William T. Padgett

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. Pathol. 2006; 34:863-78). The goals of this study were to examine effects of propiconazole, triadimefon, and myclobutanil, three triazole-containing conazoles, on the microsomal metabolism of atRA, the associated hepatic cytochrome P450 (P450) enzyme(s) involved in atRA metabolism, and their effects on hepatic atRA levels in vivo. The in vitro metabolism of atRA was quantitatively measured in liver microsomes from male CD-1 mice following four daily intraperitoneal injections of propiconazole (210 mg/kg/d), triadimefon (257 mg/kg/d) or myclobutanil (270 mg/kg/d). The formation of both 4-hydroxy-atRA and 4-oxo-atRA were significantly increased by all three conazoles. Propiconazole-induced microsomes possessed slightly greater metabolizing activities compared to myclobutanil-induced microsomes. Both propiconazole and triadimefon treatment induced greater formation of 4-hydroxy-atRA compared to myclobutanil treatment. Chemical and immuno-inhibition metabolism studies suggested that Cyp26a1, Cyp2b, and Cyp3a, but not Cyp1a1 proteins were involved in atRA metabolism. Cyp2b10/20 and Cyp3a11 genes were significantly over-expressed in the livers of both triadimefon- and propiconazole-treated mice while Cyp26a1, Cyp2c65 and Cyp1a2 genes were over-expressed in the livers of either triadimefon- or propiconazole-treated mice, and Cyp2b10/20 and Cyp3a13 genes were over-expressed in the livers of myclobutanil-treated mice. Western blot analyses indicated conazole induced-increases in Cyp2b and Cyp3a proteins. All three conazoles decreased hepatic atRA tissue levels ranging from 45-67%. The possible implications of these changes in hepatic atRA levels on cell proliferation in the mouse tumorigenesis process are discussed.

Comparison of the genotoxic activities of the K-region dihydrodiol of benzo[a]pyrene with benzo[a]pyrene in mammalian cells: morphological cell transformation; DNA damage; and stable covalent DNA adducts.

Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. While trans-B[a]P-4,5-diol and B[a]P both induce morphological cell transformation, their activities as DNA damaging agents differ, both qualitatively and quantitatively. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new mechanism/pathway for the morphological cell transforming activities of B[a]P and its metabolites.

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. We also sought to further characterize the nature and effects of ROS formation induced by propiconazole treatment in mouse liver. ROS was induced in AML12 cells by propiconazole as measured by fluorescence detection and its formation was ameliorated by N-acetylcysteine. Propiconazole induced glutathione-S-transferase (GSTα) protein levels and increased the levels of thiobarbituric acid reactive substances (TBARS) in AML12 cells. The TBARS levels were decreased by diphenylene iodonium chloride (DPIC), a cytochrome P450 (CYP) reductase inhibitor revealing the role of CYPs in ROS generation. It has been previously reported that Cyp2b and Cyp3a proteins were induced in mouse liver by propiconazole and that Cyp2b and Cyp3a proteins undergo uncoupling of their CYP catalytic cycle releasing ROS. Therefore, salicylic acid hydroxylation was used as probe for ROS formation using microsomes from mice treated with propiconazole. These studies showed that levels of 2,3-dihydroxybenzoic acid (an ROS derived metabolite) were decreased by ketoconazole, melatonin and DPIC. In vivo, propiconazole increased hepatic malondialdehyde levels and GSTα protein levels and had no effect on hepatic catalase or superoxide dismutase activities. Based on these observations we conclude that propiconazole induces ROS in mouse liver by increasing CYP protein levels leading to increased ROS levels. Our data also suggest that propiconazole induces the hydroxyl radical as a major ROS form.

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. The studies presented here sought to examine the role of stable BPQ-DNA adducts in B[a]P-induced mouse lung tumorigenesis. Male strain A/J mice were injected intraperitoneally once with BPQ or trans-7,8-dihydroxy-7,8-dihydroB[a]P (BP-7,8-diol) at 30, 10, 3, or 0mg/kg. Lungs and livers were harvested after 24h, the DNA extracted and subjected to (32)P-postlabeling analysis. Additional groups of mice were dosed once with BPQ or BP-7,8-diol each at 30 mg/kg and tissues harvested 48 and 72 h later, or with B[a]P (50mg/kg, a tumorigenic dose) and tissues harvested 72 h later. No BPQ or any other DNA adducts were observed in lung or liver tissues 24, 48, or 72 h after the treatment with 30 mg/kg BPQ. BP-7,8-diol gave BPDE-DNA adducts at all time points in both tissues and B[a]P treatment gave BPDE-DNA adducts in the lung. In each case, no BPQ-DNA adducts were detected. Mouse body weights significantly decreased over time after BPQ or BP-7,8-diol treatments suggesting that systemic toxicity was induced by both agents. Model studies with BPQ and N-acetylcysteine suggested that BPQ is rapidly inactivated by sulfhydryl-containing compounds and not available for DNA adduction. We conclude that under these treatment conditions BPQ does not form stable covalent DNA adducts in the lungs or livers of strain A/J mice, suggesting that stable BPQ-covalent adducts are not a part of the complex of mechanisms involved in B[a]P-induced mouse lung tumorigenesis.

