Prenatal Exposure to DEHP Induces Neuronal Degeneration and Neurobehavioral Abnormalities in Adult Male Mice

Prenatal Exposure to DEHP Induces Neuronal Degeneration and Neurobehavioral Abnormalities in... Abstract Phthalates are a family of synthetic chemicals that are used in producing a variety of consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is an widely used phthalate and poses a public health concern. Prenatal exposure to DEHP has been shown to induce premature reproductive senescence in animal studies. In this study, we tested the hypothesis that prenatal exposure to DEHP impairs neurobehavior and recognition memory in her male offspring and we investigated one possible mechanism—oxidative damage in the hippocampus. Pregnant CD-1 female mice were orally administered 200 μg, 500 mg, or 750 mg/kg/day DEHP or vehicle from gestational day 11 until birth. The neurobehavioral impact of the prenatal DEHP exposure was assessed at the ages of 16–22 months. Elevated plus maze and open field tests were used to measure anxiety levels. Y-maze and novel object recognition tests were employed to measure memory function. The oxidative damage in the hippocampus was measured by the levels of oxidative DNA damage and by Spatial light interference microscopic counting of hippocampal neurons. Adult male mice that were prenatally exposed to DEHP exhibited anxious behaviors and impaired spatial and short-term recognition memory. The number of hippocampal pyramidal neurons was significantly decreased in the DEHP mice. Furthermore, DEHP mice expressed remarkably high levels of cyclooxygenase-2, 8-hydroxyguanine, and thymidine glycol in their hippocampal neurons. DEHP mice also had lower circulating testosterone concentrations and displayed a weaker immunoreactivity than the control mice to androgen receptor expression in the brain. This study found that prenatal exposure to DEHP caused elevated anxiety behavior and impaired recognition memory. These behavioral changes may originate from neurodegeneration caused by oxidative damage and inflammation in the hippocampus. Decreased circulating testosterone concentrations and decreased expression of androgen receptor in the brain also may be factors contributing to the impaired neurobehavior in the DEHP mice. DEHP, endocrine disruptor, anxiety, hippocampus, aging Di-(2-ethylhexyl) phthalate (DEHP) is one of the most commonly used phthalates, and is widely used in the production of a variety of cosmetics, personal care products, food storage containers, pharmaceuticals, building material, and medical tubing (Dai et al., 2015). DEHP readily leaches from these products directly into food, beverages, water, or air (Swan et al., 2005). DEHP is known as an endocrine disruptor and has been shown to disrupt the reproductive system in both females and males (Barakat et al., 2017; Culty et al., 2008; Niermann et al., 2015). The most obvious toxicity of DEHP exposure to date is its impact on gonadal steroidogenesis (Barakat et al., 2017; Gray et al., 2009; Moore et al., 2001) and the accompanying decline in reproductive function and fertility (Andrade et al., 2006; Cho et al., 2010; Culty et al., 2008; Dalsenter et al., 2006; Gray et al., 2000; Tanaka 2005; Tang et al., 2015). Besides creating problems in the reproductive system, a strong association between DEHP exposure and neurological functions has begun to emerge. A number of epidemiological studies indicate an association between maternal urine phthalate concentrations during pregnancy and cognitive and learning disabilities in offspring (Adibi et al., 2009; Arbuckle et al., 2016; Cho et al., 2010; Engel et al., 2010; Gascon et al., 2015; Huang et al., 2017; Yolton et al., 2011). Testosterone is well known for its functions in reproduction, sexual differentiation, and sexual behavior as well as its modulating effect on anxiety (Edinger et al., 2004). In men, low circulating T concentrations hav been associated with depressive symptoms and anxiety (Almeida et al., 2008). In animal studies using rats, systemic administration of testosterone or dihydrotestosterone to gonadectomized males reduced anxiety-like behaviors (Edinger et al., 2004; Frye and Seliga, 2001). These androgens probably play this anti-anxiety role by acting directly or indirectly on neurons; androgen receptor (AR) is expressed in a variety of neurons and glial cells and regulates neuronal cell differentiation, excitability and survival (Nguyen et al., 2009; Pouliot et al., 1996; Spritzer and Galea, 2007). In support, testosterone exerts neuroprotective activity because it enhances neuronal survival and prevents neurodegeneration (Lau et al., 2015). Furthermore, decreased testosterone or dihydrotestosterone increases the accumulation of oligomeric β-amyloid peptides, accelerating neuronal cell death, and it is considered to be a risk factor for developing Alzheimer’s disease (Chu et al., 2010; Lau et al., 2014). In the hippocampal neurons, these neuroprotective roles of androgens are mediated in part by AR-dependent activation of cyclic adenosine monophosphate response element-binding protein (Nguyen et al., 2009). Age-related decline of testosterone is linked to dysfunction and disease development in several androgen-responsive tissues, including brain (Kaufman and Vermeulen, 2005). In old rodent, decline of testosterone level is associated with impaired synapse formation (Leranth et al., 2004) and development of neurodegenerative disease (Moffat et al., 2004). Importantly, prenatal exposure to endocrine disrupting chemicals (EDCs) hinders the growth and development of the fetal organs, and these adverse effects could persist throughout life (Schug et al., 2011). These findings prompted us to test the hypothesis that DEHP exposure induced decline of testosterone and leads to an adverse impact on neuro-behavioral function in adulthood. Adverse neurobehavioral changes may be provoked by oxidative stress occurring in the brain, specifically in the hippocampus (Liu et al., 2014; Murakami and Murakami, 2005). Oxidative stress is generated primarily in the form of reactive oxygen species (ROS) from mitochondrial metabolism (Finkel and Holbrook, 2000). ROS then induce oxidative damage in vital macromolecules such as DNA, proteins, and lipids, leading to negative outcomes such as accelerated aging, tumorigenesis, or neurodegeneration (Hamilton et al., 2001). Overall the antianxiety and neuroprotective roles that androgens play and our previous finding that prenatal exposure to DEHP induced a premature decline of circulating testosterone in adult male mice led us to hypothesize that maternal exposure to DEHP impairs neurobehavior and recognition memory in her male offspring via neuronal damage in the hippocampus. In this study, we investigated a possible mechanism by assessing the impact of prenatal DEHP exposure on anxiety level, memory function, brain histology, and neuro-inflammation. MATERIALS AND METHODS Chemicals DEHP (99% purity) was purchased from Sigma-Aldrich (St Louis). Tocopherol-stripped corn oil was purchased from MP Bio-Medicals (Solon, Ohio) and was used as a vehicle. Stock solutions of DEHP were prepared by diluting it in tocopherol-stripped corn oil to obtain the desired concentrations. Animals and dosing regimen CD-1 mice were used in this study and were housed at the University of Illinois at Urbana-Champaign (UIUC) animal care facility under 12-h light/dark cycles. The mice were provided with Teklad Rodent Diet 8604 (Harlan) and had ad-libitum access to food and water. Animal handling and procedures were approved by the UIUC Institutional Animal Care and Use Committee (Protocol ID No.: 14144). Pregnant dams were prepared by mating 2-month-old females with proven breeder males. The pregnant female mice were orally treated with vehicle control (tocopherol-stripped corn oil), 200 µg/kg/day, 500 mg/kg/day, or 750 mg/kg/day of DEHP from gestational day 11 to the day of birth by placing a pipette tip into the mouth as previously described in Niermann et al. (2015). DEHP doses were chosen because they are environmentally relevant. The Agency for Toxic Substances and Disease Registry estimates that the range of daily human exposure to DEHP is 3–30 µg/kg/day, and the no-observed-adverse-effect level for DEHP is 5.8 mg/kg/day (ATSDR, 2002). The lowest-observed-adverse-effect level of DEHP is 140 mg/kg/day, and potential reproductive effects occur at levels ranging from 1 to 30 µg/kg/day (Blystone et al., 2010; Gray et al., 2009). The reference dose of a daily oral exposure to DEHP in the human population is 20 µg/kg/day based on the U. S. Environmental Protection Agency published reference safe dose (https://www.epa.gov/sites/production/files/2016-09/documents/bis-2-ethylhexyl-phthalate.pdf). Specifically, DEHP at 200 μg/kg/day was used because adult exposure causes abnormal estrous cyclicity, accelerates primordial follicle recruitment in female mice, and decreased the fertility in male CD-1 mice (Barakat et al., 2017; Hannon et al., 2014). The 500 mg and 750 mg/kg/day DEHP doses were used previously in many research papers to study the different pathological effects of DEHP exposure (Andrade et al. 2006; Culty et al., 2008; Do et al., 2012; Doyle et al., 2013). Y-maze spontaneous alteration test The Y-maze spontaneous alteration test was used to examine the effects of prenatal DEHP exposure on spatial memory of prenatally exposed male mice at 16 months of age. The Y-maze was composed of 3 equal arms (120°, 41-cm long, and 15-cm high. Each mouse was placed in one of the arm compartments (A, B, or C) and was allowed to move freely for 15 min. The sequence of arm entries was manually recorded as A, B, or C. An alternation was defined as an entry into all 3 arms consecutively. The number of maximum spontaneous alternations is the total number of arms entered minus 2, and the percentage alternation is calculated as ([actual alternations/maximum alternations] × 100) (Naolapo et al., 2012). The total number of arm entries was also recorded as an indication of general locomotor activity of the mice. The apparatus was cleaned with 70% ethanol to remove odors from the previous mouse. Novel object recognition test The novel object recognition (NOR) test is one the most popular paradigms to evaluate relational memory. All treated groups were tested at 16 months of age to examine the effects of prenatal DEHP exposure on the recognition memory. In the first trial (training phase), the mouse was placed into the chamber with 2 identical objects (same color, shape, and size) for 5 min. Then the mouse was placed back into its main cage for an intertrial interval of 1 h. Objects were washed with 70% ethanol between trials to remove olfactory cues. In the second trial (testing phase), the mouse was placed back into the chamber with 2 objects, and one of the familiar objects was replaced with a new novel object. The time spent exploring the novel and familiar objects was measured for 5 min (Pascual et al., 2011). Exploration time, defined as viewing, sniffing, and/or touching at 1 cm distance or less from the object, was recorded for the familiar and novel object (Fole et al., 2015). Elevated plus maze test The elevated plus maze (EPM) is used to measure anxiety in rodent research (Walf and Frye, 2007). All treated groups were tested at 18 months of age to study the anxiety-like behavior. The EPM apparatus consists of 2 open arms alternating with 2 closed arms (30-cm high); the whole maze was elevated 50 cm above the floor, and the testing was conducted in a dimly lit room. Each mouse was placed in the center of the maze facing one of the closed arms and was allowed to explore for 10 min as described previously in Czerniczyniec et al. (2011). We used 70% ethanol to clean each arm of the maze and remove olfactory cues between trials. The EPM relies upon the mice preferring to move toward dark, enclosed spaces while fearing and avoiding heights/open spaces (Naolapo et al., 2012). Entry into the arm of the EPM was defined as the animal placing all 4 paws in the arm. The latency to enter the open arms, defined as the first time the mouse enters an open arm of the maze during the experimental period, was measured; this is expressed in seconds. The number of entries into both open and closed arms during the experimental time (10 min) was also measured. The amount of time spent in the open arms and closed arms of the maze were recorded and expressed in seconds. Open field test The open field test (OFT) is also a test used to measure anxiety and locomotor activity in rodents. The open-field apparatus consisted of a white Plexiglass box (50 × 50 × 22 cm) with the floor divided into 16 squares. The “border” was defined as the 12 outer periphery squares and the “center” as the 4 inner central squares as described previously in Davis et al. (2012) and Liu et al. (2014). At 18 months, each mouse was placed at the center of the apparatus in a dimly lit room and was allowed to move freely for 5 min. The open field was cleaned