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 (GD6) to postnatal day 92 (PND92). 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. While 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 (PPARalpha), 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.

The liver- and intestine-enriched carboxylesterase 2 (CES2) enzyme catalyzes the hydrolysis of several clinically important anti-cancer agents administered as pro-drugs. For example, irinotecan, a carbamate prodrug used in the treatment of colorectal cancer, is biotransformed in vivo by CES2 in intestine and liver, thereby producing a potent topoisomerase I inhibitor. Pregnane x receptor (PXR) and constitutive androstane receptor (CAR), two members of the nuclear receptor superfamily of ligand-activated transcription factors, mediate gene activation in response to xenobiotic stress. Together, these receptors comprise a protective response in mammals that coordinately regulate hepatic transport, metabolism, and elimination of numerous xenobiotic compounds. In the present study, microarray analysis was used to identify PXR-target genes in duodenum in mice. Here, we show that a gene encoding a member of the CES2 sub-type of liver- and intestine-enriched carboxylesterase enzymes, called Ces6, is induced following treatment with pregnenalone 16{alpha}-carbonitrile (PCN) in a PXR-dependent manner in duodenum and liver in mice. Treatment of mice with the CAR activator 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) also induced expression of Ces6 in duodenum and liver in a CAR-dependent manner, while treatment with phenobarbital (PB) produced induction of Ces6 exclusively in liver. These data identify a key role for PXR and CAR in regulating the drug-inducible expression and activity of an important carboxylesterase enzyme in vivo. Future studies should focus on determining whether these signaling pathways governing drug-inducible carboxylesterase expression in intestine and liver are conserved in humans.

Triadimefon, propiconazole and myclobutanil are conazoles, an important class of agricultural and therapeutic fungicides. Triadimefon and propiconazole are mouse liver tumorigens, while myclobutanil is not. All three conazoles are generally inactive in short-term genotoxicity tests. We studied the in vivo mutagenicity of these three conazoles using the Big Blue(R) mouse assay system. Groups of mice were fed either control diet or diet containing 1800 p.p.m. triadimefon, 2500 p.p.m. propiconazole or 2000 p.p.m. myclobutanil. After 4 days of feeding, mice were immediately euthanized, livers were removed, DNA isolated and lacI genes recovered into infectious bacteriophage lambda particles by in vitro packaging. Bacteriophage with mutations in the lacI gene was detected by infecting into Escherichia coli, and mutant frequencies were determined using a colorimetric plaque assay. Propiconazole induced a 1.97-fold increase in mutant frequency compared to concurrent controls (P=0.018) and triadimefon induced a 1.94-fold increase compared to concurrent controls (P=0.009). Myclobutanil did not induce any change in mutant frequency (P=0.548). These results provide the first evidence that the hepatotumorigenic conazoles are capable of inducing mutations in liver in vivo while the non-tumorigen myclobutanil is not, suggesting that mutagenicity may represent a key event in conazoles tumorigenic mode of action.

Ciguatoxins (CTX) are polyether neurotoxins that target voltage gated sodium channels and are responsible for ciguatera, the most common fish-borne food poisoning in humans. This study characterizes the global transcriptional response of mouse liver to a symptomatic dose (0.26 ng/g) of the highly potent Pacific ciguatoxin-1 (P-CTX-1). At 1 h post exposure 2.4% of features on a 44K whole genome array were differentially expressed (p </= 0.0001), increasing to 5.2% at 4 h and decreasing to 1.4% by 24 h post-CTX exposure. Data were filtered (|fold change|>/= 1.5 and p </= 0.0001 in at least one time point) and a trend set of 1550 genes were used for further analysis. Early gene expression was likely influenced prominently by an acute 4 degrees C decline in core body temperature by 1 h, which resolved by 8 h following exposure. An initial down-regulation of 32 different solute carriers, many involved in sodium transport, was observed. Differential gene expression in pathways involving eicosanoid biosynthesis and cholesterol homeostasis was also noted. Cytochrome P450s were of particular interest due to their role in xenobiotic metabolism. Twenty-seven genes, mostly members of Cyp2 and Cyp4 families, showed significant changes in expression. Many Cyps underwent an initial down-regulation at 1 h but were quickly and strongly up-regulated at 4 and 24 h post exposure. In addition to Cyps, increases in several glutathione S-transferases were observed, an indication that both phase I and phase II metabolic reactions are involved in the hepatic response to CTX in mice.

