TY - JOUR
AU - Dix, David, J.
AB - Abstract The mode of action for the reproductive toxicity of some triazole antifungals has been characterized as an increase in serum testosterone and hepatic response, and reduced insemination and fertility indices. In order to refine our mechanistic understanding of these potential modes of action, gene expression profiling was conducted on liver and testis from male Wistar Han IGS rats exposed to myclobutanil (500, 2000 ppm), propiconazole (500, 2500 ppm), or triadimefon (500, 1800 ppm) from gestation day six to postnatal day 92. Gene expression profiles indicated that all three triazoles significantly perturbed the fatty acid, steroid, and xenobiotic metabolism pathways in the male rat liver. In addition, triadimefon modulated expression of genes in the liver from the sterol biosynthesis pathway. Although expression of individual genes were affected, there were no common pathways modulated by all three triazoles in the testis. The pathways identified in the liver included numerous genes involved in phase I–III metabolism (Aldh1a1, Cyp1a1, Cyp2b2, Cyp3a1, Cyp3a2, Slco1a4, Udpgtr2), fatty acid metabolism (Cyp4a10, Pcx, Ppap2b), and steroid metabolism (Ugt1a1, Ugt2a1) for which expression was altered by the triazoles. These differentially expressed genes form part of a network involving lipid, sterol, and steroid homeostatic pathways regulated by the constitutive androstane (CAR), pregnane X (PXR), peroxisome proliferator–activated alpha, and other nuclear receptors in liver. These relatively high dose and long-term exposures to triazole antifungals appeared to perturb fatty acid and steroid metabolism in the male rat liver predominantly through the CAR and PXR signaling pathways. These toxicogenomic effects describe a plausible series of key events contributing to the disruption in steroid homeostasis and reproductive toxicity of select triazole antifungals. myclobutanil, propiconazole, triadimefon, toxicogenomics, steroid metabolism The ability of triazole antifungals to bind and inhibit fungal lanosterol-14α-demethylase activity (cyp51) makes this class of compounds an effective tool in controlling many species and strains of fungi (Ghannoum and Rice, 1999). Disruption in ergosterol biosynthesis leads to a build up of toxic intermediate sterols in the fungal cell membrane, increasing membrane permeability and inhibition of fungal growth (Vanden Bossche et al., 1990). Hence, triazole antifungals have been used for their target pesticidal mode of action on fungal cyp51 inhibition and have proven to be valuable in the control against multiple types of fungal disease. Cytochrome P450 51 (Cyp51) is a conserved gene in fungi, protists, mammals, and plants required for sterol biosynthesis in all eukaryotic systems. The ability of triazoles to bind to the heme protein and inhibit CYP-dependent enzymes raises concerns over triazole effects on hormone synthesis and drug metabolism (Barton et al., 2006; Goetz et al., 2007; Hester et al., 2006; Sun et al., 2007; Tully et al., 2006; Wolf et al., 2006). In rodents, reproductive toxicity has been reported following administration of myclobutanil or triadimefon, but not propiconazole; and carcinogenicity following administration of propiconazole or triadimefon, but not myclobutanil (Goetz et al., 2007; U.S. EPA, 1995, 1996, 2001, 2005a,b,c, 2006). These data prompted interest in gaining a better understanding of the modes and mechanisms of action for triazole related reproductive toxicity and whether there are common modes of actions for triazole fungicides. In a 14-day oral (gavage) toxicity study, adult male Sprague Dawley rats were administered fluconazole (0, 2, 25, or 50 mg/kg/day), myclobutanil (0, 10, 75, or 150 mg/kg/day), propiconazole (0, 10, 75, or 150 mg/kg/day) or triadimefon (0, 10, 50, or 115 mg/kg/day). Only myclobutanil (150 mg/kg/day) produced a statistically significant increase in serum testosterone levels (Tully et al., 2006). In contrast results from a reproduction and fertility study examining developmental and adult reproductive effects in Wistar Han rats exposed via feed to myclobutanil (ca. 6.1, 32.9, or 133.9 mg/kg/day), propiconazole (ca. 6.7, 31.9, or 169.7 mg/kg/day) or triadimefon (ca. 6.5, 33.1, or 139.1 mg/kg/day) from gestation day 6 to postnatal day 92, demonstrated that all three triazoles caused a significant increase in serum testosterone levels. This evidence combined with reduced fertility, hepatomegaly, and changes in pituitary weights (myclobutanil only) suggested a disruption in testosterone homeostasis was a key mode of action in the reproductive toxicity (Goetz et al., 2007). However, the modes of triazole related toxicity to the testis and liver, disruption in testosterone homeostasis, and reduced fertility were unclear. Previous research on triazoles has focused on the metabolic, hepatic and thyroid response to myclobutanil, propiconazole and triadimefon following short term (4 days) to subchronic (90 days) exposure in the adult rat and/or mouse (Allen et al., 2006; Barton et al., 2006; Chen et al., 2009; Goetz et al., 2006; Hester and Nesnow, 2008; Hester et al., 2006; Sun et al., 2007; Tully et al., 2006; Ward et al., 2006; Wolf et al., 2006). This work focused on delineating common modes of triazole toxicity in the adult animal, with an emphasis on defining biological pathways relevant for both carcinogenic and noncarcinogenic effects. Toxicogenomics studies have been conducted in liver, thyroid, and testis following 4 days to subchronic exposures to these three triazoles. The goal of the present study was to gain a better understanding of the modes and mechanisms of action for the triazole reproductive toxicity observed following gestation to adulthood exposure (Goetz et al., 2007). The specific aim was to identify key biological pathways affected by triazoles in the liver and testis following exposure from gestation through adulthood, in order to better understand the disruptions in testosterone homeostasis. Using tissue samples from the previously published study (Goetz et al., 2007), gene expression changes were assessed in the male rat liver and testis following exposure to myclobutanil, propiconazole, or triadimefon from gestational day (GD) 6 through postnatal day (PND) 92 in order to test the hypothesis that triazoles disrupt testosterone homeostasis by increasing expression of genes involved in testosterone synthesis in the testis and decrease expression of genes involved in testosterone metabolism within the liver. The PND92 time point was chosen for this study due to increased testosterone levels and compromised fertility in young adult males at this time point following exposure to triazole antifungals (Goetz et al., 2007). We also hypothesized that if triazoles share common modes of action, exposure to these various triazoles would result in similar expression profiles of steroidogenic and steroid metabolism related genes. The gene expression profiling in this study, in addition to prior toxicological assessments, was expected to provide mechanistic insights into potentially shared modes of action for myclobutanil, propiconazole, and triadimefon. MATERIALS AND METHODS Animal husbandry and dosing regimen. A full description of animal husbandry and dosing is described in Goetz et al. (2007). Briefly, animal care, handling, and treatment were conducted in an American Association for Accreditation of Laboratory Animal Care-International accredited facility, and all procedures were approved by the U.S. Environmental Protection Agency (EPA) National Human and Environmental Effects Research Laboratories Institutional Animal Care and Use Committee. Timed pregnant Wistar Han IGS rats were received from Charles River Laboratories (Raleigh, NC) on GD1–3; single housed, and allowed to acclimate for 3 days prior to the start of the treatment. Dams delivered naturally with day of delivery designated as PND0 for the F1 generation. F1 offspring were housed with their respective mothers until weaning on PND23. Males were removed from the dams and housed by treatment group in pairs until PND50. Males were single housed after PND50. Feed was prepared by Bayer CropScience (Kansas City, MO) as part of a Materials Cooperative Research and Development Agreement between the U.S. EPA and the U.S. Triazole Task Force. Control animals were fed 5002 Certified Rodent Diet with acetone vehicle added. Treatment groups used in this study received feed containing a dietary concentration of myclobutanil (M: 500 or 2000 ppm), propiconazole (P: 500 or 2500 ppm), or triadimefon (T: 500 or 1800 ppm). Dose levels used for this study were selected to match dose levels used in regulatory studies for registering these triazoles with the U.S. EPA. Dams began treated feeds on GD6, feed intake and body weights were measured weekly. The F1 generation continued on the same treated feed diets; feed intake and body weights were measured weekly until necropsy. Refer to Goetz et al. (2007) for achieved dose levels on a week by week basis. One male from each litter was necropsied on PND92 for transcriptional profiling analysis. RNA isolation. Total RNA was extracted from PND92 liver and testis of control and treatment groups using TRI Reagent (Molecular Research Center Inc., Cincinnati, OH) according to the manufacturer's protocol and subjected to quality control measures before application to microarrays. For quality control, RNA A260:A280 ratios were assessed via NanoDrop Fluorospectrometer (NanoDrop Technologies, Inc., Wilmington, DE). RNA absorbance readings with a range 1.8–2.1 were followed with DNase treatment, Total RNA Cleanup (Qiagen RNeasy), and checked for RNA quality using the model 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA). Samples with a ratio of 28S:18S rRNA > 1.6 were accepted for subsequent use in DNA microarrays. RNA was stored at −80°C until labeling for microarray hybridization. Microarray hybridization and scanning. Microarrays and reagents were provided by Affymetrix as part of a Materials Cooperative Research and Development Agreement. Microarray processing was conducted for EPA by Expression Analysis Inc. (Durham, NC). Five micrograms of purified total RNA from each liver or testis of three to seven individual rats per treatment group was hybridized to Affymetrix GeneChip Rat Genome 230 2.0 plus microarrays according to the Affymetrix GeneChip Expression