Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium.

The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression compendium that lead to alteration of CAR activity.

Concordance of transcriptional and apical benchmark dose levels for conazole-induced liver effects in mice.

The ability to anchor chemical class-based gene expression changes to phenotypic lesions and to describe these changes as a function of dose and time informs mode-of-action determinations and improves quantitative risk assessments. Previous global expression profiling identified a 330-probe cluster differentially expressed and commonly responsive to 3 hepatotumorigenic conazoles (cyproconazole, epoxiconazole, and propiconazole) at 30 days. Extended to 2 more conazoles (triadimefon and myclobutanil), the present assessment encompasses 4 tumorigenic and 1 nontumorigenic conazole.

Integration of toxicological approaches with "omic" and related technologies to elucidate mechanisms of carcinogenic action: propiconazole, an example.

The field of mechanistic chemical carcinogenesis has evolved with the advent and advances in genomic, proteomic and metabolomic technologies. These advances allow mechanistic events along the process of exposure to frank tumors to be studied in great detail. Herein is reviewed an example of this approach using, propiconazole, a triazole-containing antifungal agent that is a mouse hepatocarcinogen.

Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation.

Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole.

The hepatocarcinogenic conazoles: cyproconazole, epoxiconazole, and propiconazole induce a common set of toxicological and transcriptional responses.

Conazoles are fungicides used as agricultural pesticides and pharmaceutical products. We investigated whether a common core of toxicological and transcriptional responses underlies the observed carcinogenic effects of three conazoles: cyproconazole, epoxiconazole, and propiconazole. In studies where mice were fed diets of these conazoles for 30 days, we found a common set of toxicological effects altered by these conazoles: hepatomegaly, hepatocellular hypertrophy, decreased serum cholesterol, decreased hepatic levels of all-trans-retinoic acid, and increased hepatic cell proliferation.

Propiconazole increases reactive oxygen species levels in mouse hepatic cells in culture and in mouse liver by a cytochrome P450 enzyme mediated process.

Propiconazole induces hepatocellular carcinomas and hepatocellular adenomas in mice and promotes liver tumors in rats. Transcriptional, proteomic, metabolomic and biochemical studies of hepatic tissues from mice treated with propiconazole under the conditions of the chronic bioassay indicated that propiconazole induced oxidative stress. Here we sought to identify the source of the reactive oxygen species (ROS) induced by propiconazole using both AML12 immortalized mouse hepatocytes in culture and liver tissues from mice.

Propiconazole induces alterations in the hepatic metabolome of mice: relevance to propiconazole-induced hepatocarcinogenesis.

Propiconazole is a mouse hepatotumorigenic fungicide and has been the subject of recent investigations into its carcinogenic mechanism of action. The goals of this study were (1) to identify metabolomic changes induced in the liver by increasing doses of propiconazole in mice, (2) to interpret these results with key previously reported biochemical, transcriptomic, and proteomic findings obtained from mouse liver under the same treatment conditions, and (3) to relate these alterations to those associated with the carcinogenesis process. Propiconazole was administered to male CD-1 mice in the feed for 4 days with six mice per feed level (500, 1250, and 2500 ppm).

Lack of contribution of covalent benzo[a]pyrene-7,8-quinone-DNA adducts in benzo[a]pyrene-induced mouse lung tumorigenesis.

Benzo[a]pyrene (B[a]P) is a potent human and rodent lung carcinogen. This activity has been ascribed in part to the formation of anti-trans-7,8-dihydroxy-7,8-dihydroB[a]P-9,10-epoxide (BPDE)-DNA adducts. Other carcinogenic mechanisms have been proposed: (1) the induction of apurinic sites from radical cation processes, and (2) the metabolic formation of B[a]P-7,8-quinone (BPQ) that can form covalent DNA adducts or reactive oxygen species which can damage DNA.

Proteomic analysis of propiconazole responses in mouse liver: comparison of genomic and proteomic profiles.

We have performed for the first time a comprehensive profiling of changes in protein expression of soluble proteins in livers from mice treated with the mouse liver tumorigen, propiconazole, to uncover the pathways and networks altered by this fungicide. Utilizing two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), we identified 62 proteins that were altered. Several of these protein changes detected by 2-DE/MS were verified by Western blot analyses.

Protein carbonyl formation in response to propiconazole-induced oxidative stress.

Propiconazole, a widely used fungicide, is hepatotoxic and hepatotumorigenic in mice. Previous genomic analysis of liver tissues from propiconazole-treated mice identified genes and pathways involved in oxidative stress, suggesting that oxidative stress may play a role in propiconazole-induced toxicity. To understand the contribution of oxidative stress on toxicity at the protein level, we developed an integrated approach for the systematic measurement of protein oxidation in the livers from propiconazole-treated mice.

Discrimination of tumorigenic triazole conazoles from phenobarbital by transcriptional analyses of mouse liver gene expression.

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.

Mutagenicity, stable DNA adducts, and abasic sites induced in Salmonella by phenanthro[3,4-b]- and phenanthro[4,3-b]thiophenes, sulfur analogs of benzo[c]phenanthrene.