Cyproconazole, a triazole fungicide, causes hepatocellular adenomas and carcinomas in CD-1 mice at dose levels of 100 and 200 ppm. The constitutive androstane receptor (CAR) has been shown to play a significant role in the overall mode of action for several non-genotoxic rodent carcinogens such as phenobarbital. The liver effects of dietary cyproconazole or phenobarbital were investigated after 2, 7 or 14 days in male CD-1, C57BL/6J and C3H/HeNClrBR mice. Cyproconazole produced similar, dose-responsive effects in all three strains of mice, and the response was similar to that of phenobarbital. Subsequently, Car-null and wild type male mice on a C3H/HeNClrBR background were administered 200 ppm or 450 ppm cyproconazole, or 850 ppm phenobarbital for up to 7 days. In wild type mice, 200 ppm cyproconazole caused liver hypertrophy, increased liver weight and cell proliferation, single cell necrosis and fat vacuolation, effects generally similar to those caused by 850 ppm phenobarbital. Plasma cholesterol was decreased by both compounds, but cyproconazole had a greater effect. The higher dose (450 ppm) of cyproconazole caused similar changes, but greater evidence of liver damage was observed, including a large increase in plasma transaminases. Induction of CAR-target genes Cyp2b10 and Gadd45beta was observed with both compounds, whereas the cell cycle regulatory gene Mdm2 was unaffected. In Car-null mice, the effects noted with either cyproconazole or phenobarbital were absent or greatly diminished. These experiments demonstrate that short-term liver effects of cyproconazole in mice are CAR-dependent and similar to those of phenobarbital, a known non-genotoxic rodent liver carcinogen.

This study was undertaken to examine the inductive effects of two triazole antifungal agents, myclobutanil and triadimefon, on the expression of hepatic cytochrome P450 (CYP) genes and on the activities of CYP enzymes in male Sprague Dawley rats. Rats were dosed with the conazoles at three dose levels by gavage for 14 days: myclobutanil (150, 75, and 10mgkg(-1) body weight day(-1); triadimefon (115, 50, and 10 mg kg(-1) body weight day-'), which included their maximum tolerated dose levels (MTD). Both myclobutanil and triadimefon significantly induced pentoxyresorufin O-depentylase activities at their MTD levels: myclobutanil, 8.1-fold at 150mgkg(-1) body weight day-; and triadimefon, 18.5-fold at 115mgkg(-1) body weight day-'. Benzyloxyresorufin O-debenzylase activities were similarly increased: myclobutanil, 13.3-fold; triadimefon, 27.7-fold. Quantitative real-time reverse-transcription polymerase chain reaction assays were used to characterize the mRNA expression of specific CYP genes induced by these two conazoles. Myclobutanil and triadimefon treatment at their MTD levels significantly increased rat hepatic mRNA expression of CYP2B1 (14.3- and 54.6-fold), CYP3A23/3A1 (2.2- and 7.3-fold), and CYP3A2 (1.5- and 1.7-fold). Western immunoblots of rat hepatic microsomal proteins identified significantly increased levels of CYP isoforms after myclobutanil or triadimefon treatment at their MTD levels: CYP2BI/2 (4.8- and 5.3-fold), and CYP3A1 (2.2- and 2.9-fold). Triadimefon also increased CYP3A2 immunoreactive protein levels 1.8-fold. These results indicate that triadimefon and myclobutanil, like other triazole-containing conazoles, induced CYP2B and CYP3A families of cytochromes in rat liver.

Conazoles are environmental and pharmaceutical fungicides. The present study relates the toxicological effects of conazoles to alterations of gene and pathway transcription and identifies potential modes of tumorigenic action. In a companion study employing conventional toxicological bioassays (Allen et al., 2006), male CD-1 mice were fed triadimefon, propiconazole, or myclobutanil in a continuous oral-dose regimen for 4, 30, or 90 days. These conazoles were found to induce hepatomegaly, to induce high levels of hepatic pentoxyresorufin-O-dealkylase activity, to increase hepatic cell proliferation, to decrease serum cholesterol, and to increase serum triglycerides. Differentially expressed genes and pathways were identified using Affymetrix GeneChips. Gene-pathway associations were obtained from the Kyoto Encyclopedia of Genes and Genomes, Biocarta, and MetaCore compendia. The pathway profiles of each conazole were different at each time point. In general, the number of altered metabolism, signaling, and growth pathways increased with time and dose and were greatest with propiconazole. All conazoles had effects on nuclear receptors as evidenced by increased expression and enzymatic activities of a series of related cytochrome P450s (CYP). A subset of altered genes and pathways distinguished the three conazoles from each other. Triadimefon and propiconazole both altered apoptosis, cell cycle, adherens junction, calcium signaling, and EGFR signaling pathways. Triadimefon produced greater changes in cholesterol biosynthesis and retinoic acid metabolism genes and in selected signaling pathways. Propiconazole had greater effects on genes responding to oxidative stress and on the IGF/P13K/AKt/PTEN/mTor and Wnt-beta-catenin pathways. In conclusion, while triadimefon, propiconazole, and myclobutanil had similar effects in mouse liver on hepatomegaly, histology, CYP activities, cell proliferation, and serum cholesterol, genomic analyses revealed major differences in their gene expression profiles.