with 70% ethanol to remove odors from the previous mouse. It is well known that rodents show natural avoidance of open surfaces (Czerniczyniec et al., 2011). The time taken before the first entry to the center squares and the number of entries to the center square area were evaluated as an index of anxiety. Measurement of serum testosterone concentration Peripheral blood was collected from 22-month-old mice by cardiac puncture. The blood was centrifuged at 2000 × g, and then serum was collected and stored at −20°C until further analyses. The concentration of circulating testosterone was measured by using ELISA kits (DRG Diagnostic) with a reportable range of 0.06–25 ng/ml at 22 months of age. Tissue collection and brain weight At 22 months of age, all the F1 male mice were euthanized by CO2 asphyxiation followed by cervical dislocation. After that, the brain was collected and weighed. The tissues were fixed in 4% paraformaldehyde for 24 h and then transferred to 70% ethyl alcohol and kept at 4°C until further tissue processing. Histological examination and Nissl staining of hippocampus Brain tissue was fixed in 4% paraformaldehyde for 24 h, washed with ethanol and embedded in paraffin wax, and serially sectioned at 7-µm thickness in a sagittal orientation using a microtome. For general histological observation, slides were stained with hematoxylin and eosin (H&E). The brain tissue was also stained with Nissl staining (cresyl violet staining) to evaluate the neuronal damage. Microscopic images were obtained from pyramidal cell layers in CA1 and CA2/3 subdivisions of the hippocampus by using an Olympus BX51 microscope. Counting of hippocampal pyramidal neurons (Nissl staining and Spatial light interference microscopy) From each mouse, 4 sections of 7-µm thickness made 120 µm apart each other were selected to count the neurons in the hippocampus. The Nissl-stained hippocampus was divided into 2 subregions–Cornu Ammonis (CA) CA1 and CA2/3 regions-based on morphologic appearance and identifiable landmarks. The number of intact neurons within the CA1 and CA2/3 layers were counted using an Olympus BX51 microscope at 400× magnification (Figure 3). Spatial light interference microscopy (SLIM) comprising a commercial phase contrast microscope (Carl Zeiss, Axio Observer.Z1) combined with a SLIM module (Phi Optics, Inc., Cell Vista SLIM Pro) provided multicontrast perspectives of gross and microscopic brain anatomy (Min et al., 2016). For SLIM microscopic counting, each brain slice was deparaffinized with xylene, rehydrated with ethanol, and mounted in aqueous mounting media to recover the refractive index profile in tissue as described previously in Min et al. (2016). The hippocampus was divided into 4 subregions: CA1, CA2, CA3, and dentate gyrus (DG), and pyramidal neurons were then quantified (Figure 4). Immunohistochemistry The expression of AR, cyclooxygenase-2 (COX-2), and DNA oxidation markers such as 8-hydroxyguanine (8-OHdG) and thymidine glycol (TG) in the hippocampus were determined by immunohistochemistry. Sagittal sections of the brains from all treated groups were deparaffinized, followed by heat-induced antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) and then incubated in 3% H2O2 for 30 min at room temperature for endogenous peroxide quenching. Next, we blocked endogenous biotin with 5% goat or horse serum in avidin (200 μl/1 ml; SP-2001, Vector labs, Burlingame, CA). The slides were incubated overnight with antiAR antibody (sc-816, Santa Cruz Biotechnology), anti8-OHdG antibody (sc-66036, Santa Cruz Biotechnology), antiCOX-2 antibody (Cayman), or antiTG antibody (Institute for the Control of Aging, Shizuoka, Japan; JaICA). This was followed by incubation with a secondary biotinylated goat antirabbit antibody or horse antimouse antibody (Vectastain ABC kit, Vector labs) and avidin-biotin complex solution (Vectastain Elite ABC kit, Vector labs) at room temperature. 3, 3’-diaminobenzidine (SK-4100, Vector labs) was applied until color optimally developed. Slides were then counterstained with hematoxylin, mounted, and imaged with an Olympus BX51 microscope. Four sections with 120-µm intervals from each mouse were selected to quantitatively assess immunoreactivity of COX-2 in the hippocampus. Statistical analysis The data were analyzed using the statistical software package SPSS. Multiple comparisons between normally distributed continuous experimental groups were analyzed by 1-way analysis of variance (ANOVA) as a parametric test followed by the Tukey’s HSD (honest significant difference) test for comparisons of individual means. If data were not normally distributed, were presented as a percentage, and/or did not meet homogeneity of variance assumptions, the independent sample Kruskal-Wallis H followed by Mann-Whitney were performed. Animal used in this study were from individual litters, and the number of animals used for statistical analyses ranged from 4 to 7 mice. The data are expressed as mean ± SEM. Asterisks in figures indicate a statistically significant difference (p ≤ .05) compared with controls. RESULTS Prenatal DEHP Exposure Increases Anxiety Behavior in Adult Male Mice At the age of 16–20 months, the OFT and the EPM test were performed to assess the level of anxiety. The EPM test relies upon mice preferring to move toward dark, enclosed spaces while avoiding open spaces (Naolapo et al., 2012). The OFT relies on the natural avoidance of mice of open surfaces and fear of wide-open spaces such as the center square region (Czerniczyniec et al., 2011). The OFT showed that DEHP mice tended to take more time before making entries into the center area compared with the controls (p = .08, 750 mg/kg/day), but the difference between the DEHP and control groups did not reach statistical significance (Figure 1A). All of the DEHP-exposed groups made significantly fewer entries to the center area than the control group (p = .03, .02, and .01, respectively) (Figure 1B). The EPM test showed that DEHP mice in the highest exposure group took more time before making entries to the open arms (p = .04, 750 mg/kg/day) (Figure 1C). Interestingly, the numbers of entries into the open arms and the time spent there did not differ between the DEHP-exposed groups and the control group (Figs. 1D and 1E). Figure 1. View largeDownload slide Effect of prenatal DEHP exposure on anxiety level of F1 males measured at 18 months (open field and EPM test). A, Latency to the center square area in the OFT (seconds). B, Number of entries into the center square area in the OFT. C, Latency to the open arm in EPM (seconds), *indicates p ≤ .05 when compared with control group (Kruskal Wallis test). D, Time spent in the open arm in EPM (seconds). E, Number of open and total arm entries in EPM. Graphs show mean ± SEM; * indicates p ≤ .05 when compared with control group (1-way ANOVA and Tukey’s post hoc test). Figure 1. View largeDownload slide Effect of prenatal DEHP exposure on anxiety level of F1 males measured at 18 months (open field and EPM test). A, Latency to the center square area in the OFT (seconds). B, Number of entries into the center square area in the OFT. C, Latency to the open arm in EPM (seconds), *indicates p ≤ .05 when compared with control group (Kruskal Wallis test). D, Time spent in the open arm in EPM (seconds). E, Number of open and total arm entries in EPM. Graphs show mean ± SEM; * indicates p ≤ .05 when compared with control group (1-way ANOVA and Tukey’s post hoc test). Prenatal DEHP Exposure Impairs Spatial and Recognition Memory The impact of early exposure to DEHP on the spatial and recognition memory was assessed by the Y-maze spontaneous alteration test and NOR test, respectively. The Y-maze test measures spatial memory and locomotor activity (Naolapo et al., 2012). An animal with impaired spatial memory or locomotion tends to show less alteration behavior (tends to enter the arm that he just visited rather than visiting the next arm). The NOR test is used to measure short-term recognition memory (Pascual et al., 2011). A normal animal tends to spend more time exploring the novel object. The Y-maze test showed that the lowest DEHP dose group displayed the least alternation behavior (p = .04) and the fewest arm entries (p = .01) compared with the control group (Figs. 2A and 2B). The NOR test showed that the DEHP mice tended to spend less time exploring the new object (500 and 750 mg/kg/day groups; p = .06 and .05, respectively) than did the control group (Figs. 2C and 2D). Figure 2. View largeDownload slide Effect of prenatal DEHP exposure on spatial memory measured by the Y-maze spontaneous alteration test (A and B) and recognition memory (C and D) measured by the NOR test of F1 males at 16 months. A, Percent of alteration (A-B-C consecutive arm entry) during 15 min. B, Number of arm entries during 15 min. of experimental time; * indicates p ≤ .05 when compared with the control group (Kruskal Wallis test followed by Mann-Whitney). C, Exploration time of past and novel object (seconds; * indicates p ≤ .05 when compared with the control group (Kruskal Wallis test followed by Mann-Whitney). D, Percent of novel object exploration time during 5 min of experimental time (seconds). Graphs show mean ± SEM; * indicates p ≤ .05 when compared with the control group. Figure 2. View largeDownload slide Effect of prenatal DEHP exposure on spatial memory measured by the Y-maze spontaneous alteration test (A and B) and recognition memory (C and D) measured by the NOR test of F1 males at 16 months. A, Percent of alteration (A-B-C consecutive arm entry) during 15 min. B, Number of arm entries during 15 min. of experimental time; * indicates p ≤ .05 when compared with the control group (Kruskal Wallis test followed by Mann-Whitney). C, Exploration time of past and novel object (seconds; * indicates p ≤ .05 when compared with the control group (Kruskal Wallis test followed by Mann-Whitney). D, Percent of novel object exploration time during 5 min of experimental time (seconds). Graphs show mean ± SEM; * indicates p ≤ .05 when compared with the control group. Prenatal DEHP Exposure Induces Neurodegeneration in Hippocampal Pyramidal Neurons Memory function can be impaired by the degeneration of hippocampal pyramidal neurons (Zilka et al., 2006). To determine if hippocampal neurodegeneration was a contributing factor to the impaired memory observed in the DEHP mice, we first examined the hippocampus histologically. Brains were collected at 22 months of age, and their sections were stained by either H&E (Supplementary Figure 1) or Nissl staining (Figure 3). Histologically, the hippocampal pyramidal neurons of the DEHP mice were shrunken, loosely aligned, fewer in number and showed an enlarged inter-neuronal space between the neurons (Supplementary Figure 1) (Figure 3). A manual microscopic counting of pyramidal neurons in the Nissl-stained hippocampus showed that the hippocampi of DEHP mice contained significantly fewer neurons in a dose- and region-specific manner compared with the controls (Figure 3D). We then re-examined the hippocampi using the SLIM, a newly developed computerized microscopy (Min et al., 2016) that uses the unique physical properties of neuronal cell bodies, such as higher phase values than axons, dendrites, and extracellular matrix and an algorism that allows a computerized identification of neuronal cell bodies (Figure 4). Using SLIM, the numbers of pyramidal neurons were compared in 4 subregions of the hippocampus: CA1, CA2, CA3, and DG (Figure 4A). By SLIM analysis, we found that the hippocampi of the 500 and 750 mg/kg/day DEHP mice had fewer neurons in the regions of CA1 (p = .07 and .002, respectively) and CA2 (p = .02 and .006, respectively) than the controls (Figure 4E). Furthermore, the hippocampi of the 500 and 750 mg/kg/day DEHP mice had fewer neurons in the DG region (p = .002 and .02, respectively) (Figure 4E). Interestingly, while not statistically significant, the mean brain weight of DEHP mice was lighter than those of control (Figure 8A). Figure 3. View largeDownload slide Nissl staining of hippocampus at 22-months old. A, Hippocampus of all treatment groups at lower magnification (40×). B, CA1 pyramidal neurons of different groups at 200×. C, CA2/3 pyramidal neurons of different groups at 200×. Shows normal neurons in the control group and abnormal neurons in the DEHP-treated groups, which showed shrunken neurons (arrow heads) and enlarged intracellular space (arrow). D, Manual counting of pyramidal neurons in CA1 and CA2/3 regions in control and DEHP-treated groups. Graphs show mean ± SEM, * indicates p ≤ .05 when compared each region of DEHP-treated group with the control group (1-way ANOVA and Tukey’s post hoc test). Figure 3. View largeDownload slide Nissl staining of hippocampus at 22-months old. A, Hippocampus of all treatment groups at lower magnification (40×). B, CA1 pyramidal neurons of different groups at 200×. C, CA2/3 pyramidal neurons of different groups at 200×. Shows normal neurons in the control group and