Stephen Nesnow

Chemical carcinogens a review and analysis of the literature of selected chemicals and the establishment of the Gene-Tox carcinogen data base. A report of the U.S. environmental protection agency Gene-Tox program

The literature on 506 selected chemicals has been evaluated for evidence that these chemicals induce tumors in experimental animals and this assessment comprises the Gene-Tox Carcinogen Data Base. Three major sources of information were used to create this evaluated data base: all 185 chemicals determined by the International Agency for Research on Cancer to have Sufficient evidence of carcinogenic activity in experimental animals, 28 selected chemicals bioassayed for carcinogenic activity by the National Toxicology Program/National Cancer Institute and found to induce tumors in mice and rats, and 293 selected chemicals which had been evaluated in genetic toxicology and related bioassays as determined from previous Gene-Tox reports. The literature data on the 239 chemicals were analyzed by the Gene-Tox Carcinogenesis Panel in an organized, rational and consistent manner. Criteria were established to assess individual studies employing single chemicals and 4 categories of response were developed: Positive, Negative, Inconclusive (Equivocal) and Inconclusive. After evaluating each of the individual studies on the 293 chemicals, the Panel placed each of the 506 chemicals in an overall classification category based on the strength of the evidence indicating the presence or absence of carcinogenic effects. An 8-category decision scheme was established using a modified version of the International Agency for Research on Cancer approach. This scheme included two categories of Positive (Sufficient and Limited), two categories of Negative (Sufficient and Limited), a category of Equivocal (the evidence of carcinogenicity from well-conducted and well-reported lifetime studies had uncertain significance and was neither clearly positive nor negative), and three categories of Inadequate (the evidence of carcinogenicity was insufficient to make a decision, however, the data suggested a positive or negative indication). Of the 506 chemicals in the Gene-Tox Carcinogen Data Base, 252 were evaluated as Sufficient Positive, 99 as Limited Positive, 40 as Sufficient Negative, 21 as Limited Negative, 1 as Equivocal, 13 as Inadequate with the data suggesting a positive indication, 32 as Inadequate with the data suggesting a negative indication, and 48 Inadequate with the data not suggesting any indication of activity. This data base was analyzed and examined according to chemical class, using a 29 chemical class scheme.(ABSTRACT TRUNCATED AT 400 WORDS)

Polycyclic aromatic hydrocarbons: correlations between DNA adducts and ras oncogene mutations.

This review describes a series of studies on the tumorigenic activities of polycyclic aromatic hydrocarbons (PAHs) in various experimental animal model systems, their abilities to form PAH-DNA adducts in target tissues, and their abilities to mutate ras oncogenes in PAH-induced tumors. The review is limited to those PAHs that do not contain nitrogen, for which ras mutations have been detected in induced tumors, and for which some information is available about the structures of the DNA adducts induced in the target tissue. In general, PAHs that form DNA adducts at deoxyadenosine induce mutations at codon 61, whereas those PAHs that form DNA adducts at deoxyguanosine primarily induce mutations at codons 12 or 13. Those PAHs that induce adducts at both bases induce both types of mutations. These correlations provide evidence for the involvement of adduct-directed mutations in ras in the etiology of these tumors. The induced mutation spectra in ras may in fact point back to the identity of the type of adduct formed.

Application of short-term bioassays in the fractionation and analysis of complex environmental mixtures

Section 1: Short-Term Bioassay Systems-An Overview.- The Use of Microbial Assay Systems in the Detection of Environmental Mutagens in Complex Mixtures.- Mutagenesis of Mammalian Cells by Chemical Carcinogens After Metabolic Activation.- Oncogenic Transformation of Mammalian Cells by Chemicals and Viral-Chemical Interactions.- Higher Plant Systems as Monitors of Environmental Mutagens.- The Role of Drosophila in Chemical Mutagenesis Testing.- The Cellular Toxicity of Complex Environmental Mixtures.- Section 2: Collection and Chemical Analysis of Environmental Samples.- Atmospheric Genotoxicants-What Numbers Do We Collect?.- State-of-the-Art Analytical Techniques for Ambient Vapor Phase Organics and Volatile Organics in Aqueous Samples from Energy-Related Activities.- Strategy for Collection of Drinking Water Concentrates.- Section 3: Current Research.- Short-term Bioassay of Complex Organic Mixtures: Part I, Chemistry.- Short-term Bioassay of Complex Organic Mixtures: Part II, Mutagenicity Testing.- Quantitative Mammalian Cell Genetic Toxicology: Study of the Cytotoxicity and Mutagenicity of Seventy Individual Environmental Agents Related to Energy Technologies and Three Subfractions of a Crude Synthetic Oil in the CHO/HGPRT System.- Environmental Testing.- Integrating Microbiological and Chemical Testing into the Screening of Air Samples for Potential Mutagenicity.- Chemical and Microbiological Studies of Mutagenic Pollutants in Real and Simulated Atmospheres.- Application of Bioassay to the Characterization of Diesel Particle Emissions.- Measurement of Biological Activity of Ambient Air Mixtures Using a Mobile Laboratory for In Situ Exposures: Preliminary Results from the Tradescantia Plant Test System.- Physical and Biological Studies of Coal Fly Ash.- Mutagenicity of Shale Oil Components.- Mutagenic Analysis of Drinking Water.- In Vitro Activation of Cigarette Smoke Condensate Materials to Their Mutagenic Forms.- Mutagenic, Carcinogenic, and Toxic Effects of Residual Organics in Drinking Water.- Mutagenic Analysis of Complex Samples of Aqueous Effluents, Air Particulates, and Foods.

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.