Triazole fungicides associated with a range of reported male reproductive effects in experimental animals were selected to assess potential toxic modes of action. Wistar Han rats were fed myclobutanil (100, 500, or 2000 ppm), propiconazole (100, 500, or 2500 ppm), or triadimefon (T: 100, 500, or 1800 ppm) from gestation day 6 to postnatal day (PND) 120. One male per litter was necropsied on PND1, 22, 50, or 92. Measurements included anogenital distance (AGD) at PND0, body and organ weights, serum hormone levels, age at preputial separation (PPS), sperm morphology and motility, and fertility and fecundity. AGD was increased by the high dose of all three triazoles, indicating hypervirilization. Triadimefon delayed PPS, consistent with delayed puberty, at 1800 ppm. Relative liver weights were increased at PND1, 50, and 92 by all three triazoles. Hepatocellular hypertrophy was present at PND50 from propiconazole and triadimefon and at PND92 from all three high dose triazole treatments. Relative pituitary weights were decreased at PND92 by middle and high dose myclobutanil treatment. Absolute testis weights were increased at PND1 by myclobutanil, at PND22 by myclobutanil and triadimefon, and at PND50 by propiconazole and triadimefon treatment. Relative ventral prostate weights were increased at PND92 by myclobutanil and triadimefon treatment. Serum testosterone was increased at PND50 by triadimefon and at PND92/99 by all three triazole treatments. Insemination and fertility were impaired by myclobutanil and triadimefon treatment. In addition to the reproductive system effects, total serum thyroxine levels were decreased at PND92 by high dose triadimefon. These reproductive effects are consistent with the disruption of testosterone homeostasis as a key event in the mode of action for triazole-induced reproductive toxicity.

Four triazole fungicides were studied using toxicogenomic techniques to identify potential mechanisms of action. Adult male Sprague-Dawley rats were dosed for 14 days by gavage with fluconazole, myclobutanil, propiconazole, or triadimefon. Following exposure, serum was collected for hormone measurements, and liver and testes were collected for histology, enzyme biochemistry, or gene expression profiling. Body and testis weights were unaffected, but liver weights were significantly increased by all four triazoles, and hepatocytes exhibited centrilobular hypertrophy. Myclobutanil exposure increased serum testosterone and decreased sperm motility, but no treatment-related testis histopathology was observed. We hypothesized that gene expression profiles would identify potential mechanisms of toxicity and used DNA microarrays and quantitative real-time PCR (qPCR) to generate profiles. Triazole fungicides are designed to inhibit fungal cytochrome P450 (CYP) 51 enzyme but can also modulate the expression and function of mammalian CYP genes and enzymes. Triazoles affected the expression of numerous CYP genes in rat liver and testis, including multiple Cyp2c and Cyp3a isoforms as well as other xenobiotic metabolizing enzyme (XME) and transporter genes. For some genes, such as Ces2 and Udpgtr2, all four triazoles had similar effects on expression, suggesting possible common mechanisms of action. Many of these CYP, XME and transporter genes are regulated by xeno-sensing nuclear receptors, and hierarchical clustering of CAR/PXR-regulated genes demonstrated the similarities of toxicogenomic responses in liver between all four triazoles and in testis between myclobutanil and triadimefon. Triazoles also affected expression of multiple genes involved in steroid hormone metabolism in the two tissues. Thus, gene expression profiles helped identify possible toxicological mechanisms of the triazole fungicides.

This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods were used as measures of Cyp enzyme activities. Western analyses identified specific Cyp isoforms. Quantitative real-time reverse-transcription polymerase chain reaction (quantitative real time-RT-PCR) assays were used to quantitate the mRNA expression of specific Cyp genes induced by this conazole. Rats and mice were administered fluconazole 2, 25, or 50mg/kgbw/d by gavage daily for 14 days. In rats, fluconazole treatment (50mg/kgbw/d) significantly induced pentoxyresorufin O-dealkylation (PROD), benzyloxyresorufin O-dealkylation (BROD), and ethoxyresorufin O-dealkylation (EROD) hepatic microsomal activities. Fluconazole treatment significantly increased rat hepatic mRNA expression of CYP2B1 and CYP3A23/3A1 with dose-related responses. The highest dose of fluconazole gave a 128-fold induction of CYP2B1 and a 4.6-fold induction of CYP3A23/3A1 mRNA. CYP3A2 mRNA levels were also overexpressed 5.6-7.2-fold depending on dose. Western immunoblots of rat hepatic microsomal proteins identified Cyp isoforms: CYP1A1, CYP1A2, CYP2B1/2, CYP3A23/3A1, and Cyp3A2 with increased levels of CYP2B1/2 and CYP3A23/3A1 proteins. In mice, fluconazole induced BROD, PROD, EROD, and methoxyresorufin O-dealkylation hepatic microsomal activities after treatment with 25 and 50mg/kgbw/d. Fluconazole increased mouse hepatic mRNA expression of Cyp2b10 (1.9-fold) and Cyp3a11 (2.6-fold) in the 50mg/kgbw/d treatment group. In summary, these results indicated that fluconazole, a triazole-containing conazole, clearly induced CYP2B and CYP3A families of isoforms in rat liver and Cyp2b and Cyp3a families of isoforms in mouse liver.