abnormal neurons in the DEHP-treated groups, which showed shrunken neurons (arrow heads) and enlarged intracellular space (arrow). D, Manual counting of pyramidal neurons in CA1 and CA2/3 regions in control and DEHP-treated groups. Graphs show mean ± SEM, * indicates p ≤ .05 when compared each region of DEHP-treated group with the control group (1-way ANOVA and Tukey’s post hoc test). Figure 4. View largeDownload slide SLIM microscopic counting of hippocampal pyramidal neurons at 22 months. A, Hippocampus of all treatment groups at lower magnification (40×). B, CA1 pyramidal neurons in different groups at 200×. C, CA2 pyramidal neurons in different groups at 200×. D, CA3 pyramidal neurons in different groups at 200×. Shows that neuronal cell bodies have higher phase values than axons and dendrites, while the cell membrane, axon, and dendrites have much higher phase values than cytoplasmic regions and the extracellular matrix. E, SLIM microscopic quantification of pyramidal neurons in DG, CA1, CA2, and CA3 regions. Graphs show mean ± SEM, * indicates p ≤ .05 when compared each region of DEHP-treated group with the control group (1-way ANOVA and Tukey’s post hoc test). Figure 4. View largeDownload slide SLIM microscopic counting of hippocampal pyramidal neurons at 22 months. A, Hippocampus of all treatment groups at lower magnification (40×). B, CA1 pyramidal neurons in different groups at 200×. C, CA2 pyramidal neurons in different groups at 200×. D, CA3 pyramidal neurons in different groups at 200×. Shows that neuronal cell bodies have higher phase values than axons and dendrites, while the cell membrane, axon, and dendrites have much higher phase values than cytoplasmic regions and the extracellular matrix. E, SLIM microscopic quantification of pyramidal neurons in DG, CA1, CA2, and CA3 regions. Graphs show mean ± SEM, * indicates p ≤ .05 when compared each region of DEHP-treated group with the control group (1-way ANOVA and Tukey’s post hoc test). Prenatal DEHP Exposure Increases COX-2 Expression in Hippocampal Neurons Neuronal inflammation is one of the leading causes of neurodegeneration (Aisen, 2002). As there was an obvious sign of neurodegeneration in the DEHP mice (Figs. 3 and 4), we examined their hippocampi to see if they underwent inflammation. COX-2, (PTGES-2) expression level was measured as an inflammatory marker, as COX-2 plays a key role in neuroinflammation and pathogenesis of neurodegenerative disease. COX-2 is expressed in response to an acute inflammatory signal and converts arachidonic acid to prostaglandins (Yang and Chen, 2008). In the control tissues, a mild COX-2 immunoreactivity was observed in the CA2/3 region but not in the CA1 and DG regions (Figure 5A). In contrast, COX-2 immunoreactivity was remarkably stronger in the CA2/3 region of the DEHP mice (Figure 5A). Counting COX-2-positive neurons in the CA2/3 region showed that the 200 µg/kg/day and 750 mg/kg/day DEHP mice had more COX-2-positive neurons than the controls (p = .01 and .04; respectively) (Figure 5B). Figure 5. View largeDownload slide Immunohistochemical detection of COX-2 positive neurons in the hippocampus. A, Brown staining represents the sites of COX-2 expression (arrows) in pyramidal neurons of CA-2/3 region of the control and DEHP-treated groups at 200× magnification. B, Percent of pyramidal neurons in CA2/3 regions with positive COX-2 expression. Graphs show mean ± SEM, * indicates p ≤ .05 when compared with the control group; (Kruskal Wallis test followed by Mann-Whitney). (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Figure 5. View largeDownload slide Immunohistochemical detection of COX-2 positive neurons in the hippocampus. A, Brown staining represents the sites of COX-2 expression (arrows) in pyramidal neurons of CA-2/3 region of the control and DEHP-treated groups at 200× magnification. B, Percent of pyramidal neurons in CA2/3 regions with positive COX-2 expression. Graphs show mean ± SEM, * indicates p ≤ .05 when compared with the control group; (Kruskal Wallis test followed by Mann-Whitney). (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Prenatal DEHP Exposure Elevates DNA Oxidation Markers (8-OHdG, TG) in Hippocampal Neurons Oxidative damage to DNA is known to cause neurodegeneration (Zussy et al., 2013). Exposure to DEHP is reported to cause oxidative damage in affected tissues (Tang et al., 2015). Therefore, we looked into the possibility that the DNA of the hippocampal neurons of a DEHP mouse might be oxidatively damaged. We assessed the degree of oxidation in the hippocampus by immunohistochemical examination using 2 DNA oxidation damage markers: 8-OHdG and TG. 8-OHdG and TG are formed when DNA is attacked by hydroxyl radicals that are synthesized during oxidative stress (Lovell and Markesbery, 2007). Figure 6 shows a representative image of 8-OHdG staining in different regions of the hippocampi. DEHP mice showed stronger OHdG immunostaining in CA2, CA3, and DG regions compared with the controls (Figure 6). TG staining also revealed a higher immunostaining intensity in the CA2 and DG regions of the DEHP mice than controls (Figure 7). Figure 6. View largeDownload slide Immunohistochemical detection of 8-OHdG (DNA oxidation marker) in hippocampal pyramidal neurons. A, Brown staining represents the site of OHdG immunostaining in the CA2 regions (arrows) in different groups. B, OHdG expression in the CA3 regions (white arrows) in different groups. C, OHdG expression in DG regions (asterisks) in different groups at 200× magnification. (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Figure 6. View largeDownload slide Immunohistochemical detection of 8-OHdG (DNA oxidation marker) in hippocampal pyramidal neurons. A, Brown staining represents the site of OHdG immunostaining in the CA2 regions (arrows) in different groups. B, OHdG expression in the CA3 regions (white arrows) in different groups. C, OHdG expression in DG regions (asterisks) in different groups at 200× magnification. (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Figure 7. View largeDownload slide Immunohistochemical detection of TG (DNA oxidation marker) in hippocampal pyramidal neurons. A, Brown color represents the sites of TG immunostaining in pyramidal neurons of the CA2 region (black arrows) in different groups. B, TG expression in the CA3 region (white arrows) in different groups. C, TG expression in the DG region (asterisks) in different groups at 200× magnification. (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Figure 7. View largeDownload slide Immunohistochemical detection of TG (DNA oxidation marker) in hippocampal pyramidal neurons. A, Brown color represents the sites of TG immunostaining in pyramidal neurons of the CA2 region (black arrows) in different groups. B, TG expression in the CA3 region (white arrows) in different groups. C, TG expression in the DG region (asterisks) in different groups at 200× magnification. (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Prenatal DEHP Exposure Lowers Serum Testosterone and Neuronal AR Expression Levels Either low serum testosterone level or decreased AR expression in the neurons is associated with an elevated anxiety level (Nguyen et al., 2009). We previously showed that DEHP mice had less testosterone in sera than the controls (Barakat et al., 2017). In this study, we measured serum testosterone level at the age 22 months to determine if the trend of low testosterone level in DEHP mice continued in older males. DEHP groups (200 µg/kg/day, 500 mg/kg/day, and 750 mg/kg/day) had remarkably lower serum testosterone concentrations than the control group (p = .07, .02, and .05, respectively) (Figure 8B). This was not surprising because we and others previously showed that circulating testosterone level is significantly reduced in middle aged mice that were prenatally exposed to DEHP (Barakat et al., 2017; Culty et al., 2008). To see if AR expression was also altered in the neurons, we assessed the AR expression levels immunohistochemically in the hippocampus and other brain regions. Previous studies localized AR expression in the neurons of the prefrontal cortex, hypothalamus, and cerebellum (Jahan et al., 2015; Perez-Pouchoulen et al., 2016). Consistent with these findings, our AR immunohistochemistry localized a pronounced AR expression in those neurons of control mice (Figure 8C). The AR expression levels were not quantitatively measured but was visually obvious that DEHP mice had a remarkably decreased AR expression in those neurons than the controls (Figure 8C). Figure 8. View largeDownload slide Effect of prenatal DEHP exposure on brain weight, serum testosterone levels, and AR expression in the brain of F1 males at 22 months of age. A, Brain weight (mg). B, Serum testosterone concentration (ng/ml). Graphs show mean ± SEM; * indicates p≤ .05 when compared with the control group; (1-way ANOVA and Tukey’s post hoc test). C, AR expression in the brain of F1 control and 750 mg/kg/day DEHP-treated mice. Immunohistochemistry for androgen in the brain. Brown color represents the sites of AR localization in the pyramidal neurons in the prefrontal cortex (asterisks), Kisspeptin neurons in hypothalamus (white arrows), and Purkinje neurons in the cerebellum (black arrows) at 200× magnification. Showed that DEHP mice had a remarkably decreased AR expression in those neurons than the controls. (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Figure 8. View largeDownload slide Effect of prenatal DEHP exposure on brain weight, serum testosterone levels, and AR expression in the brain of F1 males at 22 months of age. A, Brain weight (mg). B, Serum testosterone concentration (ng/ml). Graphs show mean ± SEM; * indicates p≤ .05 when compared with the control group; (1-way ANOVA and Tukey’s post hoc test). C, AR expression in the brain of F1 control and 750 mg/kg/day DEHP-treated mice. Immunohistochemistry for androgen in the brain. Brown color represents the sites of AR localization in the pyramidal neurons in the prefrontal cortex (asterisks), Kisspeptin neurons in hypothalamus (white arrows), and Purkinje neurons in the cerebellum (black arrows) at 200× magnification. Showed that DEHP mice had a remarkably decreased AR expression in those neurons than the controls. (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) DISCUSSION The long-term deleterious effects of the prenatal DEHP exposure on male gonadal function was explored in our previous study, where we showed that DEHP induces premature decline of circulating testosterone and fertility in male mice (Barakat et al., 2017). Testosterone is the primary hormone produced by the male gonads, and it regulates reproduction. Also, this sex hormone exerts a neuroprotective and antianxiety function (Nguyen et al., 2009). Age-related decline of testosterone is linked to dysfunction and disease development in several androgen-responsive tissues, including the brain (Kaufman and Vermeulen, 2005). Rodent studies suggest that decline of testosterone level in the brain is associated with impaired synapse formation (Leranth et al., 2004), hippocampal neuron survival (Pike, 2001) and development of neurodegenerative disease such as Alzheimer’s disease (Moffat et al., 2004). It is well established that prenatal exposure to EDCs hinders the growth and development of the fetal organs, and these adverse effects could persist throughout life (Schug et al., 2011). These findings prompted us to test the possibility that prenatal DEHP exposure might have an adverse impact on neuronal development and/or function in adulthood, causing abnormal neurobehavior. In this study, we found that DEHP in fact impairs neurobehavior and recognition memory. We therefore investigated the neuronal damage in the hippocampus as a potential mechanism for the impairments. Our data showed that DEHP mice exhibited anxious behaviors and impaired recognition memory. We also found that the hippocampi of DEHP mice contained significantly fewer pyramidal neurons than those of the controls, and showed higher levels of COX-2 expression and higher 8-OHdG and TG content. DEHP mice also had lower circulating testosterone and lower AR expression in the neurons of cerebral cortex, hypothalamus, and cerebellum than the control mice. We assessed the impacts of prenatal DEHP exposure on anxiety level using the EPM test and OFT. DEHP mice took more time to enter the open center and made fewer entries into the center area in the OFT. In the EPM test, DEHP-treated mice exhibited increased latency to the open arms (Figure 1). Fewer entries and/or increased latency to enter an open arm or open field are considered to be a signs of elevated anxiety level (Naolapo et al., 2012). These results indicate that prenatal exposure to DEHP probably conditions the impacted animals to be prone to develop anxiety. Our results are reminiscent of a previous study that showed that rats exposed to DEHP postnatally exhibited higher levels of anxiety behavior during adolescence (Carbone et al., 2013). Of note, in our