Analysis Technical Manual (www.affymetrix.com). Microarray and probe set analysis. To minimize nonbiological factors, for example, total amount of target hybridized to each array, signal values from each microarray were multiplied by a scaling factor to achieve a mean intensity equal to 500. Converted .cel files were loaded into the JMP Genomics program (SAS, Inc., Cary, NC), Log2 transformed, normalized using interquartile normalization, and analyzed for significant changes in transcript levels through row-by-row modeling using one-way ANOVA. For initial exploratory analysis, principle component analysis (PCA) was applied using JMP Genomics. Comparisons were made between controls and each treatment group with statistical cut-offs applied at a p value adjusted false discovery rate (FDR) of 10% for liver (p ≤ 0.000724) or FDR of 25% for testis (p ≤ 0.000229), and an absolute difference of |1.2| or greater. Probe sets representing transcribed loci, unknown genes, and image clones were removed from the final list of each analysis; probe sets with predicted annotations were kept in the analysis. The Affymetrix .cel files can be accessed through Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo); series accession numbers GSE10411 and GSE10412. Pathway analysis. Ingenuity Pathways Analysis (IPA; Ingenuity Systems, www.ingenuity.com) was used for initial pathway level analysis. Genes from the data set that met the absolute difference cut-off of |1.2|, p value cut-off based on the data set p value adjusted FDR, and associated with a canonical pathway in the Ingenuity Pathways Knowledge Base (IPKB) were considered for the IPA-based analysis. Canonical pathways were identified from the IPA library that were impacted most significantly by the triazoles. The significance of the association between the data set and the canonical pathway was measured using the ratio of genes from the data set that mapped to the pathways divided by the total number of genes that mapped to the canonical pathway. Significance was calculated using the right-tailed Fisher's Exact Test by comparing the number of focus genes that participated in a given pathway, relative to the total number of occurrences of these genes in all pathway annotations in the IPKB. Using this methodology, over-represented pathways were identified containing more focus genes than expected by chance. Further analysis of a broader set of genes and pathways included those identified by IPA, in combination with relevant pathways from the Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/pathway.html) and other references to associate genes with the most complete biological pathways possible. For Figure 1 and the fatty acid metabolism pathway, additional references included Coleman et al. (2000), Nordlie and Foster (1999), and Sul and Wang (1998). The sterol biosynthesis pathway in Figure 2 was supplemented with information from Shibata et al. (2001) and Tansey and Shechter (2000). Figure 3, the cholesterol and bile acid biosynthetic pathway utilized Pandak et al. (2002), Russell (1999), Schwarz et al. (2001), Staudinger et al. (2001), and Wang et al. (2005). Figures 4 and 5 on nuclear receptor signaling pathways depended on Baldan et al. (2006), Dixit et al. (2005), Guzelian et al. (2006), Jigorel et al. (2005, 2006), Kretschmer and Baldwin (2005), Maglich et al. (2002), Shenoy et al. (2004), Yoshikawa et al. (2003), and You (2004). FIG. 1. Open in new tabDownload slide Effects of three triazoles on the expression of genes in rat liver from the fatty acid metabolism pathway. Key in the lower left corner indicates the order of presentation for the six treatment groups, and up- or downregulated genes. FIG. 1. Open in new tabDownload slide Effects of three triazoles on the expression of genes in rat liver from the fatty acid metabolism pathway. Key in the lower left corner indicates the order of presentation for the six treatment groups, and up- or downregulated genes. FIG. 2. Open in new tabDownload slide Triadimefon effects on the expression of genes in rat liver from the sterol biosynthesis pathway. Key in the lower left corner indicates the order of presentation for the six treatment groups, and up- or downregulated genes. FIG. 2. Open in new tabDownload slide Triadimefon effects on the expression of genes in rat liver from the sterol biosynthesis pathway. Key in the lower left corner indicates the order of presentation for the six treatment groups, and up- or downregulated genes. FIG. 3. Open in new tabDownload slide Effects of three triazoles on the expression of genes in rat liver from the steroid metabolism pathway. Key in the lower left corner indicates the order of presentation for the six treatment groups, and up- or downregulated genes. FIG. 3. Open in new tabDownload slide Effects of three triazoles on the expression of genes in rat liver from the steroid metabolism pathway. Key in the lower left corner indicates the order of presentation for the six treatment groups, and up- or downregulated genes. FIG. 4. Open in new tabDownload slide Impact of triazoles on nuclear receptor regulated gene expression in the rat liver. Key in the lower left corner indicates the order of presentation for the six treatment groups, and up- or downregulated genes. FIG. 4. Open in new tabDownload slide Impact of triazoles on nuclear receptor regulated gene expression in the rat liver. Key in the lower left corner indicates the order of presentation for the six treatment groups, and up- or downregulated genes. FIG. 5. Open in new tabDownload slide Nuclear receptor regulation of genes, enzymes, pathways, and processes in the liver. Genes listed represent effects of triazoles on expression in rat liver from this study. Expression of these genes is regulated by the nuclear receptors CAR, PXR, and PPAR-α, and the activity of these receptors, or transcription factors, is modulated by various endobiotics and xenobiotics. Perturbations of CAR, PXR, and PPAR-α signaling pathways can alter lipid and steroid homeostasis and promote hepatotoxicity. FIG. 5. Open in new tabDownload slide Nuclear receptor regulation of genes, enzymes, pathways, and processes in the liver. Genes listed represent effects of triazoles on expression in rat liver from this study. Expression of these genes is regulated by the nuclear receptors CAR, PXR, and PPAR-α, and the activity of these receptors, or transcription factors, is modulated by various endobiotics and xenobiotics. Perturbations of CAR, PXR, and PPAR-α signaling pathways can alter lipid and steroid homeostasis and promote hepatotoxicity. Quantitative PCR. TaqMan-based quantitative RT-PCR was used to determine the relative levels of Abcb1, Cyp1a1, Cyp2b1/Cyp2b2, Cyp3a1, Cyp3a2, Cyp4a1, and Ugt1a1 mRNA in the samples from each treatment group. Primer/probe sets specific for each gene were utilized from Applied Biosystems (Foster City, CA) for Abcb1 (Rn00561753_m1), Cyp1a1 (Custom assay), Cyp2b1/2 (Custom assay), Cyp3a1 (Rn01640761_g1), Cyp3a2 (Rn00756461_m1), Cyp4a1 (Rn00598510_m1), and Ugt1a1 (Rn00754947_m1). The exception to this was for Cyp2b1 and Cyp2b2, for which the primer/probe set could detect either gene transcript-that is why these results are hereafter referred to as Cyp2b1/2. A two-step RT-PCR process was performed by initial reverse transcription of ca. 200 ng of total RNA in a 60-μl reaction using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA), followed by quantitative PCR amplification with isoforms-specific primer/probe sets on 2 μl of each reverse transcribed cDNA. The reactions were characterized by the point during PCR amplification at which fluorescence of the product crossed a defined threshold (CT, automatically determined by the PE Applied Biosystems ABI 7900HT Sequencer software), and inspected to ensure all CT values were within the linear phase (log scale) of exponential growth for all targets. CT values were determined for target CYP genes and an endogenous control gene, β-actin. Each sample was normalized to both the β-actin control and to a vehicle control. A difference of one CT was considered equivalent to a twofold difference in gene expression (exponential relationship, i.e., RQ = 2−DDCt). Sample means for each replicate were determined along with the standard error of the mean if appropriate and percent of adjusted positive control. Relative fold changes in mRNA content were analyzed using the Kruskal-Wallis nonparametric ANOVA with Dunn's multiple comparisons post-test, measures with p ≤ 0.05 were considered significant. RESULTS GeneChip Quality Analysis Microarrays of poor quality, with a scaling factor of greater than 15.0 were removed from the analysis. Three GeneChips from the liver and three GeneChips from the testis data sets were removed prior to normalization and statistical analysis of the two separate data sets. Each treatment group had three to seven GeneChips available for robust analysis following removal of microarray chips with weak intensity readings. Probe Set Analysis in the Liver PCA applied to the normalized data set grouped microarrays by treatment group, with largest variation occurring within the controls, M500, and T1800 groups. It is unlikely this variation is due to sample size (Controls: 7; M500: 4; T1800: 3 microarray chips). Gene expression changes were determined using one-way ANOVA and a FDR of 10% as the multiple testing correction method to control the familywise error rate. The FDR of 10% (α = 0.10) generated a p value cut-off of 7.24E−4 and was considered an ideal cut-off in order to obtain a subset of 1,043 differentially expressed probe sets for use in pathway and gene-level analyses. Of those probe sets, 455 had an absolute difference of |1.2| or greater. Removing probe sets that interrogated unknown genes or transcribed loci, the final list of probe sets identified 308 genes up- or downregulated by myclobutanil, propiconazole, or triadimefon (Table 1). The 16 genes differentially expressed in response to all three triazoles, as detected by microarray, are listed in Table 2. Although four isoforms of Cyp2B (2b2, 2b3, 2b13, 2b15) were assessed using the Affymetrix Rat 230 2.0 GeneChip, only Cyp2b2 was induced in all the triazole treatment groups. The majority of genes modulated by all three triazoles function in lipid or fatty acid metabolism, transporter, and xenobiotic metabolism pathways; thus a pathway based approach was the focus of subsequent