Propiconazole is a N-substituted triazole used as a fungicide on fruits, grains, seeds, hardwoods, and conifers. In the present study, propiconazole was examined for its effects on the expression of hepatic cytochrome P450 genes and on the activities of P450 enzymes in male Sprague-Dawley rats and male CD-1 mice. Rats and mice were administered propiconazole by gavage daily for 14 days at doses of 10, 75, and 150 mg/kg body weight/day. Quantitative real time RT-PCR assays of rat hepatic RNA samples from animals treated at the 150 mg/kg body weight/day dose revealed significant mRNA overexpression of the following genes compared to control: CYP1A2 (1.62-fold), CYP2B1 (10.8-fold), CYP3A1/CYP3A23 (2.78-fold), and CYP3A2 (1.84-fold). In mouse liver, propiconazole produced mRNA overexpression of Cyp2b10 (2.39-fold) and Cyp3a11 (5.19-fold). mRNA expression of CYP1A1 was not detected in liver tissues from treated or controls animals from either species. Propiconazole significantly induced both pentoxyresorufin O-dealkylation (PROD) and methoxyresorufin O-dealkylation (MROD) activities in both rat and mouse liver at the 150 mg/kg body weight/day and 75 mg/kg body weight/day doses. In summary, these results indicated that propiconazole induced CYP1A2 in rat liver and CYP2B and CYP3A families of isoforms in rat and mouse liver.

Triazole fungicides associated with a range of reported male reproductive effects in experimental animals were selected to assess potential toxic modes of action. Wistar Han rats were fed myclobutanil (100, 500, or 2000 ppm), propiconazole (100, 500, or 2500 ppm), or triadimefon (T: 100, 500, or 1800 ppm) from gestation day 6 to postnatal day (PND) 120. One male per litter was necropsied on PND1, 22, 50, or 92. Measurements included anogenital distance (AGD) at PND0, body and organ weights, serum hormone levels, age at preputial separation (PPS), sperm morphology and motility, and fertility and fecundity. AGD was increased by the high dose of all three triazoles, indicating hypervirilization. Triadimefon delayed PPS, consistent with delayed puberty, at 1800 ppm. Relative liver weights were increased at PND1, 50, and 92 by all three triazoles. Hepatocellular hypertrophy was present at PND50 from propiconazole and triadimefon and at PND92 from all three high dose triazole treatments. Relative pituitary weights were decreased at PND92 by middle and high dose myclobutanil treatment. Absolute testis weights were increased at PND1 by myclobutanil, at PND22 by myclobutanil and triadimefon, and at PND50 by propiconazole and triadimefon treatment. Relative ventral prostate weights were increased at PND92 by myclobutanil and triadimefon treatment. Serum testosterone was increased at PND50 by triadimefon and at PND92/99 by all three triazole treatments. Insemination and fertility were impaired by myclobutanil and triadimefon treatment. In addition to the reproductive system effects, total serum thyroxine levels were decreased at PND92 by high dose triadimefon. These reproductive effects are consistent with the disruption of testosterone homeostasis as a key event in the mode of action for triazole-induced reproductive toxicity.

Three triazole fungicides were evaluated for effects on female rat reproductive development. Rats were exposed via feed to propiconazole (P)(100, 500, or 2500ppm), myclobutanil (M)(100, 500, or 2000ppm), or triadimefon (T)(100, 500, or 1800ppm) from gestation day 6 to postnatal day (PND) 98. Body weight (BW) and anogenital distance (AGD) at PND 0, age and BW at vaginal opening (VO), estrous cyclicity, and body and organ weight at necropsy were measured. BW at PND 0 was unaffected by treatment. AGD was increased by M2000. VO was delayed by M2000 and T1800. Estrous cyclicity was initially disrupted by P500, P2500 and T1800, but later normalized. At PND 99 there was a decrease in BW by T1800, an increase in liver weight by P2500 and T1800, and an increase in ovarian weight by M2000 and T1800. It is concluded that exposure to P, M and T adversely impacted female rodent reproductive development.

Reproductive toxicogenomic studies generate large amounts of toxicological and genomic data. On the toxicology side, a substantial quantity of data accumulates from conventional endpoints such as histology, reproductive physiology and biochemistry. The largest source of genomics data is DNA microarrays, which generate enormous amounts of information in the course of profiling gene expression. Thus, data storage and management become essential and require a more sophisticated system than lab notebooks and electronic spreadsheets. We developed a database for tracking toxicogenomic samples and procedures (TSP 1.0) for our reproductive studies based on the MIAME-Tox guidelines and relational database theory. This database stores the various types of data from both toxicological and genomic assays in a hierarchical fashion. The user-friendly interface provides easy procedures for researchers to add, edit, save, delete, and navigate different records. Finally, TSP facilitates exporting microarray data into public databases.