study the animals were exposed prenatally, and they exhibited higher anxiety behavior at the ages of 22 months, indicating that DEHP-induced damage that occurs during fetal development may lead to a life-long impact on their neurobehavior. This finding is in line with a number of epidemiological studies that showed an association between phthalate concentrations in maternal urine during pregnancy and the impairment of cognitive or behavioral development in their children (Adibi et al., 2009; Arbuckle et al., 2016; Gascon et al., 2015). We assessed the impacts of prenatal DEHP exposure on spatial and recognition memory using the Y-maze spontaneous alteration test and NOR test. The Y-maze spontaneous alteration test showed that DEHP exposure correlates to reduced spatial memory function, particularly in the 200 µg/kg/day DEHP group (Figure 2A). Interestingly, DEHP mice also made significantly fewer total entries into the arms, possibly indicating a lower locomotor activity and exploration pattern (Figure 2B). Additionally, in the NOR test, DEHP mice spent less time exploring the novel object (Figure 2A), indicating an impaired recognition memory. Previous studies showed that less novel object exploration time is associated with impaired recognition memory (Pascual et al., 2011). Taken together, our findings suggest that prenatal DEHP induced a life-long impact on recognition memory and brain function. Previous research showed that endogenous androgens during puberty are important for anxiety and memory formation, as castration of male mice led to a significant increase in anxiety and reduction in memory performance (McDermott et al., 2012), indicating a significant role that testosterone may play in controlling anxiety and memory function. In the brain, the hippocampus one of the regions involved in mediating learning and memory functions (Bird and Burgess, 2008). Based on impaired spatial and recognition memory in DEHP mice, neuronal damage was suspected in the hippocampus. Histological examination of the hippocampal tissues at 22 months of age revealed that there were fewer pyramidal neurons in the hippocampi of DEHP mice, and they were morphologically shrunken (Figure 3). Furthermore, quantitative analysis of the hippocampal neurons by SLIM analysis showed that DEHP mice had fewer neurons in the hippocampus, indicating that prenatal exposure to DEHP could lead to neuronal degeneration in the hippocampus. A study reported that acute postnatal DEHP exposure reduced the axonal markers in the CA3 distal stratum oriens in rats, indicating that the hippocampus is highly sensitive to DEHP (Smith et al., 2011). Another study showed that postnatal exposure to DEHP caused hippocampal atrophy by decreasing BDNF (brain-derived neurotrophic factor) synthesis in male rats (Smith and Holahan, 2014). Furthermore, both pre- and postnatal exposures to phthalates had an adverse impact on the developing brain, causing long-lasting neurodevelopmental damages (Miodovnik et al., 2014). Considering that the mice used in our study were prenatally exposed, and the histological examination of the brains was performed at the age of approximately 22 months, the decreased numbers of pyramidal neurons (Figure 4) may be in part an outcome of the DEHP impact on the proliferation, differentiation, or survival of pyramidal neurons during fetal brain development. However, even if there were an impact on neurogenesis, the shrunken cellular morphology of the pyramidal neurons (Figure 3) suggested to us that the impact of prenatal exposure to DEHP might continue for life, and the neurodegeneration continues later in life. The shrunken cellular morphology is often a maker of ongoing inflammation or oxidative stress (Lucas et al., 2006; Zilka et al., 2006). Therefore, we examined the tissues to see if the pyramidal neurons were undergoing inflammation and/or DNA oxidation. Immunohistochemistry showed a remarkably stronger COX-2 expression in the hippocampus of DEHP mice than in the controls. COX-2 is a rate-limiting enzyme for the synthesis prostaglandin E2, a proinflammatory prostaglandin (Yan et al., 2015). Expression of this enzyme in the neurons is linked to a number of neuronal diseases, including Alzheimer’s disease (Davis and Laroche, 2003; Rubio-Perez and Morillas-Ruiz, 2012; Zilka et al., 2006). Because of its association with ongoing diseases, COX-2 inhibitors are often used to protect neurons from inflammation-triggered neurodegeneration and also as an effort to reduce the risk of developing Alzheimer’s disease (Aisen, 2002). Therefore, the elevated expression of COX-2 in the pyramidal neurons (Figure 5) may indicate that the observed shrunken morphology and decreased neuron numbers in the hippocampus may be due to an ongoing inflammation. It will be interesting to determine how a prenatal DEHP exposure may result in the elevated COX-2 expression and neuronal inflammation in the adults. Also interesting is to see if the reduced testosterone level or AR expression (Figure 8) increased COX-expression. We also found that, possibly as a consequence of the neuroinflammation in hippocampal neurons, the immunoreactivity to DNA oxidation markers; 8-OHdG, and TG was higher in the hippocampal neurons of DEHP mice than controls (Figs. 6 and 7). Cellular oxidative stress is often caused by an excessive production of ROS that can damage a wide variety of cellular organelles and molecules such as DNA, RNA, proteins, and lipids, diminishing the cell viability (Huang et al., 2016). Notably, accumulation of oxidative DNA damage in the brain is a major driver of brain aging and a key contributing factor for the development of Alzheimer’s disease (Huang et al., 2016; Zussy et al., 2013). In particular, an increased content of 8-OHDG and/or TG is a molecular signature of brain aging and neurodegenerative disease, such as Alzheimer’s disease (Lovell and Markesbery, 2007). Overall, the elevated 8-OHdG and TG level in the pyramidal neurons of DEHP mice suggests that DNA oxidation may play a role in neurodegeneration and consequently, neurobehavioral abnormalities in the mice that were prenatally exposed to DEHP. The exact mechanisms that explain negative effects of EDCs on neurodevelopment of early life stages are unclear. However, a recent study proposes several possible mechanisms (Kim et al. 2018). Changes in lipid signal transduction pathways caused by exposure to chemicals such as DEHP may alter essential fatty acids in fetal brains that influence the development of cognitive function in the rat brain. A recent study showed that exposure to DEHP increased malondialdehyde content in the brain, which has serious consequences because normal fetal brain development relies upon adequate levels of lipids and fatty acid (Tang et al., 2015). Another study showed that the effects of DEHP on the fetal lipid metabolome was mediated by a peroxisome proliferator-activated receptor (Xu et al., 2008). The concentrations of docosahexaenoic acid and arachidonic acid have been shown to influence neurodevelopment as well (Helland et al., 2003). Furthermore, chemicals may influence sex hormone regulation and can eventually affect normal fetal brain development (Schug et al., 2015). Most importantly, antiandrogenic compounds such as DEHP and DEP are known to affect normal fetal brain development (Lottrup et al., 2006). We found that DEHP mice had considerably lower serum androgen level at the ages of 22 months than the controls (Figure 8). This finding is an extension of earlier reports that clearly linked DEHP exposure to low serum testosterone levels (Andrade et al., 2006; Barakat et al., 2017; Dombret et al., 2017; Pan et al. 2006). DEHP has been reported to decrease testosterone synthesis by interfering with the expression of steroidogenic enzymes that are involved in androgen biosynthesis (Howdeshell et al., 2007). In our study, while animals were prenatally exposed, testosterone was measured at the age of 22 months. These animals were not exposed to DEHP during the 22-month period from birth to old age. Therefore, the decreased serum testosterone level may not be an outcome of a change in the expression of steroidogenic gene(s). Rather, this hypoandrogenism may be a result of an epigenetic modification of the genes during fetal gonadal development, which causes a life-long gene alteration that may directly or indirectly influence testosterone biosynthesis. Testosterone and its derivative estradiol are known to play important roles in neuroprotection and neuroregeneration and to regulate the differentiation and function of the nervous system (Cooke et al., 1998; Farrell et al., 2015). In particular, testosterone exerts neuroprotective activity because it enhances neuronal survival and prevents neurodegeneration (Farrell et al., 2015; Nguyen et al., 2009). The reduction of testosterone is associated with increased levels of neuronal cell death. It is also considered to be a risk factor for developing many neurological disorder such as Alzheimer’s disease (Lau et al. 2014). A study showed that testosterone enhances the survival of hippocampal neurons and is necessary for maintaining learning and memory at older age (Spritzer and Galea, 2007). Taken together, our results suggest that low testosterone levels in the DEHP mice may directly or indirectly induce the anxious behavior, cognitive deficits, and neurodegeneration we observed as summarized in Figure 9. Will testosterone replacement therapy ameliorate the syndromes observed in the DEHP mice? This is possible, as DEHP-induced anxiogenic behavior has been reversed by testosterone replacement in male rats (Carbone et al., 2013). Note our unexpected discovery that DEHP mice had a remarkably lower AR expression in pyramidal neurons in prefrontal cortex, neurons in the hypothalamus, and Purkinje neurons in the cerebellum (Figure 8). Did the altered AR expression in those brain tissues exacerbate the neurodegeneration? This is an interesting question to be addressed. Figure 9. View largeDownload slide Schematic representation of the effect of prenatal DEHP exposure in the induction of oxidative stress leading to neurobehavioral abnormalities. DEHP led to an increase of DNA oxidation by inducing 8-OHdG and TG in addition to the increase in COX-2 expression, which plays a key role in the pathogenesis of neurodegeneration in hippocampal pyramidal neurons. DEHP also has indirect effects by reducing testosterone levels that decrease AR expression in brain. All these events influence the neurodegeneration and neurobehavioral abnormalities resulting from prenatal DEHP exposure. Figure 9. View largeDownload slide Schematic representation of the effect of prenatal DEHP exposure in the induction of oxidative stress leading to neurobehavioral abnormalities. DEHP led to an increase of DNA oxidation by inducing 8-OHdG and TG in addition to the increase in COX-2 expression, which plays a key role in the pathogenesis of neurodegeneration in hippocampal pyramidal neurons. DEHP also has indirect effects by reducing testosterone levels that decrease AR expression in brain. All these events influence the neurodegeneration and neurobehavioral abnormalities resulting from prenatal DEHP exposure. In summary, this study found that prenatal exposure to DEHP lead to elevated anxiety and impaired memory function in male mice. These neurobehavioral abnormalities may originate from a developmental defect in the central nervous system, a life-long neurodegeneration or a neurodegeneration that starts later in life caused by oxidative damage and/or inflammation in the hippocampus. Whether those syndromes are a direct outcome of DEHP impact or due to the observed decrease in testosterone level or AR expression in the neurons is yet to be determined. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. FUNDING This work was supported by the National Institute of Health grant NIH (P01-ES022848) and Environmental Protection Agency grant (RD-83459301) to (C.J.K.), and an Egyptian Mission Sector (JS-3041), Higher Ministry of Education Scholarship to (R.B.). ACKNOWLEDGMENTS The authors also wish to thank all the members of the Dr Flaws’ lab for dosing the animals and give special thanks to Dr Susan Schantz for her critical reading of this article. REFERENCES Adibi J. J., Hauser R., Williams P. L., Whyatt R. M., Calafat A. M., Nelson H., Herrick R., Swan S. H. ( 2009). Maternal urinary metabolites of Di-(2-ethylhexyl) phthalate in relation to the timing of labor in a US multicenter pregnancy cohort study. Am. J. Epidemiol . 169, 1015– 1024. Google Scholar CrossRef Search ADS PubMed  Aisen P. S. ( 2002). Evaluation of selective COX-2 inhibitors for the treatment of Alzheimer’s disease. J. Pain Symptom Manage . 23, S35– S40. Google Scholar CrossRef Search ADS PubMed  Almeida O.P., Yeap B. 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Alzheimer’s disease related markers, cellular toxicity and behavioral deficits induced six weeks after oligomeric amyloid-β peptide injection in rats. PLoS One  8, e53117. 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