Organ and species specificity in chemical carcinogenesis

The Nature of Organ Specificity in Chemical Carcinogenesis.- Species Differences in Response to Aromatic Amines.- Aflatoxin B1: Correlations of Patterns of Metabolism and DNA Modification with Biological Effects.- Species Specificity in Nitrosamine Carcinogenesis.- Organ Specificity and Interspecies Differences in Carcinogenesis by Metabolism-Independent Alkylating Agents.- Organ and Species Specificity in Nickel Subsulfide Carcinogenesis.- Chemical Carcinogenesis Studies in Nonhuman Primates.- Organ Specificity and Tumor Promotion.- Skin Tumor Promotion: A Comparative Study of Several Stocks and Strains of Mice.- The Role of Phorbol Ester Receptor Binding in Response to Promoters by Mouse and Human Cells.- Tissue and Species Specificity for Phorbol Ester Receptors.- Liver as a Model System for Analyzing Mechanisms of Tumor Initiation and Promotion.- Promotion of Urinary Bladder Carcinogenesis.- Species and Organ Differences in the Biotransformation of Chemical Carcinogens.- Species Heterogeneity in the Metabolic Processing of Benzo[a]pyrene.- Endocrine Regulation of Xenobiotic Conjugation Enzymes.- Multiple Effects and Metabolism of ?-Naphthoflavone in Induced and Uninduced Hepatic Microsomes.- Species Differences in the Activation of Benzo[a]pyrene in the Tracheal Epithelium of Rats and Hamsters.- Subcellular Metabolic Activation Systems: Their Utility and Limitations in Predicting Organ and Species Specific Carcinogenesis of Chemicals.- Cell-Mediated Mutagenesis, an Approach to Studying Organ Specificity of Chemical Carcinogens.- The Activation of Carcinogens by Mammary Cells: Inter-Organ and Intra-Organ Specificity.- Studies on the Mode of Action of Chemical Carcinogens in Cultured Mammalian Cells.- Comparison of the Sensitivity of Rodent and Human Cells to Chemical Carcinogens Using Viral Transformation, DNA Damage, and Cytotoxicity Assays.- Tissue-Specific Sister Chromatid Exchange Analyses in Mutagen-Carcinogen Exposed Animals.- Metabolism of Chemical Carcinogens by Tracheobronchial Tissues.- Introduction: DNA Damage and Repair.- DNA Modification and Repair In Vivo: Towards a Biochemical Basis of Organ-Specific Carcinogenesis by Methylating Agents.- Modification of DNA Repair Processes Induced by Nitrosamines.- Repair of Alkylated DNA Cell Extracts from Various Organs and Species.- Time-Dependent Differences in the Benzo[a]pyrene-DNA Adducts Present in Cell Cultures from Different Species.- DNA Binding as a Probe for Metabolic Activation in Various Systems.- Cell Specificity in DNA Damage and Repair.- DNA Adduct Formation and Removal in N-Acetoxy-2-acetylaminofluorene-Exposed Cells and in Organs from Rats Fed 2-Acetylaminofluorene.- Trans-Species and Trans-Tissue Extrapolation of Carcinogenicity Assays.- Carcinogenic Risk Assessment - The Consequences of Believing Models.- Author Index.

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.

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. To this end the cytotoxic effects of propiconazole and five of its metabolites were examined in three hepatic cell types: The mouse hepatoma Hepa1c1c7 cell line, the human hepatoma HepG2 cell line, and primary cultures of mouse hepatocytes. We initially compared the responses of propiconazole exposure in both Hepa1c1c7 and HepG2 cell lines over a concentration range of 0-200 microM using two assay systems: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the neutral red assay. Concentration-related cytotoxic responses were evident in both cell lines using both endpoints with the MTT assay providing enhanced sensitivity. The relative cytotoxic effects of propiconazole and five propiconazole metabolites were further assessed by the MTT assay using Hepa1c1c7 and HepG2 tumor cell lines. The cell cultures were exposed to various concentrations of propiconazole and five of its metabolites over a range of 0-400 microM. Propiconazole was cytotoxic in both cell lines in a dose-dependent manner. All five metabolites were less cytotoxic in both cell lines compared to the parent compound. The most cytotoxic metabolites in Hepa1c1c7 and HepG2 cells among the five were 3-(2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)propan-1-ol and 1-(2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)propan-2-ol. Propiconazole was cytotoxic in primary mouse hepatocytes; however none of the five propiconazole metabolites exerted cytotoxic activities. There was a linear relationship between the cLogP and the cytotoxic effects of propiconazole and its five metabolites in Hepa1c1c7 cells. We conclude that these propiconazole metabolites would not contribute to the propiconazole-induced cytotoxicity process in primary mouse hepatocytes. Furthermore, since in tumor cell lines the metabolites were less cytotoxic than the parent propiconazole, our results suggest that in the tumorigenesis process as tumor cells are formed they would be more susceptible to the cytotoxic effects of propiconazole compared to the metabolites.

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. Mice were administered diets containing PB (850 ppm), Pro (2500 ppm), or Tri (1800 ppm) for 4 and 30 days. 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 with 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.

An improved radiochemical assay for benzo(a)pyrene monooxygenase.

Abstract An improved assay for the determination of benzo[ a ]pyrene monooxygenase is described. This radiometric assay is a modification of the assay of DePierre, J. W., Moron, M. S., Johannesen, K. A. M., and Ernster, L. [(1975) Anal. Biochem. 63, 470] and uses trifluoroacetic acid to convert arene oxides which have been extracted into the hexane phase into phenols. These phenols are then extracted into the basic (product) phase. This assay method gives results comparable to those obtained by high-pressure liquid chromatography under a wide variety of conditions and, therefore, is generally applicable to the determination of benzo[ a ]pyrene monooxygenase, especially in the absence of arene oxide hydrase or in the presence of arene oxide hydrase inhibitors.