A byproduct of drinking water disinfection, bromochloroacetic acid (BCA), acts as a reproductive toxicant in rats. To determine if BCA produces similar reproductive toxicity in mice, juvenile and adult C57BL/6 males were exposed to 0, 8, 24, 72 or 216 mg/kg of BCA once daily for 14 days. Five of 12 animals from each dose-group were sacrificed at the end of dosing, and testes, epididymes, and seminal vesicles harvested and weighed. Seven mice from each dose-group (including juvenile-exposed mice, following a 14-week maturation period) were used in a 40-day sequential breeding assay to determine if BCA targets a particular phase of spermatogenesis. No significant effects were observed in mice exposed to BCA as juveniles, and there were no effects on fertility by 14 weeks after dosing. However, effects were observed in adult-exposed mice over the first 10 days after BCA exposure: mean number of litters/male, percentage of litters/female bred, and total number of fetuses/male were all reduced by 72 and 216 mg/kg BCA. These results in adult mice indicate BCA disrupted differentiation of spermatids during dosing and the first 10 days of mating, and are consistent with the spermatid retention and atypical residual bodies observed in animals exposed to 72 and 216 mg/kg BCA. To investigate mechanisms involved, we utilized cDNA microarrays containing 950 testis-expressed genes to profile gene expression from Control and BCA-treated mice. Statistical analyses of microarray results identified 40 well-characterized genes differentially expressed in a dose responsive manner as a result of BCA exposure. Microarray results were supplemented with quantitative real-time PCR and Westerns for several genes and proteins. The 40 genes whose expression was altered by BCA are involved in numerous biological processes including: cell communication and adhesion, cell cycle and cell proliferation, metabolism, signal transduction, stress response, and spermatogenesis and male fertility. Modulated expression of these genes, particularly the 15 expressed in Sertoli cells and spermatids, offers new insights into potential mechanisms of BCA toxicity in the mouse testis.

Environmental chemicals that alter steroid production could interfere with male reproductive development and function. Three agricultural antifungal triazoles that are known to modulate expression of cytochrome P450 (CYP) genes and enzymatic activities were tested for effects on steroidogenesis using rat in vivo (triadimefon), rat in vitro (myclobutanil and triadimefon), and human in vitro (myclobutanil, propiconazole, and triadimefon) model systems. Hormone production was measured in testis organ cultures from untreated adult and neonatal rats, following in vitro exposure to 1, 10, or 100 muM of myclobutanil or triadimefon. Myclobutanil and triadimefon reduced media levels of testosterone by 40-68% in the adult and neonatal testis culture, and altered steroid production in a manner that indicated CYP17-hydroxylase/17,20 lyase (CYP17A1) inhibition at the highest concentration tested. Rat to human comparison was explored using the H295R (human adrenal adenocarcinoma) cell line. Following 48 h exposure to myclobutanil, propiconazole, or triadimefon at 1, 3, 10, 30, or 100 muM, there was an overall decrease in estradiol, progesterone, and testosterone by all three triazoles. These data indicate that myclobutanil, propiconazole, and triadimefon are weak inhibitors of testosterone production in vitro. However, in vivo exposure of rats to triazoles resulted in increased serum and intra-testicular testosterone levels. This discordance could be due to higher concentrations of triazoles tested in vitro, and differences within an in vitro model system lacking hepatic metabolism and neuroendocrine control.

Conazoles are a class of azole based fungicides used in agriculture and as pharmaceutical products. They have a common mode of antifungal action through inhibition of ergosterol biosynthesis. Some members of this class have been shown to be hepatotoxic and will induce mouse hepatocellular tumors and/or rat thyroid follicular cell tumors. The particular mode of toxic and tumorigenic action for these compounds is not known, however it has been proposed that triadimefon-induced rat thyroid tumors arise through the specific mechanism of increased TSH. The present study was designed to identify commonalities of effects across the different conazoles and to determine unique features of the tissue responses that suggest a toxicity pathway and a mode of action for the observed thyroid response for triadimefon. Male Wistar/Han rats were treated with triadimefon (100, 500, 1800 ppm), propiconazole (100, 500, 2500 ppm), or myclobutanil (100, 500, 2000 ppm) in feed for 4, 30, or 90 days. The rats were evaluated for clinical signs, body and liver weight, histopathology of thyroid and liver, hepatic metabolizing enzyme activity, and serum T3, T4, TSH, and cholesterol levels. There was a dose-dependent increase in liver weight but not body weight for all treatments. The indication of cytochrome induction, pentoxyresorufin O-dealkylation (PROD) activity, had a dose-related increase at all time points for all conazoles. Uridine diphopho-glucuronosyl transferase (UDPGT), the T4 metabolizing enzyme measured as glucuronidation of 1-naphthol, was induced to the same extent after 30 and 90 days for all three conazoles. Livers from all high dose treated rats had centrilobular hepatocyte hypertrophy after 4 days, while only triadimefon and propiconazole treated rats had hepatocyte hypertrophy after 30 days, and only triadimefon treated rats had hepatocyte hypertrophy after 90 days. Thyroid follicular cell hypertrophy, increased follicular cell proliferation, and colloid depletion were present only after 30 days in rats treated with the high dose of triadimefon. A dose-dependent decrease in T4 was present after 4 days with all 3 compounds but only the high doses of propiconazole and triadimefon produced decreased T4 after 30 days. T3 was decreased after high-dose triadimefon after 4 days and in a dose-dependent manner for all compounds after 30 days. Thyroid hormone levels did not differ from control values after 90 days and TSH was not increased in any exposure group. A unique pattern of toxic responses was not identified for each conazole and the hypothesized mode of action for triadimefon-induced thyroid gland tumors was not supported by the data.

