ). Phthalates: toxicology and exposure . Int. J. Hyg. Environ. Health 210 , 623 – 634 . Google Scholar CrossRef Search ADS PubMed Kacew S. , Festing M. F. ( 1996 ). Role of rat strain in the differential sensitivity to pharmaceutical agents and naturally occurring substances . J. Toxicol. Environ. Health 47 , 1 – 30 . Google Scholar PubMed Kermanshahi-pour A. , Cooper D. G. , Mamer O. A. , Maric M. , Nicell J. A. ( 2009 ). Mechanisms of biodegradation of dibenzoate plasticizers . Chemosphere 77 , 258 – 263 . Google Scholar CrossRef Search ADS PubMed Kikuchi M. , Mizoroki S. , Kubo T. , Ohiwa Y. , Kubota M. , Yamada N. , Orino K. , Ohnami Y. , Watanabe K. ( 2003a ). Seminal plasma lactoferrin but not transferrin reflects gonadal function in dogs . J. Vet. Med. Sci . 65 , 679 – 684 . Google Scholar CrossRef Search ADS Kikuchi M. , Takao Y. , Tokuda N. , Ohnami Y. , Orino K. , Watanabe K. ( 2003b ). Relationship between seminal plasma lactoferrin and gonadal function in horses . J. Vet. Med. Sci . 65 , 1273 – 1274 . Google Scholar CrossRef Search ADS Kilcoyne K. R. , Smith L. B. , Atanassova N. , Macpherson S. , McKinnell C. , van den Driesche S. , Jobling M. S. , Chambers T. J. G. , De Gendt K. , Verhoeven G. et al. , . ( 2014 ). Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells . Proc. Natl Acad. Sci. USA 111 , E1924 – E1932 . Google Scholar CrossRef Search ADS Koch H. M. , Drexler H. , Angerer J. ( 2003 ). An estimation of the daily intake of di(2-ethylhexyl)phthalate (DEHP) and other phthalates in the general population . Int. J. Hyg. Environ. Health 206 , 77 – 83 . Google Scholar CrossRef Search ADS PubMed Latini G. , De Felice C. , Presta G. , Del Vecchio A. , Paris I. , Ruggieri F. , Mazzeo P. ( 2003 ). Exposure to Di(2-ethylhexyl)phthalate in humans during pregnancy. A preliminary report . Biol. Neonate 83 , 22 – 24 . Google Scholar CrossRef Search ADS PubMed Martinez-Arguelles D. B. , Culty M. , Zirkin B. R. , Papadopoulos V. ( 2009 ). In utero exposure to di-(2-ethylhexyl) phthalate decreases mineralocorticoid receptor expression in the adult testis . Endocrinology 150 , 5575 – 5585 . Google Scholar CrossRef Search ADS PubMed Nair A. B. , Jacob S. ( 2016 ). A simple practice guide for dose conversion between animals and human . J. Basic Clin. Pharm . 7 , 27 – 31 . Google Scholar CrossRef Search ADS PubMed Nardelli T. C. , Albert O. , Lalancette C. , Culty M. , Hales B. F. , Robaire B. ( 2017 ). In utero and lactational exposure study in rats to identify replacements for Di(2-ethylhexyl) phthalate . Sci. Rep . 7 , 623. Google Scholar CrossRef Search ADS PubMed Nardelli T. C. , Erythropel H. C. , Robaire B. ( 2015 ). Toxicogenomic screening of replacements for di(2-ethylhexyl) phthalate (DEHP) using the immortalized TM4 sertoli cell line . PLoS ONE 10 , e0138421. Google Scholar CrossRef Search ADS PubMed O'Donnell L. , Robertson K. M. , Jones M. E. , Simpson E. R. ( 2001 ). Estrogen and spermatogenesis . Endocr. Rev . 22 , 289 – 318 . Google Scholar CrossRef Search ADS PubMed Parks L. G. , Ostby J. S. , Lambright C. R. , Abbott B. D. , Klinefelter G. R. , Barlow N. J. , Gray L. E. ( 2000 ). The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat . Toxicol. Sci . 58 , 339 – 349 . Google Scholar CrossRef Search ADS PubMed Pezzi V. , Sirianni R. , Chimento A. , Maggiolini M. , Bourguiba S. , Delalande C. , Carreau S. , Andò S. , Simpson E. R. , Clyne C. D. ( 2004 ). Differential expression of steroidogenic factor-1/adrenal 4 binding protein and liver receptor homolog-1 (LRH-1)/fetoprotein transcription factor in the rat testis: LRH-1 as a potential regulator of testicular aromatase expression . Endocrinology 145 , 2186 – 2196 . Google Scholar CrossRef Search ADS PubMed Robaire B. , Ewing L. L. , Irby D. C. , Desjardins C. ( 1979 ). Interactions of testosterone and estradiol-17 beta on the reproductive tract of the male rat . Biol. Reprod . 