analysis and interpretation. TABLE 1 Number of Affymetrix Probe Sets Signaling Significant Treatment-Related Gene Expression Changes in Rat Liver and Testis Triazole Dose level (ppm) (mg/kg/day) Downregulated probe setsa Up regulated probe setsa Total number of probe sets Myclobutanil     Liver 500 (32.9) 4 1 5     Liver 2000 (133.9) 64 9 73     Testis 500 (32.9) 0 16 16     Testis 2000 (133.9) 0 0 0 Propiconazole     Liver 500 (31.9) 2 5 7     Liver 2500 (169.7) 44 45 89     Testis 500 (31.9) 0 0 0     Testis 2500 (169.7) 1 49 50 Triadimefon     Liver 500 (33.1) 46 8 54     Liver 1800 (139.1) 23 154 177     Testis 500 (33.1) 1 4 5     Testis 1800 (139.1) 0 6 6 Triazole Dose level (ppm) (mg/kg/day) Downregulated probe setsa Up regulated probe setsa Total number of probe sets Myclobutanil     Liver 500 (32.9) 4 1 5     Liver 2000 (133.9) 64 9 73     Testis 500 (32.9) 0 16 16     Testis 2000 (133.9) 0 0 0 Propiconazole     Liver 500 (31.9) 2 5 7     Liver 2500 (169.7) 44 45 89     Testis 500 (31.9) 0 0 0     Testis 2500 (169.7) 1 49 50 Triadimefon     Liver 500 (33.1) 46 8 54     Liver 1800 (139.1) 23 154 177     Testis 500 (33.1) 1 4 5     Testis 1800 (139.1) 0 6 6 a Probe sets significantly changed with a fold change greater than |1.2|. Open in new tab TABLE 1 Number of Affymetrix Probe Sets Signaling Significant Treatment-Related Gene Expression Changes in Rat Liver and Testis Triazole Dose level (ppm) (mg/kg/day) Downregulated probe setsa Up regulated probe setsa Total number of probe sets Myclobutanil     Liver 500 (32.9) 4 1 5     Liver 2000 (133.9) 64 9 73     Testis 500 (32.9) 0 16 16     Testis 2000 (133.9) 0 0 0 Propiconazole     Liver 500 (31.9) 2 5 7     Liver 2500 (169.7) 44 45 89     Testis 500 (31.9) 0 0 0     Testis 2500 (169.7) 1 49 50 Triadimefon     Liver 500 (33.1) 46 8 54     Liver 1800 (139.1) 23 154 177     Testis 500 (33.1) 1 4 5     Testis 1800 (139.1) 0 6 6 Triazole Dose level (ppm) (mg/kg/day) Downregulated probe setsa Up regulated probe setsa Total number of probe sets Myclobutanil     Liver 500 (32.9) 4 1 5     Liver 2000 (133.9) 64 9 73     Testis 500 (32.9) 0 16 16     Testis 2000 (133.9) 0 0 0 Propiconazole     Liver 500 (31.9) 2 5 7     Liver 2500 (169.7) 44 45 89     Testis 500 (31.9) 0 0 0     Testis 2500 (169.7) 1 49 50 Triadimefon     Liver 500 (33.1) 46 8 54     Liver 1800 (139.1) 23 154 177     Testis 500 (33.1) 1 4 5     Testis 1800 (139.1) 0 6 6 a Probe sets significantly changed with a fold change greater than |1.2|. Open in new tab TABLE 2 Expression Changes Common to all Three Triazoles for 16 Genes in Rat Liver, as Detected by Microarray Accession number Gene symbol Myclobutanil (32.9 mg/kg/day) Myclobutanil (133.9 mg/kg/day) Propiconazole (31.9 mg/kg/day) Propiconazole (169.7 mg/kg/day) Triadimefon (33.1 mg/kg/day) Triadimefon (139.1 mg/kg/day) NM_017161 Adora2b −1.24302 −1.29105 −1.20012 −1.30677 −1.16624 −1.293 BM392091 Ahctf1 −1.12196 −1.30861 −1.13063 −1.23386 −1.14095 −1.26832 NM_022407 Aldh1a1 1.60289 1.999272 1.310263 2.880486 1.532281 4.841574 NM_012737 Apoa4 −1.09288 −1.87156 −1.45817 −1.99054 −1.66115 −2.79911 BM986220 App 1.08894 1.471356 1.359173 1.753045 1.256155 1.986423 NM_133586 Ces2 1.159467 1.650327 1.347478 2.317871 1.325125 3.361062 AI137640 Cldn1 −1.172 −1.36187 −1.20889 −1.42721 −1.32804 −1.38697 BF564195 Crem −1.20309 −1.40519 −1.20393 −1.32225 −1.1299 −1.39254 AI454613 Cyp2b2 2.728842 3.292202 2.793866 4.672197 2.810611 7.625469 BG378579 Htatip2 1.047174 1.232627 1.15738 1.369729 1.120655 1.344915 BM390462 RGD1310209_pre −1.1009 −1.25043 −1.18184 −1.32877 −1.37249 −1.30658 AI010233 Ccdc126 −1.29462 −1.38855 −1.28179 −1.35011 −1.24059 −1.30313 BI296089 RGD1562101_pre −1.11631 −1.22294 −1.11001 −1.27771 −1.14867 −1.27933 U95011 Slco1a4 1.212303 1.954614 1.616718 2.129518 2.048135 1.973023 NM_031741 Slc2a5 −1.0778 −1.22666 −1.16297 −1.23688 −1.16947 −1.18458 M13506 Udpgtr2 1.688198 2.04723 1.678868 2.385029 1.614087 3.023706 Accession number Gene symbol Myclobutanil (32.9 mg/kg/day) Myclobutanil (133.9 mg/kg/day) Propiconazole (31.9 mg/kg/day) Propiconazole (169.7 mg/kg/day) Triadimefon (33.1 mg/kg/day) Triadimefon (139.1 mg/kg/day) NM_017161 Adora2b −1.24302 −1.29105 −1.20012 −1.30677 −1.16624 −1.293 BM392091 Ahctf1 −1.12196 −1.30861 −1.13063 −1.23386 −1.14095 −1.26832 NM_022407 Aldh1a1 1.60289 1.999272 1.310263 2.880486 1.532281 4.841574 NM_012737 Apoa4 −1.09288 −1.87156 −1.45817 −1.99054 −1.66115 −2.79911 BM986220 App 1.08894 1.471356 1.359173 1.753045 1.256155 1.986423 NM_133586 Ces2 1.159467 1.650327 1.347478 2.317871 1.325125 3.361062 AI137640 Cldn1 −1.172 −1.36187 −1.20889 −1.42721 −1.32804 −1.38697 BF564195 Crem −1.20309 −1.40519 −1.20393 −1.32225 −1.1299 −1.39254 AI454613 Cyp2b2 2.728842 3.292202 2.793866 4.672197 2.810611 7.625469 BG378579 Htatip2 1.047174 1.232627 1.15738 1.369729 1.120655 1.344915 BM390462 RGD1310209_pre −1.1009 −1.25043 −1.18184 −1.32877 −1.37249 −1.30658 AI010233 Ccdc126 −1.29462 −1.38855 −1.28179 −1.35011 −1.24059 −1.30313 BI296089 RGD1562101_pre −1.11631 −1.22294 −1.11001 −1.27771 −1.14867 −1.27933 U95011 Slco1a4 1.212303 1.954614 1.616718 2.129518 2.048135 1.973023 NM_031741 Slc2a5 −1.0778 −1.22666 −1.16297 −1.23688 −1.16947 −1.18458 M13506 Udpgtr2 1.688198 2.04723 1.678868 2.385029 1.614087 3.023706 Note. Values given as fold change relative to control. Bold: transcript level changes were significant. Suffix _pre represents probe sets with predicted annotation. Open in new tab TABLE 2 Expression Changes Common to all Three Triazoles for 16 Genes in Rat Liver, as Detected by Microarray Accession number Gene symbol Myclobutanil (32.9 mg/kg/day) Myclobutanil (133.9 mg/kg/day) Propiconazole (31.9 mg/kg/day) Propiconazole (169.7 mg/kg/day) Triadimefon (33.1 mg/kg/day) Triadimefon (139.1 mg/kg/day) NM_017161 Adora2b −1.24302 −1.29105 −1.20012 −1.30677 −1.16624 −1.293 BM392091 Ahctf1 −1.12196 −1.30861 −1.13063 −1.23386 −1.14095 −1.26832 NM_022407 Aldh1a1 1.60289 1.999272 1.310263 2.880486 1.532281 4.841574 NM_012737 Apoa4 −1.09288 −1.87156 −1.45817 −1.99054 −1.66115 −2.79911 BM986220 App 1.08894 1.471356 1.359173 1.753045 1.256155 1.986423 NM_133586 Ces2 1.159467 1.650327 1.347478 2.317871 1.325125 3.361062 AI137640 Cldn1 −1.172 −1.36187 −1.20889 −1.42721 −1.32804 −1.38697 BF564195 Crem −1.20309 −1.40519 −1.20393 −1.32225 −1.1299 −1.39254 AI454613 Cyp2b2 2.728842 3.292202 2.793866 4.672197 2.810611 7.625469 BG378579 Htatip2 1.047174 1.232627 1.15738 1.369729 1.120655 1.344915 BM390462 RGD1310209_pre −1.1009 −1.25043 −1.18184 −1.32877 −1.37249 −1.30658 AI010233 Ccdc126 −1.29462 −1.38855 −1.28179 −1.35011 −1.24059 −1.30313 BI296089 RGD1562101_pre −1.11631 −1.22294 −1.11001 −1.27771 −1.14867 −1.27933 U95011 Slco1a4 1.212303 1.954614 1.616718 2.129518 2.048135 1.973023 NM_031741 Slc2a5 −1.0778 −1.22666 −1.16297 −1.23688 −1.16947 −1.18458 M13506 Udpgtr2 1.688198 2.04723 1.678868 2.385029 1.614087 3.023706 Accession number Gene symbol Myclobutanil (32.9 mg/kg/day) Myclobutanil (133.9 mg/kg/day) Propiconazole (31.9 mg/kg/day) Propiconazole (169.7 mg/kg/day) Triadimefon (33.1 mg/kg/day) Triadimefon (139.1 mg/kg/day) NM_017161 Adora2b −1.24302 −1.29105 −1.20012 −1.30677 −1.16624 −1.293 BM392091 Ahctf1 −1.12196 −1.30861 −1.13063 −1.23386 −1.14095 −1.26832 NM_022407 Aldh1a1 1.60289 1.999272 1.310263 2.880486 1.532281 4.841574 NM_012737 Apoa4 −1.09288 −1.87156 −1.45817 −1.99054 −1.66115 −2.79911 BM986220 App 1.08894 1.471356 1.359173 1.753045 1.256155 1.986423 NM_133586 Ces2 1.159467 1.650327 1.347478 2.317871 1.325125 3.361062 AI137640 Cldn1 −1.172 −1.36187 −1.20889 −1.42721 −1.32804 −1.38697 BF564195 Crem −1.20309 −1.40519 −1.20393 −1.32225 −1.1299 −1.39254 AI454613 Cyp2b2 2.728842 3.292202 2.793866 4.672197 2.810611 7.625469 BG378579 Htatip2 1.047174 1.232627 1.15738 1.369729 1.120655 1.344915 BM390462 RGD1310209_pre −1.1009 −1.25043 −1.18184 −1.32877 −1.37249 −1.30658 AI010233 Ccdc126 −1.29462 −1.38855 −1.28179 −1.35011 −1.24059 −1.30313 BI296089 RGD1562101_pre −1.11631 −1.22294 −1.11001 −1.27771 −1.14867 −1.27933 U95011 Slco1a4 1.212303 1.954614 1.616718 2.129518 2.048135 1.973023 NM_031741 Slc2a5 −1.0778 −1.22666 −1.16297 −1.23688 −1.16947 −1.18458 M13506 Udpgtr2 1.688198 2.04723 1.678868 2.385029 1.614087 3.023706 Note. Values given as fold change relative to control. Bold: transcript level changes were significant. Suffix _pre represents probe sets with predicted annotation. Open in new tab Pathway Analysis in the Liver Pathway analysis identified common biological pathways and processes affected by the three triazoles in rat liver. For initial analysis of pathways the entire liver data set (31,099 probe sets) was uploaded into IPA and the absolute difference |1.2| or greater and p value ≤ 7.24E−4 was used to identify differentially expressed genes. In the liver data set, 180 of the 308 significant probe sets mapped to the IPKB. These focus genes were overlaid onto a molecular network developed from information within the IPKB. Table 3 shows the pathways identified by IPA as being affected by the three triazoles. Several metabolic pathways are common across the three triazoles including androgen and estrogen, arachidonic acid, fatty acid, glycerolipid, linoleic acid, tryptophan, and xenobiotic metabolism. Additional pathways are affected by just one or two of the triazoles. The multiple biological pathways modified by more than one triazole treatment suggest a potential for common modes of action within the rat liver. TABLE 3 Biological Pathways Containing Significant Changes in Rat Liver Gene Expression following Exposure to Triazoles Myclobutanil Propiconazole Triadimefon Aminosugars metabolism Amyloid processing Androgen and estrogen metabolism Androgen and estrogen metabolism Androgen and estrogen metabolism Antigen presentation pathway Arachidonic acid metabolism Arachidonic acid metabolism Arachidonic acid metabolism Arginine and proline metabolism Butanoate metabolism Ascorbate and aldarate metabolism Ascorbate and aldarate metabolism Cyanoamino acid metabolism Cysteine metabolism Fatty acid metabolism Fatty acid metabolism Fatty acid metabolism Fructose and mannose metabolism Fc Epsilon RI signaling Galactose metabolism Glutamate metabolism Glutathione metabolism Glutathione metabolism Glycerolipid metabolism Glycerolipid metabolism Glycine, Serine and Threonine metabolism Glycolysis/gluconeogenesis Glycolysis/gluconeogenesis Glycerophospholipid metabolism Histidine metabolism IL-6 