Conazoles comprise a class of fungicides used in agriculture and as pharmaceutical products. The fungicidal properties of conazoles are due to their inhibition of ergosterol biosynthesis. Certain conazoles are tumorigenic in rodents; both propiconazole and triadimefon are hepatotoxic and hepatotumorigenic in mice, while myclobutanil is not a mouse liver tumorigen. As a component of a large-scale study aimed at determining the mode(s) of action for tumorigenic conazoles, we report the results from comparative evaluations of liver and body weights, liver histopathology, cell proliferation, cytochrome P450 (CYP) activity, and serum cholesterol, high-density lipoprotein and triglyceride levels after exposure to propiconazole, triadimefon, and myclobutanil. Male CD-1 mice were treated in the feed for 4, 30, or 90 days with triadimefon (0, 100, 500, or 1800 ppm), propiconazole (0, 100, 500, or 2500 ppm) or myclobutanil (0, 100, 500, or 2000 ppm). Alkoxyresorufin O-dealkylation (AROD) assays indicated that all 3 chemicals induced similar patterns of dose-related increases in metabolizing enzyme activity. PROD activities exceeded those of MROD, and EROD with propiconazole inducing the highest activities of PROD. Mice had similar patterns of dose-dependent increases in hepatocyte hypertrophy after exposure to the 3 conazoles. High-dose exposures to propiconazole and myclobutanil, but not triadimefon, were associated with early (4 days) increases in cell proliferation. All the chemicals at high doses reduced serum cholesterol and high-density lipoprotein (HDL) levels at 30 days of treatment, while only triadimefon had this effect at 4 days of treatment and only myclobutanil and propiconazole at 90 days of treatment. Overall, the tumorigenic and nontumorigenic conazoles induced similar effects on mouse liver CYP enzyme activities and pathology. There was no specific pattern of tissue responses that could consistently be used to differentiate the tumorigenic conazoles, propiconazole, and triadimefon, from the nontumorigenic myclobutanil. These findings serve to anchor other transcriptional profiling studies aimed at probing differences in key events and modes of action for tumorigenic and nontumorigenic conazoles.

The mouse liver tumorigenic conazole fungicides triadimefon and propiconazole have previously been shown to be in vivo mouse liver mutagens in the Big Blue transgenic mutation assay when administered in feed at tumorigenic doses, whereas the non-tumorigenic conazole myclobutanil was not mutagenic. DNA sequencing of the mutants recovered from each treatment group as well as from animals receiving control diet was conducted to gain additional insight into the mode of action by which tumorigenic conazoles induce mutations. Relative dinucleotide mutabilities (RDMs) were calculated for each possible dinucleotide in each treatment group and then examined by multivariate statistical analysis techniques. Unsupervised hierarchical clustering analysis of RDM values segregated two independent control groups together, along with the non-tumorigen myclobutanil. The two tumorigenic conazoles clustered together in a distinct grouping. Partitioning around mediods of RDM values into two clusters also groups the triadimefon and propiconazole together in one cluster and the two control groups and myclobutanil together in a second cluster. Principal component analysis of these results identifies two components that account for 88.3% of the variability in the points. Taken together, these results are consistent with the hypothesis that propiconazole- and triadimefon-induced mutations do not represent clonal expansion of background mutations and support the hypothesis that they arise from the accumulation of reactive electrophilic metabolic intermediates within the liver in vivo.

Lipophilic pollutants can accumulate in human white adipose tissue (WAT) and consequences of this accumulation are still poorly understood. Cytochromes P450 (CYP) have recently been found in rat WAT and shown to be inducible through similar mechanisms as in the liver. Our study aimed at describing the cytochrome P450 pattern and their induction mechanisms in human WAT. Explants of subcutaneous and visceral WAT and primary culture of subcutaneous adipocytes, were used as WAT models, whereas liver biopsies and primary culture of hepatocytes were used as liver models to characterize CYP expression in both tissues. The WAT and liver models were then treated with typical CYP inducers (rifampicin, phenobarbital and 2,3,7,8-tetrachlorodibenzo-p-dioxin) and lipophilic pollutants (lindane, prochloraz and chlorpyrifos) and the effects on CYP expression were studied. CYP expression was considerably lower in WAT than in the liver, except for CYP1B1 and CYP2U1, which were the most expressed adipose CYP in all individuals. 2,3,7,8-tetrachlorodibenzo-p-dioxin and prochloraz induced CYP1A1 and CYP1B1 expression in both tissues. The Aryl hydrocarbon receptor was also present in WAT. On the contrary, nor phenobarbital or rifampicin treatment induced CYP2 or CYP3 mRNA in WAT and constitutive androstane receptor and pregnane X receptor were almost undetectable. These results suggest that the mechanisms by which CYP of family 1 are regulated in the liver are also functional in human WAT, but those regulating CYP2 and 3 expression are not.

