Sheau-Fung Thai

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. 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.

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 diphospho-glucuronosyl transferase activity. The goal of the present study was to define pathways that explain the biologic outcomes. Male Wistar/Han rats (3 per group), were exposed to the 3 conazoles in the feed for 4, 30, or 90 days of treatment at tumorigenic and nontumorigenic doses. Hepatic gene expression was determined using high-density Affymetrix GeneChips (Rat 230_2). Differential gene expression was assessed at the probe level using Robust Multichip Average analysis. Principal component analysis by treatment and time showed within group sample similarity and that the treatment groups were distinct from each other. The number of altered genes varied by treatment, dose, and time. The greatest number of altered genes was induced by triadimefon and propiconazole after 90 days of treatment, while myclobutanil had minimal effects at that time point. Pathway level analyses revealed that after 90 days of treatment the most significant numbers of altered pathways were related to cell signaling, growth, and metabolism. Pathway level analysis for triadimefon and propiconazole resulted in 71 altered pathways common to both chemicals. These pathways controlled cholesterol metabolism, activation of nuclear receptors, and N-ras and K-ras signaling. There were 37 pathways uniquely changed by propiconazole, and triadimefon uniquely altered 34 pathways. Pathway level analysis of altered gene expression resulted in a more complete description of the associated toxicological effects that can distinguish triadimefon from propiconazole and myclobutanil.

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. 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.

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.

Altered gene expression in mouse livers after dichloroacetic acid exposure

Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated that DCA exhibits hepatocarcinogenic effects in rodents when administered in drinking water. This chemical does not appear to be highly mutagenic, and the mechanism(s) involved in DCA induction of cancer are not clear. The present work was aimed at identifying changes in gene expression which may indicate critical alterations/pathways involved in this chemicals carcinogenic activities. We used cDNA microarray methods for analyses of gene expression in livers of mice treated with the tumorigenic dose of 2 g/l DCA in drinking water for 4 weeks. Total RNA samples obtained from livers of the control and DCA-treated mice were evaluated for gene expression patterns with Clontech Atlas Mouse 1.2 cDNA and Atlas mouse stress/toxicology arrays, and the data analyzed with AtlasImage 2.01 and one-way ANOVA in JMP4 software. From replicate experiments, we identified 24 genes with altered expression, of which 15 were confirmed by Northern blot analysis. Of the 15 genes, 14 revealed expression suppressed two- to five-fold; they included the following: MHR 23A, cytochrome P450 (CYP) 2C29, CYP 3A11, serum paraoxonase/arylesterase 1 (PON 1), liver carboxylesterase, alpha-1 antitrypsin, ER p72, glutathione S-transferase (GST) Pi 1, angiogenin, vitronectin precursor, cathepsin D (CTSD), plasminogen precursor (contains angiostatin), prothrombin precursor and integrin alpha 3 precursor (ITGA 3). An additional gene, CYP 2A4/5, had a two-fold elevation in expression. Further, in ancillary Northern analyses of total RNA isolated from DCA-induced hepatocellular carcinomas (from earlier reported studies of mice treated with 3.5 g/l DCA for 93 weeks), many of the same genes (11 of 15) noted above showed a similar alteration in expression. In summary, we have identified specific genes involved in the functional categories of cell growth, tissue remodeling, apoptosis, cancer progression and xenobiotic metabolism that have altered levels of expression following exposures to DCA. These findings serve to highlight new pathways in which to further probe DCA effects that may be critical to its tumorigenic activity.

Global Gene Expression Profiling of Hyperkeratotic Skin Lesions from Inner Mongolians Chronically Exposed to Arsenic

The skin is an organ that is highly sensitive to chronic arsenic (As) exposure. Skin lesions such as hyperkeratoses (HKs) are common early manifestations of arsenicosis in humans. HKs can be precursor lesions of nonmelanoma skin cancers (NMSCs), but the driving forces behind their formation and how they may ultimately progress to NMSCs are unknown. The goal of this study was to examine the global gene expression profiles of As-related HKs in an effort to better understand gene expression changes that are potentially associated with early stages of As carcinogenesis. HK biopsies were removed from individuals living in an arsenicosis-endemic region in Inner Mongolia who had been exposed to high As levels in their drinking water for >20 years. Gene expression profiling was performed on RNA isolated from 7 individuals in this group and from 4 lesion-free skin samples from healthy individuals. Consistent with the pathological characteristics of the HK lesions, major functional categories and known canonical pathways represented by altered transcripts include those involved in development, differentiation, apoptosis, proliferation, and stress response. The results of this study may help define a signature profile of gene expression changes associated with long-term As exposure in the skin.

Transcriptional Modulation of the ERK1/2 MAPK and NF-κB Pathways in Human Urothelial Cells After Trivalent Arsenical Exposure: Implications for Urinary Bladder Cancer.