signaling Keratin sulfate biosynthesis Linoleic acid metabolism Linoleic acid metabolism Linoleic acid metabolism Metabolism of xenobiotics by cytochrome P450 Metabolism of xenobiotics by cytochrome P450 Metabolism of xenobiotics by cytochrome p450 p38 MAPK signaling N-Glycan degradation Pentose and glucuronate interconversions Pentose and glucuronate interconversions Nitrogen metabolism Retinol metabolism Phospholipid degradation Propanoate metabolism Pyruvate metabolism Pyruvate metabolism Sphingolipid metabolism Sterol Biosynthesis Starch and Sucrose metabolism Starch and sucrose metabolism Toll-like receptor signaling Tryptophan metabolism Tryptophan metabolism Tryptophan metabolism Wnt/β-catenin signaling Xenobiotic metabolism signaling Xenobiotic metabolism signaling Xenobiotic metabolism signaling Myclobutanil Propiconazole Triadimefon Aminosugars metabolism Amyloid processing Androgen and estrogen metabolism Androgen and estrogen metabolism Androgen and estrogen metabolism Antigen presentation pathway Arachidonic acid metabolism Arachidonic acid metabolism Arachidonic acid metabolism Arginine and proline metabolism Butanoate metabolism Ascorbate and aldarate metabolism Ascorbate and aldarate metabolism Cyanoamino acid metabolism Cysteine metabolism Fatty acid metabolism Fatty acid metabolism Fatty acid metabolism Fructose and mannose metabolism Fc Epsilon RI signaling Galactose metabolism Glutamate metabolism Glutathione metabolism Glutathione metabolism Glycerolipid metabolism Glycerolipid metabolism Glycine, Serine and Threonine metabolism Glycolysis/gluconeogenesis Glycolysis/gluconeogenesis Glycerophospholipid metabolism Histidine metabolism IL-6 signaling Keratin sulfate biosynthesis Linoleic acid metabolism Linoleic acid metabolism Linoleic acid metabolism Metabolism of xenobiotics by cytochrome P450 Metabolism of xenobiotics by cytochrome P450 Metabolism of xenobiotics by cytochrome p450 p38 MAPK signaling N-Glycan degradation Pentose and glucuronate interconversions Pentose and glucuronate interconversions Nitrogen metabolism Retinol metabolism Phospholipid degradation Propanoate metabolism Pyruvate metabolism Pyruvate metabolism Sphingolipid metabolism Sterol Biosynthesis Starch and Sucrose metabolism Starch and sucrose metabolism Toll-like receptor signaling Tryptophan metabolism Tryptophan metabolism Tryptophan metabolism Wnt/β-catenin signaling Xenobiotic metabolism signaling Xenobiotic metabolism signaling Xenobiotic metabolism signaling Note. Pathways listed were affected by mid and/or high dose of each triazole. Bold: pathways affected by two or more triazoles. Open in new tab TABLE 3 Biological Pathways Containing Significant Changes in Rat Liver Gene Expression following Exposure to Triazoles Myclobutanil Propiconazole Triadimefon Aminosugars metabolism Amyloid processing Androgen and estrogen metabolism Androgen and estrogen metabolism Androgen and estrogen metabolism Antigen presentation pathway Arachidonic acid metabolism Arachidonic acid metabolism Arachidonic acid metabolism Arginine and proline metabolism Butanoate metabolism Ascorbate and aldarate metabolism Ascorbate and aldarate metabolism Cyanoamino acid metabolism Cysteine metabolism Fatty acid metabolism Fatty acid metabolism Fatty acid metabolism Fructose and mannose metabolism Fc Epsilon RI signaling Galactose metabolism Glutamate metabolism Glutathione metabolism Glutathione metabolism Glycerolipid metabolism Glycerolipid metabolism Glycine, Serine and Threonine metabolism Glycolysis/gluconeogenesis Glycolysis/gluconeogenesis Glycerophospholipid metabolism Histidine metabolism IL-6 signaling Keratin sulfate biosynthesis Linoleic acid metabolism Linoleic acid metabolism Linoleic acid metabolism Metabolism of xenobiotics by cytochrome P450 Metabolism of xenobiotics by cytochrome P450 Metabolism of xenobiotics by cytochrome p450 p38 MAPK signaling N-Glycan degradation Pentose and glucuronate interconversions Pentose and glucuronate interconversions Nitrogen metabolism Retinol metabolism Phospholipid degradation Propanoate metabolism Pyruvate metabolism Pyruvate metabolism Sphingolipid metabolism Sterol Biosynthesis Starch and Sucrose metabolism Starch and sucrose metabolism Toll-like receptor signaling Tryptophan metabolism Tryptophan metabolism Tryptophan metabolism Wnt/β-catenin signaling Xenobiotic metabolism signaling Xenobiotic metabolism signaling Xenobiotic metabolism signaling Myclobutanil Propiconazole Triadimefon Aminosugars metabolism Amyloid processing Androgen and estrogen metabolism Androgen and estrogen metabolism Androgen and estrogen metabolism Antigen presentation pathway Arachidonic acid metabolism Arachidonic acid metabolism Arachidonic acid metabolism Arginine and proline metabolism Butanoate metabolism Ascorbate and aldarate metabolism Ascorbate and aldarate metabolism Cyanoamino acid metabolism Cysteine metabolism Fatty acid metabolism Fatty acid metabolism Fatty acid metabolism Fructose and mannose metabolism Fc Epsilon RI signaling Galactose metabolism Glutamate metabolism Glutathione metabolism Glutathione metabolism Glycerolipid metabolism Glycerolipid metabolism Glycine, Serine and Threonine metabolism Glycolysis/gluconeogenesis Glycolysis/gluconeogenesis Glycerophospholipid metabolism Histidine metabolism IL-6 signaling Keratin sulfate biosynthesis Linoleic acid metabolism Linoleic acid metabolism Linoleic acid metabolism Metabolism of xenobiotics by cytochrome P450 Metabolism of xenobiotics by cytochrome P450 Metabolism of xenobiotics by cytochrome p450 p38 MAPK signaling N-Glycan degradation Pentose and glucuronate interconversions Pentose and glucuronate interconversions Nitrogen metabolism Retinol metabolism Phospholipid degradation Propanoate metabolism Pyruvate metabolism Pyruvate metabolism Sphingolipid metabolism Sterol Biosynthesis Starch and Sucrose metabolism Starch and sucrose metabolism Toll-like receptor signaling Tryptophan metabolism Tryptophan metabolism Tryptophan metabolism Wnt/β-catenin signaling Xenobiotic metabolism signaling Xenobiotic metabolism signaling Xenobiotic metabolism signaling Note. Pathways listed were affected by mid and/or high dose of each triazole. Bold: pathways affected by two or more triazoles. Open in new tab A more complete analysis of altered pathways and biological processes in the rat liver was undertaken; using the larger set of 308 differentially expressed genes identified at the probe set level. This extended the IPA-based analysis, to include statistically significant differentially expressed genes and altered pathways that were not captured within the IPKB. Fatty acid catabolism pathway. Figure 1 shows the triazole effects in the rat liver on gene expression in the fatty acid catabolism pathway. Myclobutanil and propiconazole decreased transcript levels of phosphatidic acid phosphatase 2B (Ppap2b), a key enzyme in glycerolipid metabolism involved in triacylglycerol formation. Propiconazole and triadimefon decreased transcript levels of pyruvate carboxylase (Pcx), the initiating enzyme in the citric acid cycle, catalyzing the carboxylation of pyruvate into oxaloacetate for later ATP synthesis. Triazoles also decreased expression of L-pyruvate kinase (Pklr), which is rate limiting in fatty acid biosynthesis and storage. Acyl-CoA synthetase (Acsl) produces the primary substrate for energy use and synthesis of fatty acids, including triacylglycerol and cholesterol esters. The triazole regulation of Acsl was complicated, with Acls5 downregulated by propiconazole, and Acls3 up regulated by triadimefon. Triadimefon also increased expression of the next enzyme, enoyl coenzyme A hydratase (Echdc1), in this latter part of fatty acid metabolism driving catalytic conversion of palmitate to acetoacetyl-coenzyme A, the precursor leading into sterol biosynthesis. Sterol biosynthesis pathway. Figure 2 presents triazole effects in the rat liver on the sterol biosynthesis pathway, and key genes involved in the biosynthesis of cholesterol. Of the three triazoles examined, only triadimefon had a significant impact on gene expression within this pathway in rat liver. Beginning with 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) and mevalonate kinase (Mvk), triadimefon increased the transcript levels of several sterol biosynthesis dependent enzymes. Squalene epoxidase (Sqle) was increased by triadimefon at both dose levels indicating Sqle is more sensitive to triadimefon relative to the other genes in this pathway. Steroid metabolism and bile acid biosynthesis pathway. Triazole exposure also altered expression in the rat liver of genes in the steroid metabolism and bile acid biosynthesis pathways (Fig. 3). Although these pathways were not identified by the IPA-based analysis, a more complete analysis based on all 308 differentially expressed genes identified seven key enzymes in these pathways. Steroid 5 alpha-reductase (Srd5a1) in the main bile acid pathway, and Cyp7b1 in the alternate bile acid pathway were decreased by triadimefon and propiconazole treatment, respectively. The two triazoles target different gene transcripts, however both enzymes’ activities are involved in primary bile acid synthesis. Propiconazole also decreased the transcript levels of inositol 1,4,5-triphosphate receptor 2 (Itpr2) which is involved in intracellular calcium homeostasis. The transcript levels for several uridine diphosphate glucuronosyltransferases (Ugt1a1, Ugt2a1, Udpgtr2) were increased by all three triazoles, indicating increased metabolism of steroids and xenobiotics. Increased expression of hydroxysteroid 17β-dehydrogenase (Hsd17b) by triadimefon also indicated elevated androgen and estrogen metabolism in the liver. Nuclear receptor regulated genes. Figure 4 shows the impact of triazoles on nuclear receptor regulated genes within the rat liver. There were several genes differentially expressed that are regulated by the constitutive androstane receptor (CAR), pregnane X receptor (PXR), aryl hydrocarbon receptor (AhR), as well as the peroxisome proliferator–activated receptor alpha (PPAR-α) and liver X receptor (LXR). The genes regulated by CAR and PXR are phase I, II, and III enzymes that are part of fatty acid and xenobiotic metabolism, sterol biosynthesis, steroid metabolism, and cell cycle pathways. Increased transcript levels of most of these CAR/PXR-regulated genes indicate activation of CAR and/or PXR. Also consistent with CAR