Prenatal Exposure to DEHP Induces Neuronal Degeneration and Neurobehavioral Abnormalities in Adult Male Mice

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Abstract

Abstract Phthalates are a family of synthetic chemicals that are used in producing a variety of consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is an widely used phthalate and poses a public health concern. Prenatal exposure to DEHP has been shown to induce premature reproductive senescence in animal studies. In this study, we tested the hypothesis that prenatal exposure to DEHP impairs neurobehavior and recognition memory in her male offspring and we investigated one possible mechanism—oxidative damage in the hippocampus. Pregnant CD-1 female mice were orally administered 200 μg, 500 mg, or 750 mg/kg/day DEHP or vehicle from gestational day 11 until birth. The neurobehavioral impact of the prenatal DEHP exposure was assessed at the ages of 16–22 months. Elevated plus maze and open field tests were used to measure anxiety levels. Y-maze and novel object recognition tests were employed to measure memory function. The oxidative damage in the hippocampus was measured by the levels of oxidative DNA damage and by Spatial light interference microscopic counting of hippocampal neurons. Adult male mice that were prenatally exposed to DEHP exhibited anxious behaviors and impaired spatial and short-term recognition memory. The number of hippocampal pyramidal neurons was significantly decreased in the DEHP mice. Furthermore, DEHP mice expressed remarkably high levels of cyclooxygenase-2, 8-hydroxyguanine, and thymidine glycol in their hippocampal neurons. DEHP mice also had lower circulating testosterone concentrations and displayed a weaker immunoreactivity than the control mice to androgen receptor expression in the brain. This study found that prenatal exposure to DEHP caused elevated anxiety behavior and impaired recognition memory. These behavioral changes may originate from neurodegeneration caused by oxidative damage and inflammation in the hippocampus. Decreased circulating testosterone concentrations and decreased expression of androgen receptor in the brain also may be factors contributing to the impaired neurobehavior in the DEHP mice. DEHP, endocrine disruptor, anxiety, hippocampus, aging Di-(2-ethylhexyl) phthalate (DEHP) is one of the most commonly used phthalates, and is widely used in the production of a variety of cosmetics, personal care products, food storage containers, pharmaceuticals, building material, and medical tubing (Dai et al., 2015). DEHP readily leaches from these products directly into food, beverages, water, or air (Swan et al., 2005). DEHP is known as an endocrine disruptor and has been shown to disrupt the reproductive system in both females and males (Barakat et al., 2017; Culty et al., 2008; Niermann et al., 2015). The most obvious toxicity of DEHP exposure to date is its impact on gonadal steroidogenesis (Barakat et al., 2017; Gray et al., 2009; Moore et al., 2001) and the accompanying decline in reproductive function and fertility (Andrade et al., 2006; Cho et al., 2010; Culty et al., 2008; Dalsenter et al., 2006; Gray et al., 2000; Tanaka 2005; Tang et al., 2015). Besides creating problems in the reproductive system, a strong association between DEHP exposure and neurological functions has begun to emerge. A number of epidemiological studies indicate an association between maternal urine phthalate concentrations during pregnancy and cognitive and learning disabilities in offspring (Adibi et al., 2009; Arbuckle et al., 2016; Cho et al., 2010; Engel et al., 2010; Gascon et al., 2015; Huang et al., 2017; Yolton et al., 2011). Testosterone is well known for its functions in reproduction, sexual differentiation, and sexual behavior as well as its modulating effect on anxiety (Edinger et al., 2004). In men, low circulating T concentrations hav been associated with depressive symptoms and anxiety (Almeida et al., 2008). In animal studies using rats, systemic administration of testosterone or dihydrotestosterone to gonadectomized males reduced anxiety-like behaviors (Edinger et al., 2004; Frye and Seliga, 2001). These androgens probably play this anti-anxiety role by acting directly or indirectly on neurons; androgen receptor (AR) is expressed in a variety of neurons and glial cells and regulates neuronal cell differentiation, excitability and survival (Nguyen et al., 2009; Pouliot et al., 1996; Spritzer and Galea, 2007). In support, testosterone exerts neuroprotective activity because it enhances neuronal survival and prevents neurodegeneration (Lau et al., 2015). Furthermore, decreased testosterone or dihydrotestosterone increases the accumulation of oligomeric β-amyloid peptides, accelerating neuronal cell death, and it is considered to be a risk factor for developing Alzheimer’s disease (Chu et al., 2010; Lau et al., 2014). In the hippocampal neurons, these neuroprotective roles of androgens are mediated in part by AR-dependent activation of cyclic adenosine monophosphate response element-binding protein (Nguyen et al., 2009). Age-related decline of testosterone is linked to dysfunction and disease development in several androgen-responsive tissues, including brain (Kaufman and Vermeulen, 2005). In old rodent, decline of testosterone level is associated with impaired synapse formation (Leranth et al., 2004) and development of neurodegenerative disease (Moffat et al., 2004). Importantly, prenatal exposure to endocrine disrupting chemicals (EDCs) hinders the growth and development of the fetal organs, and these adverse effects could persist throughout life (Schug et al., 2011). These findings prompted us to test the hypothesis that DEHP exposure induced decline of testosterone and leads to an adverse impact on neuro-behavioral function in adulthood. Adverse neurobehavioral changes may be provoked by oxidative stress occurring in the brain, specifically in the hippocampus (Liu et al., 2014; Murakami and Murakami, 2005). Oxidative stress is generated primarily in the form of reactive oxygen species (ROS) from mitochondrial metabolism (Finkel and Holbrook, 2000). ROS then induce oxidative damage in vital macromolecules such as DNA, proteins, and lipids, leading to negative outcomes such as accelerated aging, tumorigenesis, or neurodegeneration (Hamilton et al., 2001). Overall the antianxiety and neuroprotective roles that androgens play and our previous finding that prenatal exposure to DEHP induced a premature decline of circulating testosterone in adult male mice led us to hypothesize that maternal exposure to DEHP impairs neurobehavior and recognition memory in her male offspring via neuronal damage in the hippocampus. In this study, we investigated a possible mechanism by assessing the impact of prenatal DEHP exposure on anxiety level, memory function, brain histology, and neuro-inflammation. MATERIALS AND METHODS Chemicals DEHP (99% purity) was purchased from Sigma-Aldrich (St Louis). Tocopherol-stripped corn oil was purchased from MP Bio-Medicals (Solon, Ohio) and was used as a vehicle. Stock solutions of DEHP were prepared by diluting it in tocopherol-stripped corn oil to obtain the desired concentrations. Animals and dosing regimen CD-1 mice were used in this study and were housed at the University of Illinois at Urbana-Champaign (UIUC) animal care facility under 12-h light/dark cycles. The mice were provided with Teklad Rodent Diet 8604 (Harlan) and had ad-libitum access to food and water. Animal handling and procedures were approved by the UIUC Institutional Animal Care and Use Committee (Protocol ID No.: 14144). Pregnant dams were prepared by mating 2-month-old females with proven breeder males. The pregnant female mice were orally treated with vehicle control (tocopherol-stripped corn oil), 200 µg/kg/day, 500 mg/kg/day, or 750 mg/kg/day of DEHP from gestational day 11 to the day of birth by placing a pipette tip into the mouth as previously described in Niermann et al. (2015). DEHP doses were chosen because they are environmentally relevant. The Agency for Toxic Substances and Disease Registry estimates that the range of daily human exposure to DEHP is 3–30 µg/kg/day, and the no-observed-adverse-effect level for DEHP is 5.8 mg/kg/day (ATSDR, 2002). The lowest-observed-adverse-effect level of DEHP is 140 mg/kg/day, and potential reproductive effects occur at levels ranging from 1 to 30 µg/kg/day (Blystone et al., 2010; Gray et al., 2009). The reference dose of a daily oral exposure to DEHP in the human population is 20 µg/kg/day based on the U. S. Environmental Protection Agency published reference safe dose (https://www.epa.gov/sites/production/files/2016-09/documents/bis-2-ethylhexyl-phthalate.pdf). Specifically, DEHP at 200 μg/kg/day was used because adult exposure causes abnormal estrous cyclicity, accelerates primordial follicle recruitment in female mice, and decreased the fertility in male CD-1 mice (Barakat et al., 2017; Hannon et al., 2014). The 500 mg and 750 mg/kg/day DEHP doses were used previously in many research papers to study the different pathological effects of DEHP exposure (Andrade et al. 2006; Culty et al., 2008; Do et al., 2012; Doyle et al., 2013). Y-maze spontaneous alteration test The Y-maze spontaneous alteration test was used to examine the effects of prenatal DEHP exposure on spatial memory of prenatally exposed male mice at 16 months of age. The Y-maze was composed of 3 equal arms (120°, 41-cm long, and 15-cm high. Each mouse was placed in one of the arm compartments (A, B, or C) and was allowed to move freely for 15 min. The sequence of arm entries was manually recorded as A, B, or C. An alternation was defined as an entry into all 3 arms consecutively. The number of maximum spontaneous alternations is the total number of arms entered minus 2, and the percentage alternation is calculated as ([actual alternations/maximum alternations] × 100) (Naolapo et al., 2012). The total number of arm entries was also recorded as an indication of general locomotor activity of the mice. The apparatus was cleaned with 70% ethanol to remove odors from the previous mouse. Novel object recognition test The novel object recognition (NOR) test is one the most popular paradigms to evaluate relational memory. All treated groups were tested at 16 months of age to examine the effects of prenatal DEHP exposure on the recognition memory. In the first trial (training phase), the mouse was placed into the chamber with 2 identical objects (same color, shape, and size) for 5 min. Then the mouse was placed back into its main cage for an intertrial interval of 1 h. Objects were washed with 70% ethanol between trials to remove olfactory cues. In the second trial (testing phase), the mouse was placed back into the chamber with 2 objects, and one of the familiar objects was replaced with a new novel object. The time spent exploring the novel and familiar objects was measured for 5 min (Pascual et al., 2011). Exploration time, defined as viewing, sniffing, and/or touching at 1 cm distance or less from the object, was recorded for the familiar and novel object (Fole et al., 2015). Elevated plus maze test The elevated plus maze (EPM) is used to measure anxiety in rodent research (Walf and Frye, 2007). All treated groups were tested at 18 months of age to study the anxiety-like behavior. The EPM apparatus consists of 2 open arms alternating with 2 closed arms (30-cm high); the whole maze was elevated 50 cm above the floor, and the testing was conducted in a dimly lit room. Each mouse was placed in the center of the maze facing one of the closed arms and was allowed to explore for 10 min as described previously in Czerniczyniec et al. (2011). We used 70% ethanol to clean each arm of the maze and remove olfactory cues between trials. The EPM relies upon the mice preferring to move toward dark, enclosed spaces while fearing and avoiding heights/open spaces (Naolapo et al., 2012). Entry into the arm of the EPM was defined as the animal placing all 4 paws in the arm. The latency to enter the open arms, defined as the first time the mouse enters an open arm of the maze during the experimental period, was measured; this is expressed in seconds. The number of entries into both open and closed arms during the experimental time (10 min) was also measured. The amount of time spent in the open arms and closed arms of the maze were recorded and expressed in seconds. Open field test The open field test (OFT) is also a test used to measure anxiety and locomotor activity in rodents. The open-field apparatus consisted of a white Plexiglass box (50 × 50 × 22 cm) with the floor divided into 16 squares. The “border” was defined as the 12 outer periphery squares and the “center” as the 4 inner central squares as described previously in Davis et al. (2012) and Liu et al. (2014). At 18 months, each mouse was placed at the center of the apparatus in a dimly lit room and was allowed to move freely for 5 