21 , 455 – 463 . Google Scholar CrossRef Search ADS PubMed Robb G. W. , Amann R. P. , Killian G. J. ( 1978 ). Daily sperm production and epididymal sperm reserves of pubertal and adult rats . J. Reprod. Fertil . 54 , 103 – 107 . Google Scholar CrossRef Search ADS PubMed Schettler T. ( 2006 ). Human exposure to phthalates via consumer products . Int. J. Androl . 29 , 134 – 139 . discussion181–5. Google Scholar CrossRef Search ADS PubMed Shellabarger C. J. , Stone J. P. , Holtzman S. ( 1978 ). Rat differences in mammary tumor induction with estrogen and neutron radiation . J. Natl Cancer Inst . 61 , 1505 – 1508 . Google Scholar PubMed Silva M. J. , Barr D. B. , Reidy J. A. , Malek N. A. , Hodge C. C. , Caudill S. P. , Brock J. W. , Needham L. L. , Calafat A. M. ( 2004a ). Urinary levels of seven phthalate metabolites in the U.S. population from the National Health and Nutrition Examination Survey (NHANES) 1999–2000 . Environ. Health Perspect . 112 , 331 – 338 . Google Scholar CrossRef Search ADS Silva M. J. , Jia T. , Samandar E. , Preau J. L. , Calafat A. M. ( 2013 ). Environmental exposure to the plasticizer 1, 2-cyclohexane dicarboxylic acid, diisononyl ester (DINCH) in U.S. adults (2000–2012) . Environ. Res . 126 , 159 – 163 . Google Scholar CrossRef Search ADS PubMed Silva M. J. , Reidy J. A. , Herbert A. R. , Preau J. L. , Needham L. L. , Calafat A. M. ( 2004b ). Detection of phthalate metabolites in human amniotic fluid . Bull. Environ. Contam. Toxicol . 72 , 1226 – 1231 . Google Scholar CrossRef Search ADS Sirianni R. , Seely J. B. , Attia G. , Stocco D. M. , Carr B. R. , Pezzi V. , Rainey W. E. ( 2002 ). Liver receptor homologue-1 is expressed in human steroidogenic tissues and activates transcription of genes encoding steroidogenic enzymes . J. Endocrinol . 174 , R13 – R17 . Google Scholar CrossRef Search ADS PubMed Skakkebaek N. E. , Rajpert-De Meyts E. , Main K. M. ( 2001 ). Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects . Hum. Reprod . 16 , 972 – 978 . Google Scholar CrossRef Search ADS PubMed Teng C. T. ( 2006 ). Factors regulating lactoferrin gene expression . Biochem. Cell Biol . 84 , 263 – 267 . Google Scholar CrossRef Search ADS PubMed Teplyuk N. M. , Zhang Y. , Lou Y. , Hawse J. R. , Hassan M. Q. , Teplyuk V. I. , Pratap J. , Galindo M. , Stein J. L. , Stein G. S. et al. , . ( 2009 ). The osteogenic transcription factor runx2 controls genes involved in sterol/steroid metabolism, including CYP11A1 in osteoblasts . Mol. Endocrinol . 23 , 849 – 861 . Google Scholar CrossRef Search ADS PubMed Thomas J. A. , Thomas M. J. ( 1984 ). Biological effects of di-(2-ethylhexyl) phthalate and other phthalic acid esters . Crit. Rev. Toxicol . 13 , 283 – 317 . Google Scholar CrossRef Search ADS PubMed Tsai-Morris C. H. , Aquilano D. R. , Dufau M. L. ( 1985 ). Cellular localization of rat testicular aromatase activity during development . Endocrinology 116 , 38 – 46 . Google Scholar CrossRef Search ADS PubMed U.S. Food and Drug Administration ( 2002 ). Safety assessment of di(2-ethylhexyl)phthalate (DEHP) released from PVC medical devices. Available at: https://www.fda.gov/downloads/MedicalDevices/…/UCM080457.pdf. Accessed March 8, 2018. Wittassek M. , Koch H. M. , Angerer J. , Brüning T. ( 2011 ). Assessing exposure to phthalates—the human biomonitoring approach . Mol. Nutr. Food Res . 55 , 7 – 31 . Google Scholar CrossRef Search ADS PubMed Yu L. C. , Chen Y. H. ( 1993 ). The developmental profile of lactoferrin in mouse epididymis . Biochem. J . 296 , 107 – 111 . Google Scholar CrossRef Search ADS PubMed Zubkova E. V. , Robaire B. ( 2004 ). Effect of glutathione depletion on antioxidant enzymes in the epididymis, seminal vesicles, and liver and on spermatozoa motility in the aging brown Norway rat . Biol. Reprod . 71 , 1002 – 1008 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Toxicological SciencesOxford University Press

Published: Mar 21, 2018

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