Effects of Oral Administration of Non-genotoxic Hepato-hypertrophic Compounds on Metabolic Potency of Rat Liver (original) (raw)
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Environmental and Molecular Mutagenesis, 2019
To develop an improved in vitro mammalian cell gene mutation assay, it is imperative to address the known deficiencies associated with existing assays. Primary hepatocytes isolated from the MutaMouse are ideal for an in vitro gene mutation assay due to their metabolic competence, their "normal" karyotype (i.e., neither transformed nor immortalized), and the presence of the MutaMouse transgene for rapid and reliable mutation scoring. The cells were extensively characterized to confirm their utility. Freshly isolated cells were found to have a hepatocyte-like morphology, predominantly consisting of binucleated cells. These cells maintain hepatocyte-specific markers for up to 3 days in culture. Analyses revealed a normal murine hepatocyte karyotype with a modal ploidy number of 4n. Fluorescence in situ hybridization analysis confirmed the presence of the lambda shuttle vector on chromosome 3. The doubling time was determined to be 22.5 AE 3.3 h. Gene expression and enzymatic activity of key Phase I and Phase II metabolic enzymes were maintained for at least 8 and 24 h in culture, respectively. Exposure to β-naphthoflavone led to approximately 900-and 9-fold increases in Cyp1a1 and Cyp1a2 gene expression, respectively, and approximately twofold induction in cytochrome P450 (CYP) 1A1/1A2 activity. Exposure to phenobarbital resulted in an approximately twofold increase in CYP 2B6 enzyme activity. Following this characterization, it is evident that Muta-Mouse primary hepatocytes have considerable promise for in vitro mutagenicity assessment. The performance of these cells in an in vitro gene mutation assay is assessed in Part II. Environ. Mol. Mutagen. 2018.
Archives of Toxicology, 1998
Nongenotoxic rodent hepatocarcinogens do not damage DNA but cause liver tumours in the rat and mouse, associated with the induction of hepatic DNA synthesis. Previously, we have demonstrated that nongenotoxic hepatocarcinogens such as phenobarbitone and the peroxisome proliferator (PP), nafenopin, also suppress rat hepatocyte apoptosis. The nongenotoxic chemicals 1,4-dichlorobenzene (DCB) and the PP, diethylhexyl phthalate (DEHP), both induce high levels of DNA synthesis in rat liver in vivo, but only DEHP is hepatocarcinogenic in this species. Here, we investigate whether the dierence in rat carcinogenicity of these two hepatic mitogens may be due to dierences in their ability to suppress hepatocyte apoptosis. In rat hepatocytes in vitro, MEHP (the active metabolite of DEHP) induced DNA synthesis 2.5-fold (P 0.001) and suppressed 10-and 4-fold, respectively both spontaneous (P 0.0008) and transforming growth factor b1 (TGFb1)-induced (P 0.0001) apoptosis. DCB gave a small (1.7-fold) increase in DNA synthesis (P 0.03) and a small (1.7-to 2-fold) suppression of both spontaneous (P 0.022) and TGFb1-induced (P 0.015) apoptosis. We next analysed the induction of DNA synthesis and the suppression of apoptosis in rat liver in vivo. Both DEHP and DCB were able to induce DNA synthesis although, as seen in vitro, the induction by DCB (4.2-fold; P 0.023) was less marked than that with DEHP (13.4-fold; P 0.007). Similarly, DEHP and DCB were both able to suppress rat hepatocyte apoptosis in vivo but the magnitude of the suppression was comparable; apoptosis was reduced to undetectable levels in four out of ®ve animals with DCB and three out of ®ve with DEHP. Since both chemicals suppressed apoptosis and induced DNA synthesis in rat liver but, overall, DCB was less potent, the disparate hepatocarcinogenic potential of these two chemicals could arise from dierences in the magnitude of growth perturbation. To test this hypothesis, we repeated the studies in mouse, a species where both DCB and DEHP are hepatocarcinogenic. Both in vitro and in vivo, DCB and DEHP/MEHP were able to suppress apoptosis and induce hepatocyte DNA synthesis in the mouse with comparable potencies. The data support the hypothesis that the carcinogenicity of nongenotoxic hepatocarcinogens is associated strongly with the ability to perturb hepatocyte growth regulation. However, the ability to eect such changes is not unique to nongenotoxic carcinogens and is common to some noncarcinogenic chemicals, such as DCB, suggesting that the growth perturbation may need to exceed a threshold for carcinogenesis.