min. The open field was cleaned with 70% ethanol to remove odors from the previous mouse. It is well known that rodents show natural avoidance of open surfaces (Czerniczyniec et al., 2011). The time taken before the first entry to the center squares and the number of entries to the center square area were evaluated as an index of anxiety. Measurement of serum testosterone concentration Peripheral blood was collected from 22-month-old mice by cardiac puncture. The blood was centrifuged at 2000 × g, and then serum was collected and stored at −20°C until further analyses. The concentration of circulating testosterone was measured by using ELISA kits (DRG Diagnostic) with a reportable range of 0.06–25 ng/ml at 22 months of age. Tissue collection and brain weight At 22 months of age, all the F1 male mice were euthanized by CO2 asphyxiation followed by cervical dislocation. After that, the brain was collected and weighed. The tissues were fixed in 4% paraformaldehyde for 24 h and then transferred to 70% ethyl alcohol and kept at 4°C until further tissue processing. Histological examination and Nissl staining of hippocampus Brain tissue was fixed in 4% paraformaldehyde for 24 h, washed with ethanol and embedded in paraffin wax, and serially sectioned at 7-µm thickness in a sagittal orientation using a microtome. For general histological observation, slides were stained with hematoxylin and eosin (H&E). The brain tissue was also stained with Nissl staining (cresyl violet staining) to evaluate the neuronal damage. Microscopic images were obtained from pyramidal cell layers in CA1 and CA2/3 subdivisions of the hippocampus by using an Olympus BX51 microscope. Counting of hippocampal pyramidal neurons (Nissl staining and Spatial light interference microscopy) From each mouse, 4 sections of 7-µm thickness made 120 µm apart each other were selected to count the neurons in the hippocampus. The Nissl-stained hippocampus was divided into 2 subregions–Cornu Ammonis (CA) CA1 and CA2/3 regions-based on morphologic appearance and identifiable landmarks. The number of intact neurons within the CA1 and CA2/3 layers were counted using an Olympus BX51 microscope at 400× magnification (Figure 3). Spatial light interference microscopy (SLIM) comprising a commercial phase contrast microscope (Carl Zeiss, Axio Observer.Z1) combined with a SLIM module (Phi Optics, Inc., Cell Vista SLIM Pro) provided multicontrast perspectives of gross and microscopic brain anatomy (Min et al., 2016). For SLIM microscopic counting, each brain slice was deparaffinized with xylene, rehydrated with ethanol, and mounted in aqueous mounting media to recover the refractive index profile in tissue as described previously in Min et al. (2016). The hippocampus was divided into 4 subregions: CA1, CA2, CA3, and dentate gyrus (DG), and pyramidal neurons were then quantified (Figure 4). Immunohistochemistry The expression of AR, cyclooxygenase-2 (COX-2), and DNA oxidation markers such as 8-hydroxyguanine (8-OHdG) and thymidine glycol (TG) in the hippocampus were determined by immunohistochemistry. Sagittal sections of the brains from all treated groups were deparaffinized, followed by heat-induced antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) and then incubated in 3% H2O2 for 30 min at room temperature for endogenous peroxide quenching. Next, we blocked endogenous biotin with 5% goat or horse serum in avidin (200 μl/1 ml; SP-2001, Vector labs, Burlingame, CA). The slides were incubated overnight with antiAR antibody (sc-816, Santa Cruz Biotechnology), anti8-OHdG antibody (sc-66036, Santa Cruz Biotechnology), antiCOX-2 antibody (Cayman), or antiTG antibody (Institute for the Control of Aging, Shizuoka, Japan; JaICA). This was followed by incubation with a secondary biotinylated goat antirabbit antibody or horse antimouse antibody (Vectastain ABC kit, Vector labs) and avidin-biotin complex solution (Vectastain Elite ABC kit, Vector labs) at room temperature. 3, 3’-diaminobenzidine (SK-4100, Vector labs) was applied until color optimally developed. Slides were then counterstained with hematoxylin, mounted, and imaged with an Olympus BX51 microscope. Four sections with 120-µm intervals from each mouse were selected to quantitatively assess immunoreactivity of COX-2 in the hippocampus. Statistical analysis The data were analyzed using the statistical software package SPSS. Multiple comparisons between normally distributed continuous experimental groups were analyzed by 1-way analysis of variance (ANOVA) as a parametric test followed by the Tukey’s HSD (honest significant difference) test for comparisons of individual means. If data were not normally distributed, were presented as a percentage, and/or did not meet homogeneity of variance assumptions, the independent sample Kruskal-Wallis H followed by Mann-Whitney were performed. Animal used in this study were from individual litters, and the number of animals used for statistical analyses ranged from 4 to 7 mice. The data are expressed as mean ± SEM. Asterisks in figures indicate a statistically significant difference (p ≤ .05) compared with controls. RESULTS Prenatal DEHP Exposure Increases Anxiety Behavior in Adult Male Mice At the age of 16–20 months, the OFT and the EPM test were performed to assess the level of anxiety. The EPM test relies upon mice preferring to move toward dark, enclosed spaces while avoiding open spaces (Naolapo et al., 2012). The OFT relies on the natural avoidance of mice of open surfaces and fear of wide-open spaces such as the center square region (Czerniczyniec et al., 2011). The OFT showed that DEHP mice tended to take more time before making entries into the center area compared with the controls (p = .08, 750 mg/kg/day), but the difference between the DEHP and control groups did not reach statistical significance (Figure 1A). All of the DEHP-exposed groups made significantly fewer entries to the center area than the control group (p = .03, .02, and .01, respectively) (Figure 1B). The EPM test showed that DEHP mice in the highest exposure group took more time before making entries to the open arms (p = .04, 750 mg/kg/day) (Figure 1C). Interestingly, the numbers of entries into the open arms and the time spent there did not differ between the DEHP-exposed groups and the control group (Figs. 1D and 1E). Figure 1. View largeDownload slide Effect of prenatal DEHP exposure on anxiety level of F1 males measured at 18 months (open field and EPM test). A, Latency to the center square area in the OFT (seconds). B, Number of entries into the center square area in the OFT. C, Latency to the open arm in EPM (seconds), *indicates p ≤ .05 when compared with control group (Kruskal Wallis test). D, Time spent in the open arm in EPM (seconds). E, Number of open and total arm entries in EPM. Graphs show mean ± SEM; * indicates p ≤ .05 when compared with control group (1-way ANOVA and Tukey’s post hoc test). Figure 1. View largeDownload slide Effect of prenatal DEHP exposure on anxiety level of F1 males measured at 18 months (open field and EPM test). A, Latency to the center square area in the OFT (seconds). B, Number of entries into the center square area in the OFT. C, Latency to the open arm in EPM (seconds), *indicates p ≤ .05 when compared with control group (Kruskal Wallis test). D, Time spent in the open arm in EPM (seconds). E, Number of open and total arm entries in EPM. Graphs show mean ± SEM; * indicates p ≤ .05 when compared with control group (1-way ANOVA and Tukey’s post hoc test). Prenatal DEHP Exposure Impairs Spatial and Recognition Memory The impact of early exposure to DEHP on the spatial and recognition memory was assessed by the Y-maze spontaneous alteration test and NOR test, respectively. The Y-maze test measures spatial memory and locomotor activity (Naolapo et al., 2012). An animal with impaired spatial memory or locomotion tends to show less alteration behavior (tends to enter the arm that he just visited rather than visiting the next arm). The NOR test is used to measure short-term recognition memory (Pascual et al., 2011). A normal animal tends to spend more time exploring the novel object. The Y-maze test showed that the lowest DEHP dose group displayed the least alternation behavior (p = .04) and the fewest arm entries (p = .01) compared with the control group (Figs. 2A and 2B). The NOR test showed that the DEHP mice tended to spend less time exploring the new object (500 and 750 mg/kg/day groups; p = .06 and .05, respectively) than did the control group (Figs. 2C and 2D). Figure 2. View largeDownload slide Effect of prenatal DEHP exposure on spatial memory measured by the Y-maze spontaneous alteration test (A and B) and recognition memory (C and D) measured by the NOR test of F1 males at 16 months. A, Percent of alteration (A-B-C consecutive arm entry) during 15 min. B, Number of arm entries during 15 min. of experimental time; * indicates p ≤ .05 when compared with the control group (Kruskal Wallis test followed by Mann-Whitney). C, Exploration time of past and novel object (seconds; * indicates p ≤ .05 when compared with the control group (Kruskal Wallis test followed by Mann-Whitney). D, Percent of novel object exploration time during 5 min of experimental time (seconds). Graphs show mean ± SEM; * indicates p ≤ .05 when compared with the control group. Figure 2. View largeDownload slide Effect of prenatal DEHP exposure on spatial memory measured by the Y-maze spontaneous alteration test (A and B) and recognition memory (C and D) measured by the NOR test of F1 males at 16 months. A, Percent of alteration (A-B-C consecutive arm entry) during 15 min. B, Number of arm entries during 15 min. of experimental time; * indicates p ≤ .05 when compared with the control group (Kruskal Wallis test followed by Mann-Whitney). C, Exploration time of past and novel object (seconds; * indicates p ≤ .05 when compared with the control group (Kruskal Wallis test followed by Mann-Whitney). D, Percent of novel object exploration time during 5 min of experimental time (seconds). Graphs show mean ± SEM; * indicates p ≤ .05 when compared with the control group. Prenatal DEHP Exposure Induces Neurodegeneration in Hippocampal Pyramidal Neurons Memory function can be impaired by the degeneration of hippocampal pyramidal neurons (Zilka et al., 2006). To determine if hippocampal neurodegeneration was a contributing factor to the impaired memory observed in the DEHP mice, we first examined the hippocampus histologically. Brains were collected at 22 months of age, and their sections were stained by either H&E (Supplementary Figure 1) or Nissl staining (Figure 3). Histologically, the hippocampal pyramidal neurons of the DEHP mice were shrunken, loosely aligned, fewer in number and showed an enlarged inter-neuronal space between the neurons (Supplementary Figure 1) (Figure 3). A manual microscopic counting of pyramidal neurons in the Nissl-stained hippocampus showed that the hippocampi of DEHP mice contained significantly fewer neurons in a dose- and region-specific manner compared with the controls (Figure 3D). We then re-examined the hippocampi using the SLIM, a newly developed computerized microscopy (Min et al., 2016) that uses the unique physical properties of neuronal cell bodies, such as higher phase values than axons, dendrites, and extracellular matrix and an algorism that allows a computerized identification of neuronal cell bodies (Figure 4). Using SLIM, the numbers of pyramidal neurons were compared in 4 subregions of the hippocampus: CA1, CA2, CA3, and DG (Figure 4A). By SLIM analysis, we found that the hippocampi of the 500 and 750 mg/kg/day DEHP mice had fewer neurons in the regions of CA1 (p = .07 and .002, respectively) and CA2 (p = .02 and .006, respectively) than the controls (Figure 4E). Furthermore, the hippocampi of the 500 and 750 mg/kg/day DEHP mice had fewer neurons in the DG region (p = .002 and .02, respectively) (Figure 4E). Interestingly, while not statistically significant, the mean brain weight of DEHP mice was lighter than those of control (Figure 8A). Figure 3. View largeDownload slide Nissl staining of hippocampus at 22-months old. A, Hippocampus of all treatment groups at lower magnification (40×). B, CA1 pyramidal neurons of different groups at 200×. C, CA2/3 pyramidal neurons of different groups at 200×. Shows normal neurons in the control group and abnormal neurons in the DEHP-treated groups, which showed shrunken neurons (arrow heads) and enlarged intracellular space (arrow). D, Manual counting of pyramidal neurons in CA1 and CA2/3 regions in control and DEHP-treated groups. Graphs show mean ± SEM, * indicates p ≤ .05 when compared each region of DEHP-treated group with the control group (1-way ANOVA and Tukey’s post hoc test). Figure 3. View largeDownload slide Nissl staining of hippocampus at 22-months old. A, Hippocampus of all treatment groups at lower magnification (40×). B, CA1 pyramidal neurons of different groups at 200×. C, CA2/3 pyramidal neurons of different groups at 200×. Shows normal