activation, the genes Pcx and Ppap2b were downregulated. Results for Cyp2b2 from the arrays and for Cyp2b1/Cyp2b2 from PCR are both listed (Table 4). CAR-specific, PXR-specific, and genes coregulated by CAR and PXR are identified in Figure 4. Additional genes with overlapping regulation by multiple receptors, modulated by triazoles in rat liver; included Alas1 expression regulated by CAR, PXR, and LXR, and Cyp1a1 expression regulated by AhR and CAR. Effects on nuclear receptor regulated genes included increased expression of steroid and xenobiotic metabolism genes aldehyde dehydrogenase (Aldh1a1), Cyp1a1, and Cyp2b2, the previously mentioned glucuronide and glucoside conjugation genes Ugt1a1, Ugt2a1, and Udpgtr2, glutathione conjugator Gstm4 and phase III transporter Abcc3; all changes were likely to have altered metabolism and excretion of steroids and xenobiotics. TABLE 4 Comparisons between Microarray and Quantitative PCR Measurement of Gene Expression in Rat Liver following Triazole Exposure Abcb1 Cyp1a1 Cyp2b2 Cyp3a1 Cyp3a2 Cyp4a1 Ugt1a1 Treatment mg/kg/day Array qPCR Array qPCR Arraya qPCR Array qPCR Arrayb qPCR Arrayc qPCR Array qPCR Myclobutanil 134 1.24 −2.37 1.76 5.82 3.29 64.57 −1.12 2.05 3.22 −1.73 −1.91 1.50 1.44 Propiconazole 170 1.48 1.64 3.02 21.31 4.67 132.12 1.08 2.04 4.01 −1.59 −1.53 1.82 1.32 Triadimefon 139 1.41 −4.99 5.60 79.99 7.63 63.13 1.98 18.20 1.57 −1.30 −2.70 1.78 7.51 Abcb1 Cyp1a1 Cyp2b2 Cyp3a1 Cyp3a2 Cyp4a1 Ugt1a1 Treatment mg/kg/day Array qPCR Array qPCR Arraya qPCR Array qPCR Arrayb qPCR Arrayc qPCR Array qPCR Myclobutanil 134 1.24 −2.37 1.76 5.82 3.29 64.57 −1.12 2.05 3.22 −1.73 −1.91 1.50 1.44 Propiconazole 170 1.48 1.64 3.02 21.31 4.67 132.12 1.08 2.04 4.01 −1.59 −1.53 1.82 1.32 Triadimefon 139 1.41 −4.99 5.60 79.99 7.63 63.13 1.98 18.20 1.57 −1.30 −2.70 1.78 7.51 Note. Values given as fold change relative to control. Bold: significant transcript level or fold changes. a Probe set representing Cyp2b2 on GeneChip. b No representative probe set on GeneChip. c Probe set representing Cyp4a10 on GeneChip. Open in new tab TABLE 4 Comparisons between Microarray and Quantitative PCR Measurement of Gene Expression in Rat Liver following Triazole Exposure Abcb1 Cyp1a1 Cyp2b2 Cyp3a1 Cyp3a2 Cyp4a1 Ugt1a1 Treatment mg/kg/day Array qPCR Array qPCR Arraya qPCR Array qPCR Arrayb qPCR Arrayc qPCR Array qPCR Myclobutanil 134 1.24 −2.37 1.76 5.82 3.29 64.57 −1.12 2.05 3.22 −1.73 −1.91 1.50 1.44 Propiconazole 170 1.48 1.64 3.02 21.31 4.67 132.12 1.08 2.04 4.01 −1.59 −1.53 1.82 1.32 Triadimefon 139 1.41 −4.99 5.60 79.99 7.63 63.13 1.98 18.20 1.57 −1.30 −2.70 1.78 7.51 Abcb1 Cyp1a1 Cyp2b2 Cyp3a1 Cyp3a2 Cyp4a1 Ugt1a1 Treatment mg/kg/day Array qPCR Array qPCR Arraya qPCR Array qPCR Arrayb qPCR Arrayc qPCR Array qPCR Myclobutanil 134 1.24 −2.37 1.76 5.82 3.29 64.57 −1.12 2.05 3.22 −1.73 −1.91 1.50 1.44 Propiconazole 170 1.48 1.64 3.02 21.31 4.67 132.12 1.08 2.04 4.01 −1.59 −1.53 1.82 1.32 Triadimefon 139 1.41 −4.99 5.60 79.99 7.63 63.13 1.98 18.20 1.57 −1.30 −2.70 1.78 7.51 Note. Values given as fold change relative to control. Bold: significant transcript level or fold changes. a Probe set representing Cyp2b2 on GeneChip. b No representative probe set on GeneChip. c Probe set representing Cyp4a10 on GeneChip. Open in new tab A more integrated representation of the genes, enzymes, pathways and processes regulated by nuclear receptors in the liver is presented in Figure 5. Various agonists and antagonists of the CAR, PXR, and PPAR-α receptors are indicated, as well as the regulated genes which in this study were differentially expressed in response to triazole exposure. This included up regulation of the antiapoptotic genes Gadd45b and Mdm2. Fatty acid metabolism, sterol biosynthesis, steroid metabolism, bile acid metabolism, and cellular growth pathways are all coordinately regulated by these nuclear receptors. Based on these findings, it is postulated that alterations in these pathways from exposure to xenobiotics like the triazoles, mediated by these nuclear receptors, resulted in effects on biological processes including hepatomegaly, detoxification and elimination, and plasma lipid transport. Quantitative PCR To further examine and confirm changes in liver gene expression, a set of genes modulated by all three triazoles, as well as additional CAR regulated genes, were analyzed by PCR. Quantitative PCR confirmed the increased expression of Cyp1a1 and Cyp2b1/Cyp2b2, and yielded more definitive results for Cyp3a1 and Ugt1a1 (Table 4). Based on the PCR results, Cyp3a1 is the 17th gene for which expression is modulated by all three triazoles (see Table 2 for other 16). Cyp3a2 was not represented on the microarray. PCR detected an increase in Cyp3a2 mRNA in response to myclobutanil and propiconazole, and with the increased Cyp3a1 mRNA content by all three triazoles, suggests PXR activation by triazoles in the rat liver. The magnitude of increased Cyp2b1/Cyp2b2 indicated a strong activation of CAR consistent with a maladaptive toxic response as a result of long term triazole exposure. The one clear case of discordance between microarray and PCR results, for Abcb1 and triadimefon, may be due to the sequences used for probes in the separate assays. Probe Set Analysis in the Testis Gene expression changes were determined using a one-way ANOVA and a FDR of 25% (α = 0.25) which generated a p value cut-off of 2.29E−4 yielding 169 differentially expressed probe sets. Of those probe sets, 77 had an absolute difference of |1.2| or greater. Removal of probe sets interrogating unknown or transcribed loci, the final list of probe sets equaled 70 (Table 1). An ANOVA analysis using a FDR of 10% defined one unknown gene; a FDR of 15 or 20% defined a list of 10 genes. The liberal cut-off was used in order to obtain a subset of genes for pathway analysis. It is clear, however, from the ANOVA that there were either large variations within treatment groups or very small changes in gene expression within the testis. Pathway Analysis in the Testis IPA was used to identify common pathways affected by the three triazoles. From the entire data set (31,099 probe sets), the absolute difference of |1.2| or greater and p value ≤ 2.29E−4 was used to identify genes whose expression was differentially regulated. In the testis data set, 50 of the 72 significant probe sets mapped to the IPKB. There were no common pathways affected by all three triazoles, however, there were five pathways modulated by at least two triazoles (Table 5). The IPA-based analysis of altered pathways in the testis did identify 11 potentially significant matches to pathways affected by triazoles in the liver. Many of these pathways common to testis and liver are critical to reproduction, including androgen and estrogen metabolism, C21-Steroid hormone metabolism, and sterol biosynthesis. The other common pathways are significant in relation to how testis and liver respond to triazole exposures: metabolism of xenobiotics by cytochrome P450, and xenobiotic metabolism signaling. Several additional pathways recognized in the testis, were common and robust responders to all three triazoles in the liver: arachidonic acid metabolism, fatty acid metabolism, linoleic acid metabolism, and tryptophan metabolism. TABLE 5 Biological Pathways Containing Significant Changes in Rat Testis Gene Expression following Exposure to Triazoles Myclobutanil Propiconazole Triadimefon Androgen and estrogen metabolism Androgen and estrogen metabolism Arachidonic acid metabolism C21-Steroid hormone metabolism Complement and Coagulation cascades Cysteine metabolism Fatty acid metabolism Fatty acid metabolism Linoleic acid metabolism Metabolism of xenobiotics by cytochrome P450 Nitrogen metabolism Methionine metabolism Neuregulin signaling Pentose and glucuronate interconversions Propanoate metabolism Phenylalanine metabolism Propanoate metabolism Retinol metabolism Sterol biosynthesis Selenoamino acid metabolism Starch and sucrose metabolism Taurine and hypotaurine metabolism Tryptophan metabolism Valine, leucine, and isoleucine degradation Urea cycle and metabolism of amino groups Valine, leucine, and isoleucine degradation β-Alanine metabolism Xenobiotic metabolism signaling β-Alanine metabolism Myclobutanil Propiconazole Triadimefon Androgen and estrogen metabolism Androgen and estrogen metabolism Arachidonic acid metabolism C21-Steroid hormone metabolism Complement and Coagulation cascades Cysteine metabolism Fatty acid metabolism Fatty acid metabolism Linoleic acid metabolism Metabolism of xenobiotics by cytochrome P450 Nitrogen metabolism Methionine metabolism Neuregulin signaling Pentose and glucuronate interconversions Propanoate metabolism Phenylalanine metabolism Propanoate metabolism Retinol metabolism Sterol biosynthesis Selenoamino acid metabolism Starch and sucrose metabolism Taurine and hypotaurine metabolism Tryptophan metabolism Valine, leucine, and isoleucine degradation Urea cycle and metabolism of amino groups Valine, leucine, and isoleucine degradation β-Alanine metabolism Xenobiotic metabolism signaling β-Alanine metabolism Note. Pathways listed were affected by mid and/or high dose of each triazole. Bold: pathways affected by two or more triazoles. Open in new tab TABLE 5 Biological Pathways Containing Significant Changes in Rat Testis Gene Expression following Exposure to Triazoles Myclobutanil Propiconazole Triadimefon Androgen and estrogen metabolism Androgen and estrogen metabolism Arachidonic acid metabolism C21-Steroid hormone metabolism Complement and Coagulation cascades Cysteine metabolism Fatty acid metabolism Fatty acid metabolism Linoleic acid metabolism Metabolism of xenobiotics by cytochrome P450 Nitrogen metabolism Methionine metabolism Neuregulin signaling Pentose and glucuronate interconversions Propanoate metabolism Phenylalanine metabolism Propanoate metabolism Retinol metabolism Sterol biosynthesis Selenoamino acid metabolism Starch and sucrose metabolism Taurine and hypotaurine metabolism Tryptophan metabolism Valine, leucine, and isoleucine degradation Urea cycle and metabolism of amino groups Valine, leucine, and isoleucine degradation β-Alanine metabolism Xenobiotic metabolism signaling β-Alanine metabolism Myclobutanil Propiconazole Triadimefon Androgen and estrogen metabolism Androgen and estrogen metabolism Arachidonic acid metabolism C21-Steroid hormone metabolism Complement and Coagulation cascades Cysteine