Cytotoxic and genotoxic effects of five in primary cultures of rat and human hepatocytes
Mutation Research Letters, 1994
Five n-alkanals were examined for cytotoxicity, as evaluated by the trypan blue exclusion test, and for genotoxicity, as evaluated by the induction of unscheduled DNA synthesis (UDS), in primary cultures of rat and human hepatocytes. After 20 h exposure, cytotoxicity was similar in cells of the two species, and increased with the length of the carbon chain. In rat hepatocytes, propanal (10-100 mM), butanal (10-100 mM), pentanal (3-30 mM) and hexanal (3-30 mM) induced a modest but significant and dose-dependent increase of net nuclear grain counts, while in human hepatocytes this effect was not detected. Nonanal (3-30 mM), which showed the highest cytotoxic effect, failed to induce UDS in both cell types. These results seem to suggest that at the concentrations which are presumably attained after ingestion with food or generated by lipid peroxidation processes the five n-alkanals tested are presumably unable to induce genotoxic effects in the human liver.
Initiation/promotion versus complete carcinogenicity in the rodent liver
Environmental Health Perspectives, 1983
(4N) is a close structural analog of the rodent liver carcinogen 4-dimethylaminoazobenzene (DAB). This structural similarity led us to evaluate it for genotoxic activity in vitro. We observed activity for 4N and DAB in the BHK cell transformation assay and subsequently in the Salmonella mutation assay of Ames. By a curious chance, Scribner, Miller and Miller, probably prompted by the same structural similarity, had synthesized 4N in the 1960s and found it to be noncarcinogenic to the rodent liver using a bioassay test protocol that detected DAB as carcinogenic. These findings were only described following the publication of our observations made in vitro. The conflict that apparently exists between these data can be interpreted in two separate ways. (a) Scribner et al. have suggested that 4N may be a carcinogenic initiator as opposed to a complete carcinogen like DAB. They also suggested that promotion of 4N-treated rodents with phenobarbitone might lead to the production of liver tumors. (b) We have evaluated the simpler concept that the activities observed for 4N in vitro define a carcinogenic potential that is not realized in vivo due to its rapid detoxification, at least in rodents. The first of these explanations implies that pure carcinogenic initiators may form a separate class of genotoxic agents from complete carcinogens, and perhaps of greater interest, that 4N might provide a valuable model compound for the study of carcinogenic promotion in the rodent liver. The second explanation regards potential carcinogenicity as a single property that can be defined in vitro and which may or may not be expressed in vivo depending on the enzymic environments encountered by the test chemical. It is clearly important to evaluate these different propositions in order to aid progress in the study of carcinogenic promotion, especially in the rodent liver. The presentation will describe our recent studies in vitro and in vivo in this connection.
Environmental Mutagenesis, 1981
The autoradiographic identification of unscheduled DNA synthesis (UDS) in primary cultures of adult rat hepatocytes (HPC) has been proposed as a predictive test for mutagens/carcinogens. To assess the predictive value of this test, results in the hepatocyte UDS assay were compared with data for bacterial mutagenicity using a modified Ames test. Over 200 compounds representing a variety of chemical classes consisting of procarcinogens, ultimate carcinogens, and noncarcinogens were tested in each system. The accurate discrimination of many carcinogens/noncarcinogens was demonstrated by both systems. The induction of UDS in hepatocytes showed an excellent correlation with bacterial mutagenesis in response to polycyclic aromatic hydrocarbons, aromatic amines, biphenyls, nitrosamines, carbamates, azo‐compounds, acridines, halogenated compounds, nitrosureas, quinolines, pyridines, purines, pyrimidines, esters and carbamates. Nitrocompounds, although active in bacteria, were poor inducers ...