Chronic exposure to drinking water contaminated with inorganic arsenic (iAs) is associated with an increased risk of urinary bladder (UB) cancers in humans. The exact role of specific iAs metabolite(s) in As-mediated carcinogenesis remains largely unknown. Experimental evidence suggests that trivalent arsenicals, namely arsenite (iAsIII) and two of its metabolites, monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII), are possible proximate UB carcinogens. Here, we used a transcriptomics approach to examine perturbed molecular pathways in a human urothelial cell line (UROtsa) after short-term exposure to iAsIII, MMAIII and DMAIII. Molecular pathways containing genes that encode proteins implicated in UB cancer development were perturbed by both MMAIII and DMAIII. These pathways included those of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK 1/2 MAPK) and nuclear factor kappa beta (NF-κB). Together, these results may inform the current understanding of effects in the UB induced by acute As exposure and the relationship of these effects with As-mediated carcinogenesis.

A comprehensive framework for evaluating the environmental health and safety implications of engineered nanomaterials

Abstract Engineered nanomaterials (ENM) are a growing aspect of the global economy, and their safe and sustainable development, use, and eventual disposal requires the capability to forecast and avoid potential problems. This review provides a framework to evaluate the health and safety implications of ENM releases into the environment, including purposeful releases such as for antimicrobial sprays or nano-enabled pesticides, and inadvertent releases as a consequence of other intended applications. Considerations encompass product life cycles, environmental media, exposed populations, and possible adverse outcomes. This framework is presented as a series of compartmental flow diagrams that serve as a basis to help derive future quantitative predictive models, guide research, and support development of tools for making risk-based decisions. After use, ENM are not expected to remain in their original form due to reactivity and/or propensity for hetero-agglomeration in environmental media. Therefore, emphasis is placed on characterizing ENM as they occur in environmental or biological matrices. In addition, predicting the activity of ENM in the environment is difficult due to the multiple dynamic interactions between the physical/chemical aspects of ENM and similarly complex environmental conditions. Others have proposed the use of simple predictive functional assays as an intermediate step to address the challenge of using physical/chemical properties to predict environmental fate and behavior of ENM. The nodes and interactions of the framework presented here reflect phase transitions that could be targets for development of such assays to estimate kinetic reaction rates and simplify model predictions. Application, refinement, and demonstration of this framework, along with an associated knowledgebase that includes targeted functional assay data, will allow better de novo predictions of potential exposures and adverse outcomes.

Differential Genomic Effects of Six Different TiO2 Nanomaterials on Human Liver HepG2 Cells.

Human HepG2 cells were exposed to six TiO2 nanomaterials (with dry primary particle sizes ranging from 22 to 214 nm, either 0.3, 3, or 30 μg/mL) for 3 days. Some of these canonical pathways changed by nano‐TiO2 in vitro treatments have been already reported in the literature, such as NRF2‐mediated stress response, fatty acid metabolism, cell cycle and apoptosis, immune response, cholesterol biosynthesis, and glycolysis. But this genomic study also revealed some novel effects such as protein synthesis, protein ubiquitination, hepatic fibrosis, and cancer‐related signaling pathways. More importantly, this genomic analysis of nano‐TiO2 treated HepG2 cells linked some of the in vitro canonical pathways to in vivo adverse outcomes: NRF2‐mediated response pathways to oxidative stress, acute phase response to inflammation, cholesterol biosynthesis to steroid hormones alteration, fatty acid metabolism changes to lipid homeostasis alteration, G2/M cell checkpoint regulation to apoptosis, and hepatic fibrosis/stellate cell activation to liver fibrosis.

Abstract 2814: Comparative DNA damage and transcriptomic effects of engineered nanoparticles in human lung cells in vitro

A series of six titanium dioxide and two cerium oxide engineered nanomaterials were assessed for their ability to induce cytotoxicity, reactive oxygen species (ROS), various types of DNA damage, and transcriptional changes in human respiratory BEAS-2B cells exposed in vitro at several concentrations for 72 hours. Only limited cytotoxicity was observed at concentrations up to 300 μg/ml for all of the nanomaterials. Small increases in 8-oxo-deoxyguanosine were induced by some of the nanomaterials, but did not achieve statistical significance. No increases in ethenoadenosine or ethenocytidine were detected by ELISA assays for any of the tested nanomaterials. Several of the nanomaterials exhibited concentration related increases in levels of apurinic/apyrimidinic sites, endogenous DNA adducts measured by 32 P-postlabeling, lipid peroxidation, and ROS. Consistent with these findings, several of the nanomaterials also affected expression of genes involved in p53, ATM, and mismatch repair pathways. Integrin signaling pathways were also altered by a majority of the nanomaterials tested. There was general agreement between activity in DNA damage assays and extent of pathway transcriptional alteration. One out of the cerium oxide nanomaterials tested did not induce a high enough incidence of differentially expressed genes relative to controls to allow analysis at the pathway level, and also elicited the lowest response in multiple DNA damage assays. Taken together, these data are consistent with the contribution of DNA damage induced by reactive oxygen species as mediators of potentially adverse biological effects following exposure to engineered titanium and cerium oxide nanomaterials, and suggests the utility of short term in vitro tests to predict relative potencies of these particles. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy. Citation Format: Garret B. Nelson, Sheau-Fung Y. Thai, Carlton P. Jones, Audrey Barbee, Micaela Killius, Jeffrey A. Ross. Comparative DNA damage and transcriptomic effects of engineered nanoparticles in human lung cells in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2814.