metabolism Fatty acid metabolism Fatty acid metabolism Linoleic acid metabolism Metabolism of xenobiotics by cytochrome P450 Nitrogen metabolism Methionine metabolism Neuregulin signaling Pentose and glucuronate interconversions Propanoate metabolism Phenylalanine metabolism Propanoate metabolism Retinol metabolism Sterol biosynthesis Selenoamino acid metabolism Starch and sucrose metabolism Taurine and hypotaurine metabolism Tryptophan metabolism Valine, leucine, and isoleucine degradation Urea cycle and metabolism of amino groups Valine, leucine, and isoleucine degradation β-Alanine metabolism Xenobiotic metabolism signaling β-Alanine metabolism Note. Pathways listed were affected by mid and/or high dose of each triazole. Bold: pathways affected by two or more triazoles. Open in new tab Expanding on the pathways highlighted by the IPA analysis, the larger set of 72 differentially expressed genes were examined based on relevant biological processes or pathways. No one major biological process or pathway stood out in this analysis. This set of genes did map to the lipid, fatty acid and C21-steroid hormone metabolism, inflammatory response, intracellular signaling, or xenobiotic metabolism pathways. However, due to the liberal threshold (FDR of 25%) used during the ANOVA, and the limited number of genes differentially expressed, it was difficult to have confidence in these results for defining specific modes of triazole related toxicity within the testis. This was exacerbated by the fact that propiconazole, which demonstrated no overt reproductive toxicity, caused a greater number of differentially expressed genes in the testis compared with the two reproductive toxicants, myclobutanil and triadimefon. DISCUSSION Toxicological endpoints from our previous assessment of the reproductive toxicity of myclobutanil, propiconazole, and triadimefon identified a potential mode of action for the reproductive toxicity of triazole antifungals (Goetz et al., 2007). The combination of increased serum testosterone levels by all three triazoles, increased anogenital distance and testis weight, hepatomegaly, and decreased insemination and fertility indices strongly suggested disruption in testosterone homeostasis as a mode of action for triazole toxicity. However, the molecular mechanisms underlying these effects remained indeterminate. This study was designed to test the hypothesis that disruption in testosterone homeostasis was a result of changes in gene expression leading to increased steroidogenesis in the testis and decreased steroid metabolism in the liver. Furthermore, it was designed to identify whether this putative mode of action was common to the triazoles, and if so, to gain mechanistic understanding of the common biological pathways perturbed by triazoles that lead to toxicity. Analyses based on biological pathways was used to interpret the significant gene expression changes in the liver and testis and to provide context for interpreting these changes. Numerous common gene transcripts and biological pathways were identified for triazoles in the liver defining common biological processes modulated by all three triazoles and supporting the interpretation of a common mode of action. In contrast, the small number of differentially expressed genes and affected pathways in the testis indicated that the observed reproductive effects were not due to modulations of gene expression within the testis, and that the testis was not a target organ for triazole reproductive toxicity. Common metabolic pathways for all three triazoles included androgen and estrogen, arachidonic acid, fatty acid, glycerolipid, linoleic acid, tryptophan, and xenobiotic. Several of the perturbed pathways common to the triazoles formed a large interconnected network between glycolysis and fatty acid catabolism, sterol biosynthesis and bile acid biosynthesis or steroid metabolism. All three triazoles had a significant impact on lipid metabolism, including fatty acid and steroid metabolism, as well as lipid transport. The genomic data in this study demonstrates triazole disruption of pathways of key biological functions in the liver; including energy homeostasis, biological membrane fluidity, and CYP and other metabolic activities. Moreover, these pathways are critical to liver-mediated steroid homeostasis, and we propose that it is the perturbation of these critical pathways that leads to the observed reproductive and hepatic toxicity of the triazoles. Hepatic Fatty Acid Metabolism Many phase I, II, and III metabolic genes perturbed by triazole exposures are regulated by the nuclear receptors PPAR, CAR, PXR, LXR, FXR, and AhR. Nuclear receptors within the liver regulate specific and overlapping subsets of genes (Honkakoski and Negishi, 2000; Wei et al., 2002; Yoshinari et al., 2008), and respond to a variety of endogenous metabolites. As an example, fatty acids activate PPAR-α through a ligand-induced conformational structure change. Down regulation of Cyp4a10 and Cyp4a1, and up regulation of Cyp4a12 indicates changes in fatty acid levels and PPAR-α modulation by the triazoles (Gonzalez and Shah, 2008). Triazoles affected multiple fatty acid metabolic genes, such as Acsl3 and Acsl5, which encode enzymes residing in different subcellular locations and regulating different steps in the metabolic pathway (Lewin et al., 2001). Overall, there appeared to be a shift from insulin-stimulated glucose metabolism and fatty acid synthesis and storage (lipogenesis), over to fatty acid oxidation—similar to what has been reported for PPAR-α agonists (Xu et al., 2006). The triazoles also seemed to modulate LXR regulated genes and pathways, the up regulation of Alas1, Ces2, and Scd1 expression caused by triazoles indicated oxysterol activation of LXRα (Chu et al., 2006) and promotion of bile acid biosynthesis and secretion. Constitutive Androstance Receptor The most robust nuclear receptor mediated response to triazoles in the rat liver is the induction of Cyp2b2, which is regulated by CAR (Wei et al., 2000). Cyp2b2 was the one gene differentially expressed in all the triazole treatment groups in the rat liver. Expression levels of Cyp2b, like Cyp4a, are increased by ketone bodies and fatty acids, and decrease following mitochondrial β-oxidation due to decreased intercellular fatty acids. Cyp2b2 catalyzes the oxidation of testosterone, arachidonic acid, lauric acid, and numerous environmental agents. Of the five Cyp2b isoforms assessed by microarray (2b2, 2b3, 2b13, 2b15) or PCR (2b1 and 2b2) in the present study, only Cyp2b2 was clearly and strongly induced by all three triazoles. The biological functions of Cyp2b3 and Cyp2b13 have not been determined; however Cyp2b3 is not phenobarbital inducible (Jean et al., 1994). Cyp2b2 and Cyp2b15 are both induced by phenobarbital and regulated by CAR, yet only the array probe set designed for Cyp2b2 was consistently positive. PCR confirmed that Cyp2b2 was highly induced by the triazoles, and the sensitivity of Cyp2b2 to xenobiotics, elevated levels of testosterone and fatty acids suggests multiple functions across several metabolic pathways. It should be noted that triazoles may not be metabolized by rat Cyp2b1 or human CYP2B6 (Barton et al., 2006). However, Barton et al. did not test rat Cyp2b2 metabolism of triazoles to determine if it might be directly involved in triazole biotransformation. It is likely that Cyp2b2 ability to metabolize androgens and its impact on related metabolic pathways in the liver is more critical to understanding triazole reproductive and hepatic toxicity. CAR regulates multiple metabolic enzyme and transporter genes modulated by triazoles, including Alas1, Cyp1a1, Cyp2b2, Lss, Abcc3, Slco1a4, Pcx, and Ugt1a1. Results for these and other genes are consistent with CAR activation by triazoles, demonstrating the multifunctionality of this receptor and a direct or indirect responsiveness to triazoles. Other CAR activators, like triazoles, also induce hepatomegaly, hepatocyte hypertrophy, and induction of CYP and other xenobiotic metabolizing enzymes in rodent liver. In the case of this triazole study in rats, repeated exposures to triazoles also led to disruption of steroid homeostasis and infertility. Chronic, high-dose exposures to these and other triazoles can also lead to hepatic tumors and carcinogenesis in rodents, similar to what has been reported for other CAR activators (Huang et al., 2005). The mode of action behind these CAR mediated tumor and cancer outcomes appears to be increased cell proliferation and suppression of apoptosis (Huang et al., 2005). Up regulation of cell growth and antiapoptotic genes, as well as well established CAR regulated genes such as Cyp2b2, following the triazole treatments in this study suggest activation of rat CAR is a key event in the observed hepatomegaly and other hepatotoxicity reported (Goetz et al., 2007). Using CAR knockout mice, Yamamoto et al. (2004) have demonstrated that CAR is essential for at least some cases of mouse hepatotoxicity and tumor formation, and additional studies have proven these CAR-dependent mechanisms relevant to at least cyproconazole (Peffer et al., 2007). It is worth noting that the antiapoptotic gene Gadd45b, which was up regulated by triadimefon in the present study, has recently been reported as a CAR coactivator (Yamamoto and Negishi, 2008). It appears that triazole modulation of Gadd45b is dependent on CAR (Peffer et al., 2007). Pregnane X Receptor Activation of genes regulated by PXR was not as robust as the effects on CAR genes, but it was another common effect of triazoles in rat liver. Cyp3a1 and Cyp3a2, Aldh1a1, as well as some of the genes coregulated by CAR or other nuclear receptors were induced by the triazoles. Rat Cyp3a1 and Cyp3a2 both metabolize myclobutanil, and perhaps triadimefon also (Barton et al., 2006). Human CYP3A4 appears to do the same, so this PXR mediated induction of Cyp3ais likely enhancing triazole biotransformation. However, because of the many ligands shared by PXR and CAR (Moore et al., 2000), overlapping regulation of hepatic metabolism by these receptors (Maglich et al., 2002; Tien and Negishi, 2006), and the promiscuity of PXR binding to many xenobiotics (Orans et al., 2005), it was difficult to determine the specificity and significance of PXR in the liver following triazole exposure. PXR regulates numerous metabolic pathways and genes, including several altered