PubMed · 367444
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A Sivak. 1979-02-04. Cocarcinogenesis.. https://doi.org/10.1016/0304-419x(79)90003-9
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Despite an increasing incidence of human breast cancer, its etiology remains unknown. Since some environmental chemicals are stored in human breast fat and are rodent mammary carcinogens, determining the genotoxic potential of environmental agents in this key target tissue is important. An assay was developed for detecting genotoxic activity, as unscheduled DNA synthesis (UDS), induced by chemicals and UV radiation in early passage cultures of normal human mammary epithelial cells (HMEC) derived from 5 different women. In order to measure UDS in culture, reduction in the percentage of cells in S-phase was accomplished either by depriving the cells of epidermal growth factor and bovine pituitary extract or by contact inhibition of growth. Cultures were incubated with test chemicals for 24 h in the presence of [3H]-thymidine. UDS was quantitated autoradiographically as net grains per nucleus (nuclear grains minus cytoplasmic background, population average) with > or = 6 net nuclear grains considered in repair for any individual cell. A positive response was observed with UV radiation, benzo(a)-pyrene, aflatoxin B1, ethylmethanesulfonate, 1,6-dinitropyrene, 2-acetylaminofluorene, and tobacco smoke condensate but not 7,12-dimethylbenz(a)anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin. These results demonstrate that HMEC from all 5 women examined have the ability to metabolize a variety of environmental chemicals to DNA-reactive forms. Furthermore, some chemicals known either to cause mammary cancer in rodents or to be contaminants in human breast tissue are genotoxic in HMEC. A positive response in passage 9 cultures was observed only with direct acting agents, suggesting that HMEC may lose their metabolic capabilities in longer-term cultures. The HMEC UDS assay may be used to address the role of environmental agents in human breast cancer by determining whether chemicals are DNA reactive or metabolized to DNA reactive species in this critical target tissue.
A complementary DNA (cDNA) for rat hepatic aryl sulfotransferase IV (AST IV) was isolated, characterized, and used as a hybridization probe to evaluate the molecular basis for the differential expression of AST IV during 2-acetylaminofluorine (2AAF)-induced hepatocarcinogensis. The AST IV cDNA clone was obtained by immunochemical screening of a male Sprague-Dawley rat liver cDNA library. The AST IV cDNA was found to be 1.3 kilobases long and to encode a fusion protein which was reactive with an antibody to AST IV and enzymatically able to generate the sulfuric acid ester of N-hydroxy-2AAF. Sequence analysis of the AST IV cDNA showed it to be 1127 residues in length and to have essentially complete homology with PST-I cDNA, a previously reported (S. Ozawa, et al., Nucleic Acids Res., 18: 4001, 1990), 1028-base cDNA for an uncharacterized rat liver aryl sulfotransferase. Comparison of the PST-I/AST IV cDNA-deduced amino acid sequence with data from a partial (51%) amino acid sequence analysis of purified AST IV showed complete amino acid homology, confirming the identity of the cDNA and establishing that AST IV was an N-blocked, 291-amino acid protein with a molecular mass of 33,909 daltons. The AST IV cDNA sequence differed from the PST-I cDNA in two principal ways: the 5' end lacked 18 coding bases, and the 3' end contained a 190-base extention in the untranslated region, including a consensus sequence for signalling polyadenylation. Studies of AST IV gene transcript levels showed that the livers of rats fed 2AAF for 3 wk (early stage hepatocarcinogenesis) and hyperplastic nodules from the livers of rats fed 2AAF for 19 wk (intermediate stage hepatocarcinogenesis) displayed transcript levels similar to those of livers from normal rats. This contrasted with the 60 to 70% lower than normal capacity of the mRNA fractions to express AST IV observed during in vitro translation. These results indicated that modulation of AST IV expression at early and intermediate stages of hepatocarcinogenesis involved regulatory mechanisms at the translational level. In contrast, mRNA fractions isolated from some 2AAF-induced liver tumors or from known chemical carcinogen-derived rat hepatoma cell lines showed losses of both AST IV transcript level and in vitro translation capacity, suggesting that regulation at the transcriptional level may become important at late stages of 2AAF-induced hepatocarcinogenesis. These results indicated that the molecular mechanisms for the 2AAF-mediated down regulation of AST IV expression during 2AAF-induced hepatocarcinogenesis involved alterations in regulation at both translational and transcriptional levels.
The metabolism and DNA binding of acetylaminofluorene (AAF) was investigated in human hepatocytes that were isolated from donor liver tissue by collagenase perfusion. Hepatocytes were treated with 0.01 microM pentachlorophenol (PCP), as a sulfotransferase inhibitor, to investigate the role of sulfotransferase in human bioactivation of aromatic amines. Concentrations of PCP greater than 0.1 microM resulted in cytotoxicity as noted by detachment of cells and atypical morphology. The metabolites of AAF were identified by HPLC as aminofluorene, 7-OH-AAF, 9-OH-AAF, 5-OH-AAF, N-OH-AAF, 1-OH-AAF and 3-OH-AAF. No consistent alteration in the metabolites produced occurred with PCP treatment compared to controls. PCP treatment increased total DNA binding of AAF metabolites compared with controls, suggesting that sulfotransferase does not activate AAF in human hepatocytes. Inhibition of sulfotransferase in human hepatocytes does not decrease DNA binding of AAF metabolites as noted previously with rat hepatocytes. Therefore, PCP may inhibit a detoxication pathway. This study supports N,O-acyltransferase as the critical enzyme for the formation of the major reactive metabolite in human liver.