Understanding the fate and effects of organic chemicals in animals requires knowledge of cytochrome P450 (CYP) genes, which thus far are poorly known in bivalve mollusks. We searched for CYP sequences in EST databases for Mytilus and Crassostrea species, lophotrochozoan representatives of the protostomes. From ESTs averaging ca. 924bp, we identified 58 CYP genes in Mytilus californianus and 39 CYP genes in Crassostrea gigas. The sequences fell in all known animal CYP clans, and collectively they clustered in phylogenetic analysis with vertebrate CYP families 1, 2, 3, 4, 17, 20, 26 and 27. As in deuterostomes, a majority of the sequences fell in Clan 2. The CYP sequences found thus far in bivalves suggest a diversity consistent with that found in many other animal species. The present description of mollusk genes provides the overall framework for classification of any additional bivalve sequences. The sequences identified also will be useful in obtaining full-length sequences and in designing primers for analysis of expression of mussel and oyster CYP genes, or for recombinant protein expression to identify potential substrates for the bivalve CYP proteins, and understand their roles in xenobiotic detoxification and physiology of bivalves.

ABSTRACT: BACKGROUND: Hepatotoxicity is a form of liver injury caused by exposure to stressors. Genomic-based approaches have been used to detect changes in transcription in response to hepatotoxicants. However, there are no straightforward ways of using co-expressed genes anchored to a phenotype or constrained by the experimental design for discerning mechanisms of a biological response. RESULTS: Through the analysis of a gene expression dataset containing 318 liver samples from rats exposed to hepatotoxicants and leveraging alanine aminotransferase (ALT), a serum enzyme indicative of liver injury as the phenotypic marker, we identified biological processes and molecular pathways that may be associated with mechanisms of hepatotoxicity. Our analysis used an approach called Coherent Co-expression Biclustering (cc-Biclustering) for clustering a subset of genes through a coherent (consistency) measure within each group of samples representing a subset of experimental conditions. Supervised biclustering identified 87 genes co-expressed and correlated with ALT in all the samples exposed to the chemicals. None of the over-represented pathways related to liver injury. However, biclusters with subsets of samples exposed to one of the 7 hepatotoxicants, but not to a non-toxic isomer, contained co-expressed genes that represented pathways related to a stress response. Unsupervised biclustering of the data resulted in 1) four to five times more genes within the bicluster containing all the samples exposed to the chemicals, 2) biclusters with co-expression of genes that discerned 1,4 dichlorobenzene (a non-toxic isomer at low and mid doses) from the other chemicals, pathways and biological processes that underlie liver injury and 3) a bicluster with genes up-regulated in an early response to toxic exposure. CONCLUSIONS: We obtained clusters of co-expressed genes that over-represented biological processes and molecular pathways related to hepatotoxicity in the rat. The mechanisms involved in the response of the liver to the exposure to 1,4-dichlorobenzene suggest non-genotoxicty whereas the exposure to the hepatotoxicants could be DNA damaging leading to overall genomic instability and activation of cell cycle check point signaling. In addition, key pathways and biological processes representative of an inflammatory response, energy production and apoptosis were impacted by the hepatotoxicant exposures that manifested liver injury in the rat.

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 (GD6) to postnatal day 92 (PND92). 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. While 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 (PPARalpha), 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.

The triazole antifungals myclobutanil, propiconazole and triadimefon cause varying degrees of hepatic toxicity and disrupt steroid hormone homeostasis in rodent in vivo models. To identify biological pathways consistently modulated across multiple time-points and various study designs, gene expression profiling was conducted on rat livers from three separate studies with triazole treatment groups ranging from 6 h after a single oral gavage exposure, to prenatal to adult exposures via feed. To explore conservation of responses across species, gene expression from the rat liver studies were compared to in vitro data from rat and human primary hepatocytes exposed to the triazoles. Toxicogenomic data on triazoles from 33 different treatment groups and 135 samples (microarrays) identified thousands of probe sets and dozens of pathways differentially expressed across time, dose, and species - many of these were common to all three triazoles, or conserved between rodents and humans. Common and conserved pathways included androgen and estrogen metabolism, xenobiotic metabolism signaling through CAR and PXR, and CYP mediated metabolism. Differentially expressed genes included the phase I xenobiotic, fatty acid, sterol and steroid metabolism genes Cyp2b2 and CYP2B6, Cyp3a1 and CYP3A4, and Cyp4a22 and CYP4A11; phase II conjugation enzyme genes Ugt1a1 and UGT1A1; and phase III ABC transporter genes Abcb1 and ABCB1. Gene expression changes caused by all three triazoles in liver and hepatocytes were concentrated in biological pathways regulating lipid, sterol and steroid homeostasis, identifying a potential common mode of action conserved between rodents and humans. Modulation of hepatic sterol and steroid metabolism is a plausible mode of action for changes in serum testosterone and adverse reproductive outcomes observed in rat studies, and may be relevant to human risk assessment.