Carcinogen Induced Unscheduled DNA Synthesis in Mouse Hepatocytes
Toxicologic Pathology, 1984
Mouse primary liver cell cultures were examined for evidence of unscheduled DNA synthesis (UDS) following treatment with the carcinogens; dimethylnitrosamine (DMNA), diethylnitrosamine (DENA), 2-acetylaminofluorene (2-AAF), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), benzo(a)pyrene (BP), dimethylbenzanthracene (DMBA), l,l,-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), safrole, diethylstilbestrol (DES), aflatoxin Bl (AFBl), and dieldrin and the noncarcinogens; dimethylformamide (DMF), fluorene, and pyrene. Mouse hepatocyte cultures were simultaneously treated with three concentrations of each compound and 'H-thymidine. After 24 hrs, cells were fixed and processed for autoradiography. 'H-thymidine incorporation in both experimental and control cell nuclei, as evidenced by autoradiographic grains, was quantitated microscopically. DMNA, DENA, 2-AAF, MNNC, BP, AFB, and DMBA significantly increased UDS over untreated cells at all concentrations studied. DDT, DMF, fluorene, pyrene, safrole, DES, and dieldrin were negative for UDS in all concentrations examined. DMNA, 2-AAF and MNNG were also studied for UDS induction in 2 hr old, 1 day old and 4 day old cultures. A progressive decrease in UDS with increased time after plating was found in DMNA and 2-AAF treated cultures. After 4 days DMNA and 2-AAF induced UDS only at the highest concentrations examined (lo-' M and M respectively). MNNG induced UDS at all time periods and concentrations sampled. An attempt to enhance the sensitivity of the UDS assay by inducing the mixed function oxidative enzyme activity in the hepatocytes with phenobarbital administered in vivo resulted in no statistically significant increase in UDS with DMNA, 2-AAF, MNNG, DDT, and dieldrin when compared with cells from non-induced animals. ' To whom reprints should be addrcsscd at hiedical College of Ohio. Dcpartmcnt of I'athology. 3000 Arlington Avenue.
Cancer research, 1982
The metabolism of the mutagen and carcinogen, 5-nitroacenaphthene, by the 9000 x g supernatant from the livers of Aroclor-pretreated rats was studied. The major primary metabolites were 1-hydroxy-5-nitroacenaphthene and 2-hydroxy-5-nitroacenaphthene. These metabolites were oxidized to 1-oxo-5-nitroacenaphthene and 2-oxo-5-nitroacenaphthene, hydroxylated to cis-1,2-dihydroxy-5-nitroacenaphthene and trans-1,2-dihydroxy-5-nitroacenaphthene, and reduced to 1-hydroxy-5-aminoacenaphthene and 2-hydroxy-5-aminoacenaphthene. Reduction of 1- and 2-oxo-5-nitroacenaphthene to 1-oxo- and 2-oxo-5-aminoacenaphthene was also observed. When incubations were carried out in a N2-enriched atmosphere (10% O2 in N2), the major metabolites were 1-hydroxy- and 2-hydroxy-5-nitroacenaphthene and 2-oxo-5-aminoacenaphthene. Selected metabolites were tested for mutagenicity toward Salmonella typhimurium TA 98. The most mutagenic of the metabolites tested, in the presence or absence of rat liver 9000 x g superna...
The Egyptian Journal of Forensic Sciences and Applied Toxicology, 2022
Many pesticides are used together or in a method, eventually resulting in multiple exposures. These mixtures can lead to unpredicted adverse health consequences in the exposed population. Numerous studies on individual risk assessments are available, but combined usage's toxicity is still to estimate. So, the current study investigated hepatotoxicity induced by exposure to deltamethrin (DLT) and dimethoate (DM) and their mixture in adult male albino rats. Methods: Forty adult male albino rats were randomized into five equal groups (n=8); Group I: control, Group II: the vehicle group received 1ml corn oil. Group III: received DLT (5 mg/kg b.w.), Group IV: received DM (20 mg/kg b.w.), Group V: received DLT (5 mg/kg b.w) and DM (20 mg/kg b.w). Treatments were orally gavaged once-daily dose for twelve weeks. Results: Separate DLT and DM exposure significantly induced an increase in serum liver enzymes, including aspartate aminotransferase, alanine aminotransferase, alkaline transferase, and lactate dehydrogenase, bilirubin, and liver malondialdehyde levels accompanied by a decrease in serum total protein, plasma cholinesterase enzyme, liver superoxide dismutase, catalase, and reduced glutathione levels. These biochemical alterations were supported by the lesions observed in histological sections, decreasing the expression of Bcl-2 and DNA damage and genotoxicity detected by the comet assay. Conclusion: Co-administration of DLT and DM aggravated hepatic dysfunction, exhausting the endogenous antioxidant status and down-regulating the expression of Bcl-2. Thus, these mixtures can increase the harmful effects of each compound on the liver.