Sulfur-containing polycyclic aromatic hydrocarbons (thia-PAHs or thiaarenes) are common constituents of air pollution and cigarette smoke, but only a few have been studied for health effects. We evaluated the mutagenicity in Salmonella TA98, TA100, and TA104 of two sulfur-containing derivatives of benzo[c]phenanthrene, phenanthro[3,4-b]thiophene (P[3,4-b]T), and phenanthro[4,3-b]thiophene (P[4,3-b]T) as well as their dihydrodiol and sulfone derivatives. In addition, we assessed levels of stable DNA adducts (by (32)P-postlabeling) as well as abasic sites (by an aldehydic-site assay) produced by six of these compounds in TA100.

Three conazoles increase hepatic microsomal retinoic acid metabolism and decrease mouse hepatic retinoic acid levels in vivo.

Conazoles are fungicides used in agriculture and as pharmaceuticals. In a previous toxicogenomic study of triazole-containing conazoles we found gene expression changes consistent with the alteration of the metabolism of all trans-retinoic acid (atRA), a vitamin A metabolite with cancer-preventative properties (Ward et al., Toxicol.

Cytotoxic effects of propiconazole and its metabolites in mouse and human hepatoma cells and primary mouse hepatocytes.

Propiconazole is a triazole-containing fungicide that is used agriculturally on grasses, fruits, grains, seeds, hardwoods, and conifers. Propiconazole is a mouse liver hepatotoxicant and a hepatocarcinogen that has adverse reproductive and developmental toxicities in experimental animals. The goal of this study was to investigate the cytotoxic responses of propiconazole and its metabolites to determine if metabolism of this agent differentially affected its cytotoxic activities in hepatic tumor cell lines and in primary hepatocytes.

Transcriptional responses in thyroid tissues from rats treated with a tumorigenic and a non-tumorigenic triazole conazole fungicide.

Conazoles are azole-containing fungicides that are used in agriculture and medicine. Conazoles can induce follicular cell adenomas of the thyroid in rats after chronic bioassay. The goal of this study was to identify pathways and networks of genes that were associated with thyroid tumorigenesis through transcriptional analyses.

Induction of cytochrome P450 enzymes in rat liver by two conazoles, myclobutanil and triadimefon.

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.

Toxicity profiles in rats treated with tumorigenic and nontumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil.

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.

Transcriptional profiles in liver from rats treated with tumorigenic and non-tumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil.

Conazoles are a class of fungicides used as pharmaceutical and agricultural agents. In chronic bioassays in rats, triadimefon was hepatotoxic and induced follicular cell adenomas in the thyroid gland, whereas, propiconazole and myclobutanil were hepatotoxic but had no effect on the thyroid gland. These conazoles administered in the feed to male Wistar/Han rats were found to induce hepatomegaly, induce high levels of pentoxyresorufin-O-dealkylase, increase cell proliferation in the liver, increase serum cholesterol, decrease serum T3 and T4, and increase hepatic uridine diphosphoglucuronosyl transferase activity.

Transcriptional profiles in liver from mice treated with hepatotumorigenic and nonhepatotumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil.

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.

Toxicity profiles in mice treated with hepatotumorigenic and non-hepatotumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil.

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.

Characterization of naphtho[1,2-a]pyrene and naphtho[1,2-e]pyrene DNA adducts in C3H10T1/2 fibroblasts.

Polycyclic aromatic hydrocarbons (PAHs) are a class of carcinogenic chemicals that are ubiquitous in the environment. Fjord-region naphthopyrene isomers are structurally similar to the potent fjord-region PAH carcinogen dibenzo[a,l]pyrene and thus have the potential to be potent carcinogens. Naphtho[1,2-a]pyrene (N[1,2-a]P) exhibited similar bacterial mutagenicity and morphological cell transforming activity when compared to benzo[a]pyrene (B[a]P), whereas the structural isomer, naphtho[1,2-e]pyrene (N[1,2-e]P) was inactive is these bioassays.

Benzo[a]pyrene-7,8-quinone-3'-mononucleotide adduct standards for 32P postlabeling analyses: detection of benzo[a]pyrene-7,8-quinone-calf thymus DNA adducts.

Benzo[a]pyrene-7,8-quinone (BPQ) is one of the reactive metabolites of the widely distributed archetypal polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). The formation of BPQ from B[a]P through trans-7,8-dihydroxy-7,8-dihydroB[a]P by the mediation of aldo-keto reductases and its role in the genotoxicity and carcinogenesis of B[a]P currently are under extensive investigation. Toxicity pathways related to BPQ are believed to include both stable and unstable (depurinating) DNA adduct formation as well as reactive oxygen species.

Fluconazole-induced hepatic cytochrome P450 gene expression and enzymatic activities in rats and mice.

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.

Role of cell signaling in B[a]P-induced apoptosis: characterization of unspecific effects of cell signaling inhibitors and apoptotic effects of B[a]P metabolites.

Here we show that several cell signaling inhibitors have effect on cyp1a1 expression and the metabolism of benzo[a]pyrene (B[a]P) in Hepa1c1c7 cells. The CYP1A1 inhibitor alpha-naphthoflavone (alpha-NF), the p53 inhibitor pifithrin-alpha (PFT-alpha), the ERK inhibitors PD98059 and U0126, and the p38 MAPK inhibitors SB202190 and PD169316 induced the expression and level of cyp1a1 protein. On the other hand, during the first h the inhibitors appeared to reduce the metabolism of B[a]P as measured by the generation of tetrols and by covalent binding of B[a]P to macromolecules.