Identification of 5-(deoxyguanosin-N2-yl)-1,2-dihydroxy-1,2-dihydro-6-aminochrysene as the major DNA lesion in the mammary gland of rats treated with the environmental pollutant 6-nitrochrysene

The environmental pollutant 6-nitrochrysene (6-NC) is a potent carcinogen in several animal models including the rat mammary gland. 6-NC can be activated to intermediates that can damage DNA by simple nitroreduction, ring oxidation, or a combination of ring oxidation and nitroreduction. Only the first pathway (nitroreduction) has been clearly established, and DNA adducts derived from this pathway have been fully characterized in in vitro systems. We also showed previously that the second pathway, ring oxidation leading to the formation of the bay region diol epoxide of 6-NC, is not responsible for the formation of the major DNA adduct in the mammary gland of rats treated with 6-NC. Therefore, in the present study, we explored the validity of the third pathway that involves the combination of both ring oxidation and nitroreduction of 6-NC to form trans-1,2-dihydroxy-1,2-dihydro-6-hydroxylaminochrysene (1,2-DHD-6-NHOH-C). During the course of this study, we synthesized for the first time 1,2-DHD-6-NHOH-C, N-(deoxyguanosin-8-yl)-6-aminochrysene, and N-(deoxyguanosin-8-yl)-1,2-dihydroxy-1,2-dihydro-6-aminochrysene. Incubation of 1,2-DHD-6-NHOH-C with calf thymus DNA resulted in the formation of three adducts. Upon LC/MS combined with 1H NMR analyses, the first eluting adduct was identified as 5-(deoxyguanosin-N2-yl)-1,2-dihydroxy-1,2-dihydro-6-aminochrysene [5-(dG-N2-yl)-1,2-DHD-6-AC], the second eluting adduct was identified as N-(deoxyguanosin-8-yl)-1,2-dihydroxy-1,2-dihydro-6-aminochrysene, and the last was identified as N-(deoxyinosin-8-yl)-1,2-dihydroxy-1,2-dihydro-6-aminochrysene. We also report here for the first time that among those adducts identified in vitro, only 5-(dG-N2-yl)-1,2-DHD-6-AC is the major DNA lesion detected in the mammary glands of rats treated with 6-NC.

Engineering human cell spheroids to model embryonic tissue fusion in vitro

Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human in vitro assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior in vitro using human cells. We engineered size-controlled human Wharton’s Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications.

Biotransformation and DNA Adduct Formation of Trans-8,9-Dihydroxy-8,9-Dihydrodibenzo[a, l]Pyrene by Induced Rat Liver and Human CYP1A1 Microsomes

Abstract In order to explain the adduct patterns observed from the human CYP1A1-mediated binding of dibenzo[a, l]pyrene (DB[a, l]P) to DNA, we have investigated the further metabolism and DNA adduct activity of trans-DB[a, l]P-8,9-diol by induced rat liver and human CYP1A1 microsomes. trans-DB[a, l]P-8,9-diol was synthesized and metabolic studies with β-naphthoflavone-induced rat liver microsomes indicated three major metabolites: 2 diastereomers of trans,trans-8,9,11,12-tetrahydro-8,9,11,12-tetrahydroxy-DB[a, l]P and 8,9,13,14-tetrahydro-8,9,13,14-tetrahydroxy-DB[a, l]P. DB[a, l]P when activated by CYP1A1/epoxide hydrase (EH) and calf thymus DNA gave a complex pattern of DNA adducts most of which cochromatograph with syn- and anti-DB[a, l]P fjord region diol epoxide-DNA standards. Two highly polar eluting adducts were also observed, one which cochromatographs with the single major DNA adduct obtained from the CYP1A1/EH activation of trans-DB[a, l]P-8,9-diol. The relative retention time of this adduct suggests either a bis-diol epoxide adduct or a more polar diol epoxide adduct.