Conazoles are fungicides used to control fungal growth in environmental settings and to treat humans with fungal infections. Mouse hepatotumorigenic conazoles display many of the same hepatic toxicologic responses as the mouse liver carcinogen phenobarbital (PB): constitutive androstane receptor (CAR) activation, hypertrophy, Cyp2b induction, and increased cell proliferation. The goal of this study was to apply transcriptional analyses to hepatic tissues from mice exposed to PB, propiconazole (Pro) or triadimefon (Tri) at tumorigenic exposure levels to reveal similarities and differences in response among these treatments. Mice were administered diets containing PB (850 ppm), Pro (2500 ppm) or Tri (1800 ppm) for 4 and 30d. Targeted transcriptomic analyses were conducted at the gene level examining differentially expressed genes (DEGs), and subsets of DEGs: cell cycle genes, and transcription factors. Analyses were also conducted on function, pathway and network levels examining Ingenuity Pathway Analysis Tox Lists and Canonical Pathways, and Gene-Go MetaCore dynamic networks and their central hubs. Genes expressed by PB or the two conazoles were also compared to those genes associated with human hepatocellular cancer. The results from these analyses indicated greater differences between PB and the two conazoles than similarities. Significant commonalities between the two conazole treatments were also noted. We posit that the transcriptional profiles of tissues exposed to toxic chemicals inherently contain their mechanisms of toxicity. We conclude that although PB and these 2 conazoles induce mouse liver tumors and exhibit similar toxicological responses, their transcriptional profiles are significantly different and thus their mechanisms of tumorigenic action are likely to differ.

In order to determine the effects of intestinal flora on the expression of cytochrome P450 (CYP), the mRNA expression of CYP was compared between specific pathogen-free (SPF) and germ-free (GF) mice. Most of the major CYP isozymes showed higher expression in the livers of SPF mice compared with GF mice. Nuclear factors such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR), as well as transporters and conjugation enzymes involved in the detoxification of lithocholic acid (LCA), also showed higher expression in SPF mice. The findings suggest that in the livers of SPF mice, LCA produced by intestinal flora increases the expression of CYPs via activation of PXR and CAR. Drugs such as antibiotics, some diseases and ageing, etc. are known to alter intestinal flora. The present findings suggest that such changes also affect CYP and are one of the factors responsible for individual differences in pharmacokinetics.

Technical grade dichlorodiphenyltrichloroethane (DDT) is an agricultural pesticide and malarial vector control agent that has been designated a potential human hepatocarcinogen. The o,p'-enantiomer exhibits estrogenic activity that has been associated with the carcinogenicity of DDT. The temporal and dose-dependent hepatic estrogenicity of o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT) was investigated using cDNA microarrays in immature, ovariectomized Sprague Dawley rats with complementary histopathology and tissue level analysis. Animals were gavaged with 300 mg/kg o,p'-DDT either once or once daily for three consecutive days. Liver samples were examined 2, 4, 8, 12, 18 or 24 h after a single dose or following three daily doses. For dose response studies, a single dose of 3, 10, 30, 100 or 300 mg/kg b.w o,p'-DTT was administered for three consecutive days. Genes associated with drug metabolism (Cyp2b2, Cyp3a2), the nuclear receptors CAR and PXR, cell proliferation (Ccnd1, Ccnb1, Ccnb2, Stmn1) and oxidative stress (Gclm, Hmox1) were significantly induced. Cyp2b2 exhibited dose-dependent regulation and was significantly induced across all time points, while cell proliferation- and oxidative stress-related genes exhibited transient induction. The induction of Cyp2b2 and Cyp3a2 mRNA levels suggest PXR/CAR activation, consistent with expression of genes associated with oxidative stress. Few genes known to be estrogen receptor (ER) regulated were differentially expressed when compared to the hepatic gene expression profile elicited by ethynyl estradiol in immature ovariectomized C57BL/6 mice using the same study design and analysis methods. These data indicate that o,p'-DDT elicits PXR/CAR, not ER, mediated gene expression in the rat liver. Based on the species-specific differences in CAR regulation, the extrapolation of rodent DDT hepatocarcinogenicity to humans warrants further investigation.

Xenobiotic induced liver injury is a leading cause for drug withdrawal and toxicogenomics may help to identify molecular causes. Here we report studies with cultures of human hepatocytes to detect early responses of liver toxicity upon treatment with the hepatotoxin Aroclor 1254. We studied transcript abundance of 302 genes with biological functions in detoxification, cell proliferation, tumor development, stress response, signal transduction, apoptosis and cell cycle regulation. More than 40 genes were regulated by at least twofold change, the majority coding for xenobiotic defense. In the case of CYP monooxygenases induction of coded protein was confirmed by Western immunoblotting whereas catalytic activities were determined by assaying for testosterone hydroxylase activity. There was good agreement between gene and protein expression and enzyme activity of CYP monooxygenases. In conclusion, xenobiotic induced hepatotoxicity may arise via several mechanisms and gene expression profiling helps to pinpoint location and pathways perturbed by drugs and chemicals.