by triazoles in the present study. Results with transgenic mice have provided evidence that PXR response to xenobiotics is an important determinant in both rodent and human hepatocarcinogenesis (Ma et al., 2007). Future studies will need to clearly distinguish the functions of PXR from those of CAR and other nuclear receptors in regulating genes and pathways. These studies will be crucial in defining triazole modes of action relative to hepatic and reproductive toxicity, and determining the relevance of these mechanistic insights to human health risk. Hepatic Steroid Metabolism Many of the genes in the steroid metabolism pathway perturbed by triazoles in the present study are regulated by CAR and PXR. This genomic response indicated an attempt by the rat liver to respond to increased serum testosterone levels following long term and relatively high-dose exposures to triazoles. Many of these triazole-induced changes are likely to have altered metabolism and excretion of steroids and xenobiotics. As an example, changes in Hsd17b were likely to be an adaptive response to the elevated circulating testosterone seen with all three triazoles; part of a hepatic attempt to regain steroid homeostasis (Mustonen et al., 1997). In contrast, down regulation of Srd5a1, which helps eliminate excess androgens and is typically positively regulated by testosterone and dihydrotestosterone (Torres and Ortega, 2003), appears to be a maladaptative response to triazole exposure. Conclusions The molecular events measured in this toxicogenomic study demonstrate that myclobutanil, propiconazole, and triadimefon perturb common biological pathways, many of which are regulated by nuclear receptors. By defining the common changes in gene expression and their associated biological pathways, strong inferences can be made about the causative factors leading up to a disruption in testosterone homeostasis and associated reproductive toxicity of triazoles: triazoles increased fatty acid catabolism, reduced bile acid biosynthesis, induced cholesterol biosynthesis, and impaired steroid metabolism. The induction of CAR and PXR nuclear receptors drove many of these changes in gene expression, and subsequent changes in fatty acid, steroid and xenobiotic metabolism. It is likely that modulation of CAR and PXR by the triazoles in this long term exposure study led to the observed hepatomegaly. The observed disruption in testosterone homeostasis by triazoles was not due to modulation of steroidogenic genes in the testis. Instead, there appeared to be disruption of normal hepatic testosterone metabolism, leading to increased expression of genes in the steroid metabolism and sterol biosynthesis pathways as an adaptive response. For reasons not currently understood, negative feedback mechanisms in the hypothalamus-pituitary-gonadal axis did not compensate for these increases in circulating steroids, and changes in hepatic metabolism were not able to maintain steroid homeostasis. In this rat model where exposure started gestationally and continued to adulthood, disruption of systemic steroid homeostasis was accompanied by reproductive toxicity and infertility. The gene expression profiles in this study have provided strong support for a mode of action for reproductive and hepatic toxicity, mediated through the CAR and PXR signaling pathways that is common to the triazole antifungals. FUNDING EPA/North Carolina State University Cooperative Training Agreement (#CT826512010) supported A.K.G. We thank Drs Hongzu Ren and Indira Thillainadarajah (EPA) for excellent technical support. We also thank Dr Douglas Wolf (EPA) for technical review of this manuscript. Microarrays and reagents for a portion of this study were provided by Affymetrix as part of a Materials Cooperative Research and Development Agreement with EPA. References Allen JW , Wolf DC , George MH , Hester SD , Sun G , Thai S-F , Delker DA , Moore T , Jones C , Nelson G , et al. Toxicity profiles in mice treated with hepatotumorigenic and non-hepatotumorigenic triazole conazoles fungicides: Propiconazole, triadimefon, and myclobutanil , Toxicol. Pathol. , 2006 , vol. 34 (pg. 853 - 862 ) Google Scholar Crossref Search ADS PubMed WorldCat Baldan A , Tarr P , Lee R , Edwards PA . ATP-binding cassette transporter G1 and lipid homeostasis , Curr. Opin. Lipidol. , 2006 , vol. 17 (pg. 227 - 232 ) Google Scholar Crossref Search ADS PubMed WorldCat Barton HA , Tang J , Sey YM , Stanko JP , Murrell RN , Rockett JC , Dix DJ . Metabolism of myclobutanil and triadimefon by human and rat cytochrome P450 enzymes and liver microsomes , Xenobiotica , 2006 , vol. 36 (pg. 793 - 806 ) Google Scholar Crossref Search ADS PubMed WorldCat Chen P-J , Padgett WT , Moore T , Winnik W , Lambert GR , Thai S-F , Hester SD , Nesnow S . Three conazoles increase hepatic microsomal retinoic acid metabolism and decrease mouse hepatic retinoic acid levels in vivo , Toxicol. Appl. Pharmacol. , 2009 , vol. 234 (pg. 143 - 155 ) Google Scholar Crossref Search ADS PubMed WorldCat Chu K , Miyazaki M , Man WC , Ntambi JM . Stearoyl-coenzyme A desaturase 1 deficiency protects against hypertriglyceridemia and increases plasma high-density lipoprotein cholesterol induced by liver X receptor activation , Mol. Cell Biol. , 2006 , vol. 26 (pg. 6786 - 6798 ) Google Scholar Crossref Search ADS PubMed WorldCat Coleman RS , Lewin TM , Muoio DM . Physiological and nutritional regulation of enzymes of triacylglycerol synthesis , Annu. Rev. Nutr. , 2000 , vol. 20 (pg. 77 - 103 ) Google Scholar Crossref Search ADS PubMed WorldCat Dixit SG , Tirona RG , Kim RB . Beyond CAR and PXR , Curr. Drug Metab. , 2005 , vol. 6 (pg. 385 - 397 ) Google Scholar Crossref Search ADS PubMed WorldCat Ghannoum MA , Rice LB . Antifungal agents: Mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance , Clin. Microbiol. Rev. , 1999 , vol. 12 (pg. 501 - 517 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Goetz AK , Bao W , Ren H , Schmid JE , Tully DB , Wood C , Rockett JC , Narotsky MG , Sun G , Lambert GR , et al. Gene expression profiling in the liver of CD-1 mice to characterize the hepatotoxicity of triazole fungicides , Toxicol. Appl. Pharmacol. , 2006 , vol. 215 (pg. 274 - 284 ) Google Scholar Crossref Search ADS PubMed WorldCat Goetz AK , Ren H , Schmid JE , Blystone CR , Thillainadarajah I , Best DS , Nichols HP , Strader LF , Wolf DC , Narotsky MG , et al. Disruption of testosterone homeostasis as a mode of action for the reproductive toxicity of triazole fungicides in the male rat , Toxicol. Sci. , 2007 , vol. 95 (pg. 227 - 239 ) Google Scholar Crossref Search ADS PubMed WorldCat Gonzalez FJ , Shah YM . PPARa: Mechanism of species differences and hepatocarcinogenesis of peroxisome proliferators , Toxicology , 2008 , vol. 246 (pg. 2 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Guzelian J , Barwick JL , Hunter L , Phang TL , Quattrochi LC , Guxelian PS . Identification of genes controlled by the pregnane X receptor by microarray analysis of mRNAs from pregnenolone 16alpha-carbonitrole-treated rats , Toxicol. Sci. , 2006 , vol. 94 (pg. 379 - 387 ) Google Scholar Crossref Search ADS PubMed WorldCat Hester SD , Nesnow S . Transcriptional responses in thyroid tissues from rats treated with a tumorigenic and a non-tumorigenic conazoles fungicide , Toxicol. Appl. Pharmacol. , 2008 , vol. 227 (pg. 357 - 369 ) Google Scholar Crossref Search ADS PubMed WorldCat Hester SD , Wolf DC , Nesnow S , Thai SF . Transcriptional profiles in liver from rats treated with tumorigenic and non-tumorigenic triazoles conazole fungicides: Propiconazole, triadimefon, and myclobutanil , Toxicol. Pathol. , 2006 , vol. 34 (pg. 879 - 894 ) Google Scholar Crossref Search ADS PubMed WorldCat Honkakoski P , Negishi M . Regulation of cytochrome P450 (CYP) genes by nuclear receptors , Biochem. J. , 2000 , vol. 347 (pg. 321 - 337 ) Google Scholar Crossref Search ADS PubMed WorldCat Huang W , Zhang J , Washington M , Liu J , Parant JM , Lozano G , Moore DD . Xenobiotic stress induces hepatomegaly and liver tumors via the nuclear receptor constitutive androstane receptor , Mol. Endocrinol. , 2005 , vol. 19 (pg. 1646 - 1653 ) Google Scholar Crossref Search ADS PubMed WorldCat Jean A , Reiss A , Desrochers M , Dubois S , Trottier E , Trottier Y , Wirtanen L , Adesnik M , Waxman DJ , Anderson A . Rat liver cytochrome P450 2B3: Structure of the CYP2B3 gene and immunological identification of a constitutive P450 2B3-like protein in rat liver , DNA Cell Biol. , 1994 , vol. 13 (pg. 781 - 792 ) Google Scholar Crossref Search ADS PubMed WorldCat Jigorel E , Le Vee M , Boursier-Neyret C , Bertrand M , Fardel O . Functional expression of sinusoidal drug transporters in primary human and rat hepatocytes , Drug Metab. Dispos. , 2005 , vol. 33 (pg. 1418 - 1422 ) Google Scholar Crossref Search ADS PubMed WorldCat Jigorel E , Le Vee M , Boursier-Neyret C , Bertrand M , Fardel O . Differential regulation of sinusoidal and canalicular hepatic drug transporter expression by xenobiotics activating drug-sensing receptors in primary human hepatocytes , Drug Metab. Dispos. , 2006 , vol. 34 (pg. 1756 - 1763 ) Google Scholar Crossref Search ADS PubMed WorldCat Kretschmer XC , Baldwin WS . CAR and PXR: Xenosensors of endocrine disrupters? , Chem. Biol. Interact. , 2005 , vol. 155 (pg. 111 - 128 ) Google Scholar Crossref Search ADS PubMed WorldCat Lewin TM , Kim JH , Granger DA , Vance JE , Coleman RA . Acyl-CoA synthetase isoforms 1,4, and 5 are present in different subcellular membranes in rat liver and can be inhibited independently , J. Biol. Chem. , 2001 , vol. 276 (pg. 24674 - 24679 ) Google Scholar Crossref Search ADS PubMed WorldCat Ma X , Shah Y , Cheung C , Guo GL , Feigenbaum L , Krausz KW , Idle JR , Gonzalez FJ . The pregnane X receptor gene-humanized mouse: A model for investigating drug-drug-interactions mediated by cytochromes P450 3A , Drug Metab. Dispos. , 2007 , vol. 35 (pg. 194 - 200 ) Google Scholar Crossref Search ADS PubMed WorldCat Maglich JM , Stoltz CM , Goodwin B , Hawkins-Brown D , Moore JT , Kliewer SA . Nuclear pregnane X receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification , Mol. Pharmacol. , 2002 , vol. 62 (pg. 638 - 646 ) Google Scholar Crossref Search ADS PubMed WorldCat Moore LB , Parks DJ , Jones SA , Bledsoe RK , Consler TG , Stimmel JB , Goodwin B , Liddle C , Blanchard SG , Willson TM , et al. Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands , J. Biol. Chem. , 2000 , vol. 275 (pg. 15122 - 15127 ) Google Scholar Crossref Search ADS PubMed WorldCat Mustonen MVJ , Poutanen MH , Isomaa VV , Vihko RK . Cloning of mouse 17β-hydroxysteroid dehydrogenase type 2, and analyzing expression of the mRNAs for types 1, 2, 3, 4 and 5 in mouse embryos and adult tissues , J. Biochem. , 1997 , vol. 325 (pg. 199 - 205 ) Google Scholar Crossref Search ADS WorldCat Nordlie RC , Foster JD . Regulation of glucose production by the liver , Annu. Rev. Nutr. , 1999 , vol. 19 (pg. 379 - 406 ) Google Scholar Crossref Search ADS PubMed WorldCat Orans J , Teotico DG , Redinbo MR . The nuclear xenobiotic receptor pregnane X receptor: Recent insights and new challenges , Mol. Endocrinol. , 2005 , vol. 19 (pg. 2891 - 2900 ) Google Scholar Crossref Search ADS PubMed WorldCat Pandak WM , Hylemon PB , Ren S , Marques D , Gil G , Redford K , Mallonee D , Valhcevic ZR . Regulation of oxysterol 7alpha-hydroxylase (CYP7B1) in primary cultures of rat hepatocytes , Hepatology , 2002 , vol. 35 (pg. 1400 - 1408 ) Google Scholar Crossref Search ADS PubMed WorldCat Peffer RC , Moggs JG , Pastoor T , Currie RA , Wright J , Milburn G , Waechter F , Rusyn I . Mouse liver effects of cyproconazole, a triazole fungicide: Role of the constitutive androstane receptor , Toxicol. Sci. , 2007 , vol. 99 (pg. 315 - 325 ) Google Scholar Crossref Search ADS PubMed WorldCat Russell DW . Nuclear orphan receptors control cholesterol catabolism , Cell , 1999 , vol. 97 (pg. 539 - 542 ) Google Scholar Crossref Search ADS PubMed WorldCat Schwarz M , Russell DW , Dietschy JM , Turley SD . Alternate pathways of bile acid synthesis in the cholesterol 7alpha-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding , J. Lipid Res. , 2001 , vol. 42 (pg. 1594 - 1603 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Shenoy SD , Spencer TA , Mercer-Haines NA , Alipour M , Gargano MD , Runge-Morris M , Kocarek TA . CYP3A Induction by liver X receptor ligands in primary cultured rat and mouse hepatocytes is mediated by the pregnane X receptor , Drug Metab. Dispos. , 2004 , vol. 32 (pg. 66 - 71 ) Google Scholar Crossref Search ADS PubMed WorldCat Shibata N , Arita M , Misaki Y , Dohmae N , Takio K , Ono T , Inoue K , Arai H . Supernatant protein factor, which stimulates the conversion of squalene to lanosterol, is a cytosolic squalene transfer protein and enhances cholesterol biosynthesis , Proc. Natl. Acad. Sci. U. S. A. , 2001 , vol. 98 (pg. 2244 - 2249 ) Google Scholar Crossref Search ADS PubMed WorldCat Staudinger J , Liu Y , Madan A , Habeebu S , Klaassen CD . Coordinate regulation of xenobiotic and bile acid homeostasis by pregnane X receptor , Drug Metab. Dispos. , 2001 , vol. 29 (pg. 1467 - 1472 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Sul HS , Wang D . Nutritional and hormonal regulation of enzymes in fat synthesis: Studies of fatty acid synthase and mitochondrial glycerol-3-phosphate acyltransferase gene transcription , Annu. Rev. Nutr. , 1998 , vol. 18 (pg. 331 - 351 ) Google Scholar Crossref Search ADS PubMed WorldCat Sun G , Grindstaff RD , Thai SF , Lambert GR , Tully DB , Dix DJ , Nesnow S . Induction of cytochrome P450 enzymes in rat liver by two conazoles, myclobutanil and triadimefon , Xenobiotica , 2007 , vol. 37 (pg. 180 - 193 ) Google Scholar Crossref Search ADS PubMed WorldCat Tansey TR , Shechter I . Structure and regulation of mammalian squalene synthase , Biochim. Biophys. Acta , 2000 , vol. 1529 (pg. 49 - 62 ) Google Scholar Crossref Search ADS PubMed WorldCat Tien ES , Negishi M . Nuclear receptors CAR and PXR in the regulation of hepatic metabolism , Xenobiotica , 2006 , vol. 36 (pg. 1152 - 1163 ) Google Scholar Crossref Search ADS PubMed WorldCat Torres JM , Ortega E . Precise quantitation of 5α-reductase type 1 mRNA by RT-PCR in rat liver and its positive regulation by testosterone and dihydrotestosterone , Biochem. Biophys. Res. Commun. , 2003 , vol. 308 (pg. 469 - 473 ) Google Scholar Crossref Search ADS PubMed WorldCat Tully DB , Bao W , Goetz AK , Blystone CR , Ren H , Schmid JE , Strader LF , Wood CR , Best DS , Narotsky MG , et al. Gene expression profiling in liver and testis of rats to characterize the toxicity of triazole fungicides , Toxicol. Appl. Pharmacol. , 2006 , vol. 215 (pg. 260 - 273 ) Google Scholar Crossref Search ADS PubMed WorldCat U.S. Environmental Protection Agency, Office of Prevention, Pesticides and Toxic Substances Myclobutanil; Pesticide tolerances , Fed. Regist. , 1995 , vol. 60 (pg. 40500 - 40503 ) OpenURL Placeholder Text WorldCat U.S. Environmental Protection Agency, Office of Prevention, Pesticides and Toxic Substances , Carcinogenicity Peer Review of Bayleton , 1996 Washington, DC U.S. EPA Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC U.S. Environmental Protection Agency, Office of Prevention, Pesticides and Toxic Substances Myclobutanil, pesticide tolerance for emergency exemptions , Fed. Regist. , 2001 , vol. 66 (pg. 298 - 306 ) OpenURL Placeholder Text WorldCat U.S. Environmental Protection Agency, Office of Prevention, Pesticides and Toxic Substances Myclobutanil; pesticide tolerances for emergency exemptions , Fed. Regist. , 2005a , vol. 70 (pg. 49499 - 49507 ) OpenURL Placeholder Text WorldCat U.S. Environmental Protection Agency, Office of Prevention, Pesticides and Toxic Substances Propiconazole; pesticide tolerances for emergency exemptions , Fed. Regist. , 2005b , vol. 70 (pg. 43284 - 43292 ) OpenURL Placeholder Text WorldCat U.S. Environmental Protection Agency, Office of Prevention, Pesticides and Toxic Substances , Memorandum. Triadimefon: Occupational and Residential Exposure Assessment for the Registration Eligibility Decision Document. PC Code 109901, DP Barcode 314814, 315040 , 2005c Washington, DC Office of Prevention, Pesticides, and Toxic Substances Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC U.S. Environmental Protection Agency, Office of Prevention, Pesticides and Toxic Substances , Triadimefon. Preliminary Human Health Risk Assessment revised , 2006 Washington, DC Office of Prevention, Pesticides, and Toxic Substances Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Vanden Bossche H , Marichal P , Gorrens J , Coene M-C . Biochemical basis for the activity and selectivity of oral antifungal drugs , Br. J. Clin. Pract. Suppl. , 1990 , vol. 71 (pg. 41 - 46 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Wang XJ , Chamberlain M , Vassieva O , Henderson CJ , Wolf CR . Relationship between hepatic phenotype and changes in gene expression in cytochrome P450 reductase (POR) null mice , Biochem. J. , 2005 , vol. 388 (pg. 857 - 867 ) Google Scholar Crossref Search ADS PubMed WorldCat Ward WO , Delker DA , Hester SD , Thai S-F , Wolf DC , Allen JW , Nesnow S . Transcriptional profiles in liver from mice treated with hepatotumorigenic and nonhepatotumorigenic triazole conazoles fungicides: Propiconazole, triadimefon, and myclobutanil , Toxicol. Pathol. , 2006 , vol. 34 (pg. 863 - 878 ) Google Scholar Crossref Search ADS PubMed WorldCat Wei P , Zhang J , Egan-Hafley M , Liang S , Moore DD . The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism , Nature , 2000 , vol. 407 (pg. 920 - 923 ) Google Scholar Crossref Search ADS PubMed WorldCat Wei P , Zhang J , Dowhan DH , Han Y , Moore DD . Specific and overlapping functions of the nuclear hormone receptors CAR and PXR in xenobiotic response , Pharmacogenomics J. , 2002 , vol. 2 (pg. 117 - 126 ) Google Scholar Crossref Search ADS PubMed WorldCat Wolf DC , Allen JW , George MH , Hester SD , Sun G , Moore T , Thai SF , Delker D , Winkfield E , Leavitt S , et al. Toxicity profiles in rats treated with tumorigenic and nontumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil , Toxicol. Pathol. , 2006 , vol. 34 (pg. 895 - 902 ) Google Scholar Crossref Search ADS PubMed WorldCat Xu J , Christian B , Jump DB . Regulation of rat hepatic L-pyruvate kinase promoter composition and activity by glucose, n-3 polyunsaturated fatty acids, and peroxisome proliferator-activated receptor-α agonist , J. Biol. Chem. , 2006 , vol. 281 (pg. 18351 - 18362 ) Google Scholar Crossref Search ADS PubMed WorldCat Yamamoto Y , Moore R , Goldsworthy TL , Negishi M , Maronpot RR . The orphan nuclear receptor constitutive active/androstane receptor is essential for liver tumor promotion by phenobarbital in mice , Cancer Res. , 2004 , vol. 64 (pg. 7197 - 7200 ) Google Scholar Crossref Search ADS PubMed WorldCat Yamamoto Y , Negishi M . The antiapoptotic factor growth arrest and DNA-damage-inducible 45 beta regulates the nuclear receptor constitutive active/androstane receptor-mediated transcription , Drug Metab. Dispos. , 2008 , vol. 36 (pg. 1189 - 1193 ) Google Scholar Crossref Search ADS PubMed WorldCat Yoshikawa T , Ide T , Shimano H , Yahagi N , Amemiya-Kudo M , Matsuzaka T , Yatoh S , Kitamine T , Okazaki H , Tamura Y , et al. Cross-talk between peroxisome proliferator-activated receptor (PPAR) α and liver x receptor (LXR) in nutritional regulation of fatty acid metabolism. I. PPARs suppress sterol regulatory element binding protein-1c promoter through inhibition of LXR signaling , Mol. Endocrinol. , 2003 , vol. 17 (pg. 1240 - 1254 ) Google Scholar Crossref Search ADS PubMed WorldCat Yoshinari K , Tien E , Negishi M , Honkakoski P . Ioannides C . Receptor-mediated regulation of cytochromes P450 , Cytochromes P450: Role in the Metabolism and Toxicity of Drugs and Other Xenobiotics , 2008 Cambridge RSC Publishing (pg. 417 - 448 ) Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC You L . Steroid hormone biotransformation and xenobiotic induction of hepatic steroid metabolizing enzymes , Chem. Biol. Interact. , 2004 , vol. 147 (pg. 233 - 246 ) Google Scholar Crossref Search ADS PubMed WorldCat Author notes Disclaimer: The United States Environmental Protection Agency through its Office of Research and Development funded and managed the research described here. It has been subjected to Agency administrative review and approved for publication. Published by Oxford University Press 2009.
TI - Mode of Action for Reproductive and Hepatic Toxicity Inferred from a Genomic Study of Triazole Antifungals
JO - Toxicological Sciences
DO - 10.1093/toxsci/kfp098
DA - 2009-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/mode-of-action-for-reproductive-and-hepatic-toxicity-inferred-from-a-xJAd60v00i
SP - 449
EP - 462
VL - 110
IS - 2
DP - DeepDyve
ER -