neurons in the control group and abnormal neurons in the DEHP-treated groups, which showed shrunken neurons (arrow heads) and enlarged intracellular space (arrow). D, Manual counting of pyramidal neurons in CA1 and CA2/3 regions in control and DEHP-treated groups. Graphs show mean ± SEM, * indicates p ≤ .05 when compared each region of DEHP-treated group with the control group (1-way ANOVA and Tukey’s post hoc test). Figure 4. View largeDownload slide SLIM microscopic counting of hippocampal pyramidal neurons at 22 months. A, Hippocampus of all treatment groups at lower magnification (40×). B, CA1 pyramidal neurons in different groups at 200×. C, CA2 pyramidal neurons in different groups at 200×. D, CA3 pyramidal neurons in different groups at 200×. Shows that neuronal cell bodies have higher phase values than axons and dendrites, while the cell membrane, axon, and dendrites have much higher phase values than cytoplasmic regions and the extracellular matrix. E, SLIM microscopic quantification of pyramidal neurons in DG, CA1, CA2, and CA3 regions. Graphs show mean ± SEM, * indicates p ≤ .05 when compared each region of DEHP-treated group with the control group (1-way ANOVA and Tukey’s post hoc test). Figure 4. View largeDownload slide SLIM microscopic counting of hippocampal pyramidal neurons at 22 months. A, Hippocampus of all treatment groups at lower magnification (40×). B, CA1 pyramidal neurons in different groups at 200×. C, CA2 pyramidal neurons in different groups at 200×. D, CA3 pyramidal neurons in different groups at 200×. Shows that neuronal cell bodies have higher phase values than axons and dendrites, while the cell membrane, axon, and dendrites have much higher phase values than cytoplasmic regions and the extracellular matrix. E, SLIM microscopic quantification of pyramidal neurons in DG, CA1, CA2, and CA3 regions. Graphs show mean ± SEM, * indicates p ≤ .05 when compared each region of DEHP-treated group with the control group (1-way ANOVA and Tukey’s post hoc test). Prenatal DEHP Exposure Increases COX-2 Expression in Hippocampal Neurons Neuronal inflammation is one of the leading causes of neurodegeneration (Aisen, 2002). As there was an obvious sign of neurodegeneration in the DEHP mice (Figs. 3 and 4), we examined their hippocampi to see if they underwent inflammation. COX-2, (PTGES-2) expression level was measured as an inflammatory marker, as COX-2 plays a key role in neuroinflammation and pathogenesis of neurodegenerative disease. COX-2 is expressed in response to an acute inflammatory signal and converts arachidonic acid to prostaglandins (Yang and Chen, 2008). In the control tissues, a mild COX-2 immunoreactivity was observed in the CA2/3 region but not in the CA1 and DG regions (Figure 5A). In contrast, COX-2 immunoreactivity was remarkably stronger in the CA2/3 region of the DEHP mice (Figure 5A). Counting COX-2-positive neurons in the CA2/3 region showed that the 200 µg/kg/day and 750 mg/kg/day DEHP mice had more COX-2-positive neurons than the controls (p = .01 and .04; respectively) (Figure 5B). Figure 5. View largeDownload slide Immunohistochemical detection of COX-2 positive neurons in the hippocampus. A, Brown staining represents the sites of COX-2 expression (arrows) in pyramidal neurons of CA-2/3 region of the control and DEHP-treated groups at 200× magnification. B, Percent of pyramidal neurons in CA2/3 regions with positive COX-2 expression. Graphs show mean ± SEM, * indicates p ≤ .05 when compared with the control group; (Kruskal Wallis test followed by Mann-Whitney). (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Figure 5. View largeDownload slide Immunohistochemical detection of COX-2 positive neurons in the hippocampus. A, Brown staining represents the sites of COX-2 expression (arrows) in pyramidal neurons of CA-2/3 region of the control and DEHP-treated groups at 200× magnification. B, Percent of pyramidal neurons in CA2/3 regions with positive COX-2 expression. Graphs show mean ± SEM, * indicates p ≤ .05 when compared with the control group; (Kruskal Wallis test followed by Mann-Whitney). (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Prenatal DEHP Exposure Elevates DNA Oxidation Markers (8-OHdG, TG) in Hippocampal Neurons Oxidative damage to DNA is known to cause neurodegeneration (Zussy et al., 2013). Exposure to DEHP is reported to cause oxidative damage in affected tissues (Tang et al., 2015). Therefore, we looked into the possibility that the DNA of the hippocampal neurons of a DEHP mouse might be oxidatively damaged. We assessed the degree of oxidation in the hippocampus by immunohistochemical examination using 2 DNA oxidation damage markers: 8-OHdG and TG. 8-OHdG and TG are formed when DNA is attacked by hydroxyl radicals that are synthesized during oxidative stress (Lovell and Markesbery, 2007). Figure 6 shows a representative image of 8-OHdG staining in different regions of the hippocampi. DEHP mice showed stronger OHdG immunostaining in CA2, CA3, and DG regions compared with the controls (Figure 6). TG staining also revealed a higher immunostaining intensity in the CA2 and DG regions of the DEHP mice than controls (Figure 7). Figure 6. View largeDownload slide Immunohistochemical detection of 8-OHdG (DNA oxidation marker) in hippocampal pyramidal neurons. A, Brown staining represents the site of OHdG immunostaining in the CA2 regions (arrows) in different groups. B, OHdG expression in the CA3 regions (white arrows) in different groups. C, OHdG expression in DG regions (asterisks) in different groups at 200× magnification. (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Figure 6. View largeDownload slide Immunohistochemical detection of 8-OHdG (DNA oxidation marker) in hippocampal pyramidal neurons. A, Brown staining represents the site of OHdG immunostaining in the CA2 regions (arrows) in different groups. B, OHdG expression in the CA3 regions (white arrows) in different groups. C, OHdG expression in DG regions (asterisks) in different groups at 200× magnification. (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Figure 7. View largeDownload slide Immunohistochemical detection of TG (DNA oxidation marker) in hippocampal pyramidal neurons. A, Brown color represents the sites of TG immunostaining in pyramidal neurons of the CA2 region (black arrows) in different groups. B, TG expression in the CA3 region (white arrows) in different groups. C, TG expression in the DG region (asterisks) in different groups at 200× magnification. (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Figure 7. View largeDownload slide Immunohistochemical detection of TG (DNA oxidation marker) in hippocampal pyramidal neurons. A, Brown color represents the sites of TG immunostaining in pyramidal neurons of the CA2 region (black arrows) in different groups. B, TG expression in the CA3 region (white arrows) in different groups. C, TG expression in the DG region (asterisks) in different groups at 200× magnification. (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Prenatal DEHP Exposure Lowers Serum Testosterone and Neuronal AR Expression Levels Either low serum testosterone level or decreased AR expression in the neurons is associated with an elevated anxiety level (Nguyen et al., 2009). We previously showed that DEHP mice had less testosterone in sera than the controls (Barakat et al., 2017). In this study, we measured serum testosterone level at the age 22 months to determine if the trend of low testosterone level in DEHP mice continued in older males. DEHP groups (200 µg/kg/day, 500 mg/kg/day, and 750 mg/kg/day) had remarkably lower serum testosterone concentrations than the control group (p = .07, .02, and .05, respectively) (Figure 8B). This was not surprising because we and others previously showed that circulating testosterone level is significantly reduced in middle aged mice that were prenatally exposed to DEHP (Barakat et al., 2017; Culty et al., 2008). To see if AR expression was also altered in the neurons, we assessed the AR expression levels immunohistochemically in the hippocampus and other brain regions. Previous studies localized AR expression in the neurons of the prefrontal cortex, hypothalamus, and cerebellum (Jahan et al., 2015; Perez-Pouchoulen et al., 2016). Consistent with these findings, our AR immunohistochemistry localized a pronounced AR expression in those neurons of control mice (Figure 8C). The AR expression levels were not quantitatively measured but was visually obvious that DEHP mice had a remarkably decreased AR expression in those neurons than the controls (Figure 8C). Figure 8. View largeDownload slide Effect of prenatal DEHP exposure on brain weight, serum testosterone levels, and AR expression in the brain of F1 males at 22 months of age. A, Brain weight (mg). B, Serum testosterone concentration (ng/ml). Graphs show mean ± SEM; * indicates p≤ .05 when compared with the control group; (1-way ANOVA and Tukey’s post hoc test). C, AR expression in the brain of F1 control and 750 mg/kg/day DEHP-treated mice. Immunohistochemistry for androgen in the brain. Brown color represents the sites of AR localization in the pyramidal neurons in the prefrontal cortex (asterisks), Kisspeptin neurons in hypothalamus (white arrows), and Purkinje neurons in the cerebellum (black arrows) at 200× magnification. Showed that DEHP mice had a remarkably decreased AR expression in those neurons than the controls. (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) Figure 8. View largeDownload slide Effect of prenatal DEHP exposure on brain weight, serum testosterone levels, and AR expression in the brain of F1 males at 22 months of age. A, Brain weight (mg). B, Serum testosterone concentration (ng/ml). Graphs show mean ± SEM; * indicates p≤ .05 when compared with the control group; (1-way ANOVA and Tukey’s post hoc test). C, AR expression in the brain of F1 control and 750 mg/kg/day DEHP-treated mice. Immunohistochemistry for androgen in the brain. Brown color represents the sites of AR localization in the pyramidal neurons in the prefrontal cortex (asterisks), Kisspeptin neurons in hypothalamus (white arrows), and Purkinje neurons in the cerebellum (black arrows) at 200× magnification. Showed that DEHP mice had a remarkably decreased AR expression in those neurons than the controls. (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.) DISCUSSION The long-term deleterious effects of the prenatal DEHP exposure on male gonadal function was explored in our previous study, where we showed that DEHP induces premature decline of circulating testosterone and fertility in male mice (Barakat et al., 2017). Testosterone is the primary hormone produced by the male gonads, and it regulates reproduction. Also, this sex hormone exerts a neuroprotective and antianxiety function (Nguyen et al., 2009). Age-related decline of testosterone is linked to dysfunction and disease development in several androgen-responsive tissues, including the brain (Kaufman and Vermeulen, 2005). Rodent studies suggest that decline of testosterone level in the brain is associated with impaired synapse formation (Leranth et al., 2004), hippocampal neuron survival (Pike, 2001) and development of neurodegenerative disease such as Alzheimer’s disease (Moffat et al., 2004). It is well established that prenatal exposure to EDCs hinders the growth and development of the fetal organs, and these adverse effects could persist throughout life (Schug et al., 2011). These findings prompted us to test the possibility that prenatal DEHP exposure might have an adverse impact on neuronal development and/or function in adulthood, causing abnormal neurobehavior. In this study, we found that DEHP in fact impairs neurobehavior and recognition memory. We therefore investigated the neuronal damage in the hippocampus as a potential mechanism for the impairments. Our data showed that DEHP mice exhibited anxious behaviors and impaired recognition memory. We also found that the hippocampi of DEHP mice contained significantly fewer pyramidal neurons than those of the controls, and showed higher levels of COX-2 expression and higher 8-OHdG and TG content. DEHP mice also had lower circulating testosterone and lower AR expression in the neurons of cerebral cortex, hypothalamus, and cerebellum than the control mice. We assessed the impacts of prenatal DEHP exposure on anxiety level using the EPM test and OFT. DEHP mice took more time to enter the open center and made fewer entries into the center area in the OFT. In the EPM test, DEHP-treated mice exhibited increased latency to the open arms (Figure 1). Fewer entries and/or increased latency to enter an open arm or open field are considered to be a signs of elevated anxiety level (Naolapo et al., 2012). These results indicate that prenatal exposure to DEHP probably conditions the impacted animals to be prone to develop anxiety. Our results are reminiscent of a previous study that showed that rats exposed to DEHP postnatally exhibited higher levels of anxiety behavior during adolescence (Carbone et al., 2013). Of note, in our study the animals were exposed prenatally, and they exhibited higher anxiety behavior at the ages of 22 months, indicating that DEHP-induced damage that occurs during fetal development may lead to a life-long impact on their neurobehavior. This finding is in line with a number of epidemiological studies that showed an association between phthalate concentrations in maternal urine during pregnancy and the impairment of cognitive or behavioral development in their children (Adibi et al., 2009; Arbuckle et al., 2016; Gascon et al., 2015). We assessed the impacts of prenatal DEHP exposure on spatial and recognition memory using the Y-maze spontaneous alteration test and NOR test. The Y-maze spontaneous alteration test showed that DEHP exposure correlates to reduced spatial memory function, particularly in the 200 µg/kg/day DEHP group (Figure 2A). Interestingly, DEHP mice also made significantly fewer total entries into the arms, possibly indicating a lower locomotor activity and exploration pattern (Figure 2B). Additionally, in the NOR test, DEHP mice spent less time exploring the novel object (Figure 2A), indicating an impaired recognition memory. Previous studies showed that less novel object exploration time is associated with impaired recognition memory (Pascual et al., 2011). Taken together, our findings suggest that prenatal DEHP induced a life-long impact on recognition memory and brain function. Previous research showed that endogenous androgens during puberty are important for anxiety and memory formation, as castration of male mice led to a significant increase in anxiety and reduction in memory performance (McDermott et al., 2012), indicating a significant role that testosterone may play in controlling anxiety and memory function. In the brain, the hippocampus one of the regions involved in mediating learning and memory functions (Bird and Burgess, 2008). Based on impaired spatial and recognition memory in DEHP mice, neuronal damage was suspected in the hippocampus. Histological examination of the hippocampal tissues at 22 months of age revealed that there were fewer pyramidal neurons in the hippocampi of DEHP mice, and they were morphologically shrunken (Figure 3). Furthermore, quantitative analysis of the hippocampal neurons by SLIM analysis showed that DEHP mice had fewer neurons in the hippocampus, indicating that prenatal exposure to DEHP could lead to neuronal degeneration in the hippocampus. A study reported that acute postnatal DEHP exposure reduced the axonal markers in the CA3 distal stratum oriens in rats, indicating that the hippocampus is highly sensitive to DEHP (Smith et al., 2011). Another study showed that postnatal exposure to DEHP caused hippocampal atrophy by decreasing BDNF (brain-derived neurotrophic factor) synthesis in male rats (Smith and Holahan, 2014). Furthermore, both pre- and postnatal exposures to phthalates had an adverse impact on the developing brain, causing long-lasting neurodevelopmental damages (Miodovnik et al., 2014). Considering that the mice used in our study were prenatally exposed, and the histological examination of the brains was performed at the age of approximately 22 months, the decreased numbers of pyramidal neurons (Figure 4) may be in part an outcome of the DEHP impact on the proliferation, differentiation, or survival of pyramidal neurons during fetal brain development. However, even if there were an impact on neurogenesis, the shrunken cellular morphology of the pyramidal neurons (Figure 3) suggested to us that the impact of prenatal exposure to DEHP might continue for life, and the neurodegeneration continues later in life. The shrunken cellular morphology is often a maker of ongoing inflammation or oxidative stress (Lucas et al., 2006; Zilka et al., 2006). Therefore, we examined the tissues to see if the pyramidal neurons were undergoing inflammation and/or DNA oxidation. Immunohistochemistry showed a remarkably stronger COX-2 expression in the hippocampus of DEHP mice than in the controls. COX-2 is a rate-limiting enzyme for the synthesis prostaglandin E2, a proinflammatory prostaglandin (Yan et al., 2015). Expression of this enzyme in the neurons is linked to a number of neuronal diseases, including Alzheimer’s disease (Davis and Laroche, 2003; Rubio-Perez and Morillas-Ruiz, 2012; Zilka et al., 2006). Because of its association with ongoing diseases, COX-2 inhibitors are often used to protect neurons from inflammation-triggered neurodegeneration and also as an effort to reduce the risk of developing Alzheimer’s disease (Aisen, 2002). Therefore, the elevated expression of COX-2 in the pyramidal neurons (Figure 5) may indicate that the observed shrunken morphology and decreased neuron numbers in the hippocampus may be due to an ongoing inflammation. It will be interesting to determine how a prenatal DEHP exposure may result in the elevated COX-2 expression and neuronal inflammation in the adults. Also interesting is to see if the reduced testosterone level or AR expression (Figure 8) increased COX-expression. We also found that, possibly as a consequence of the neuroinflammation in hippocampal neurons, the immunoreactivity to DNA oxidation markers; 8-OHdG, and TG was higher in the hippocampal neurons of DEHP mice than controls (Figs. 6 and 7). Cellular oxidative stress is often caused by an excessive production of ROS that can damage a wide variety of cellular organelles and molecules such as DNA, RNA, proteins, and lipids, diminishing the cell viability (Huang et al., 2016). Notably, accumulation of oxidative DNA damage in the brain is a major driver of brain aging and a key contributing factor for the development of Alzheimer’s disease (Huang et al., 2016; Zussy et al., 2013). In particular, an increased content of 8-OHDG and/or TG is a molecular signature of brain aging and neurodegenerative disease, such as Alzheimer’s disease (Lovell and Markesbery, 2007). Overall, the elevated 8-OHdG and TG level in the pyramidal neurons of DEHP mice suggests that DNA oxidation may play a role in neurodegeneration and consequently, neurobehavioral abnormalities in the mice that were prenatally exposed to DEHP. The exact mechanisms that explain negative effects of EDCs on neurodevelopment of early life stages are unclear. However, a recent study proposes several possible mechanisms (Kim et al. 2018). Changes in lipid signal transduction pathways caused by exposure to chemicals such as DEHP may alter essential fatty acids in fetal brains that influence the development of cognitive function in the rat brain. A recent study showed that exposure to DEHP increased malondialdehyde content in the brain, which has serious consequences because normal fetal brain development relies upon adequate levels of lipids and fatty acid (Tang et al., 2015). Another study showed that the effects of DEHP on the fetal lipid metabolome was mediated by a peroxisome proliferator-activated receptor (Xu et al., 2008). The concentrations of docosahexaenoic acid and arachidonic acid have been shown to influence neurodevelopment as well (Helland et al., 2003). Furthermore, chemicals may influence sex hormone regulation and can eventually affect normal fetal brain development (Schug et al., 2015). Most importantly, antiandrogenic compounds such as DEHP and DEP are known to affect normal fetal brain development (Lottrup et al., 2006). We found that DEHP mice had considerably lower serum androgen level at the ages of 22 months than the controls (Figure 8). This finding is an extension of earlier reports that clearly linked DEHP exposure to low serum testosterone levels (Andrade et al., 2006; Barakat et al., 2017; Dombret et al., 2017; Pan et al. 2006). DEHP has been reported to decrease testosterone synthesis by interfering with the expression of steroidogenic enzymes that are involved in androgen biosynthesis (Howdeshell et al., 2007). In our study, while animals were prenatally exposed, testosterone was measured at the age of 22 months. These animals were not exposed to DEHP during the 22-month period from birth to old age. Therefore, the decreased serum testosterone level may not be an outcome of a change in the expression of steroidogenic gene(s). Rather, this hypoandrogenism may be a result of an epigenetic modification of the genes during fetal gonadal development, which causes a life-long gene alteration that may directly or indirectly influence testosterone biosynthesis. Testosterone and its derivative estradiol are known to play important roles in neuroprotection and neuroregeneration and to regulate the differentiation and function of the nervous system (Cooke et al., 1998; Farrell et al., 2015). In particular, testosterone exerts neuroprotective activity because it enhances neuronal survival and prevents neurodegeneration (Farrell et al., 2015; Nguyen et al., 2009). The reduction of testosterone is associated with increased levels of neuronal cell death. It is also considered to be a risk factor for developing many neurological disorder such as Alzheimer’s disease (Lau et al. 2014). A study showed that testosterone enhances the survival of hippocampal neurons and is necessary for maintaining learning and memory at older age (Spritzer and Galea, 2007). Taken together, our results suggest that low testosterone levels in the DEHP mice may directly or indirectly induce the anxious behavior, cognitive deficits, and neurodegeneration we observed as summarized in Figure 9. Will testosterone replacement therapy ameliorate the syndromes observed in the DEHP mice? This is possible, as DEHP-induced anxiogenic behavior has been reversed by testosterone replacement in male rats (Carbone et al., 2013). Note our unexpected discovery that DEHP mice had a remarkably lower AR expression in pyramidal neurons in prefrontal cortex, neurons in the hypothalamus, and Purkinje neurons in the cerebellum (Figure 8). Did the altered AR expression in those brain tissues exacerbate the neurodegeneration? This is an interesting question to be addressed. Figure 9. View largeDownload slide Schematic representation of the effect of prenatal DEHP exposure in the induction of oxidative stress leading to neurobehavioral abnormalities. DEHP led to an increase of DNA oxidation by inducing 8-OHdG and TG in addition to the increase in COX-2 expression, which plays a key role in the pathogenesis of neurodegeneration in hippocampal pyramidal neurons. DEHP also has indirect effects by reducing testosterone levels that decrease AR expression in brain. All these events influence the neurodegeneration and neurobehavioral abnormalities resulting from prenatal DEHP exposure. Figure 9. View largeDownload slide Schematic representation of the effect of prenatal DEHP exposure in the induction of oxidative stress leading to neurobehavioral abnormalities. DEHP led to an increase of DNA oxidation by inducing 8-OHdG and TG in addition to the increase in COX-2 expression, which plays a key role in the pathogenesis of neurodegeneration in hippocampal pyramidal neurons. DEHP also has indirect effects by reducing testosterone levels that decrease AR expression in brain. All these events influence the neurodegeneration and neurobehavioral abnormalities resulting from prenatal DEHP exposure. In summary, this study found that prenatal exposure to DEHP lead to elevated anxiety and impaired memory function in male mice. These neurobehavioral abnormalities may originate from a developmental defect in the central nervous system, a life-long neurodegeneration or a neurodegeneration that starts later in life caused by oxidative damage and/or inflammation in the hippocampus. Whether those syndromes are a direct outcome of DEHP impact or due to the observed decrease in testosterone level or AR expression in the neurons is yet to be determined. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. FUNDING This work was supported by the National Institute of Health grant NIH (P01-ES022848) and Environmental Protection Agency grant (RD-83459301) to (C.J.K.), and an Egyptian Mission Sector (JS-3041), Higher Ministry of Education Scholarship to (R.B.). ACKNOWLEDGMENTS The authors also wish to thank all the members of the Dr Flaws’ lab for dosing the animals and give special thanks to Dr Susan Schantz for her critical reading of this article. REFERENCES Adibi J. J., Hauser R., Williams P. L., Whyatt R. M., Calafat A. M., Nelson H., Herrick R., Swan S. H. ( 2009). Maternal urinary metabolites of Di-(2-ethylhexyl) phthalate in relation to the timing of labor in a US multicenter pregnancy cohort study. Am. J. Epidemiol . 169, 1015– 1024. Google Scholar CrossRef Search ADS PubMed  Aisen P. S. ( 2002). Evaluation of selective COX-2 inhibitors for the treatment of Alzheimer’s disease. J. Pain Symptom Manage . 23, S35– S40. Google Scholar CrossRef Search ADS PubMed  Almeida O.P., Yeap B. 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Toxicological SciencesOxford University Press

Published: Apr 23, 2018

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