Evaluation of auramine genotoxicity in primary rat and human hepatocytes and in the intact rat.

Auramine, a dye previously found to be a liver carcinogen in both mice and rats, was evaluated for its DNA-damaging and clastogenic activities in primary cultures of rats and human hepatocytes and for the induction of DNA single-strand breaks in the liver and urinary bladder mucosa of intact rats. A similar dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic concentrations ranging from 10 to 32 microM. In contrast, neither rat nor human hepatocytes displayed an increased frequency of micronuclei after a 48-h exposure to the same auramine concentrations. In rats given a single oral dose of 125, 250 or 500 mg kg-1 auramine, the Comet assay revealed a significant increase in the frequency of DNA lesions in the liver and in the urinary bladder mucosa, the effect being slightly more marked in the liver. Taken as a whole and compared with previous findings, these results suggest that auramine is biotransformed into reactive species in target organs of both rats and humans, and that this dye might play by itself the main role in the increased incidence of bladder cancer which has been judged as causally related to its manufacture.

Testing of p-dichlorobenzene and hexachlorobenzene for their ability to induce DNA damage and micronucleus formation in primary cultures of rat and human hepatocytes.

The genotoxicity of p-dichlorobenzene (DCB) and hexachlorobenzene (HCB) was evaluated in primary cultures of rat and human hepatocytes. DNA fragmentation was measured by the alkaline elution technique and clastogenic activity by the increase in micronucleus formation. In rat hepatocytes, exposure to concentrations of DCB ranging from 0.56 to 3.2 mM and of HCB from 0.1 to 0.56 mM did not induce any significant increase in the frequency of DNA breaks but consistently produced a statistically significant increase in the frequency of micronucleated cells. In human hepatocytes, under the same experimental conditions, the response to DCB was negative in terms of both DNA fragmentation and clastogenic effect, whereas HCB produced a significant increase in the frequency of both DNA breaks and micronuclei. Taken as a whole these results suggest that of the two pesticides examined only HCB should be considered as a weak genotoxic carcinogen.

In vitro and in vivo testing of hydralazine genotoxicity.

Hydralazine (HDZ), a p.o. effective antihypertensive drug, was evaluated for its genotoxic effects in both rodent and human cultured cells and in the intact rat. Dose-dependent amounts of DNA fragmentation, as measured by the alkaline elution technique, and of DNA repair synthesis, as revealed by autoradiography, were produced in primary cultures of metabolically competent rat hepatocytes by subtoxic HDZ concentrations ranging from 0.32 to 1.0 mM. A similar potency in inducing DNA repair synthesis was displayed by HDZ in primary cultures of hepatocytes from four human donors. A modest reduction of both DNA fragmentation (-13%) and DNA repair (approximately -50%) was observed in hepatocytes obtained from rats pretreated with indomethacin in order to reduce prostaglandin synthetase activity. In contrast, neither in rat nor in human hepatocytes, differences in N-acetyltransferase activity resulted in meaningful changes of the same end points. V79 cells, which are essentially deficient of monooxygenases catalyzing the biotransformation of xenobiotics, were as sensitive as hepatocytes to the DNA-damaging activity of HDZ. Moreover, after exposure to 0.1 to 0.3 mM HDZ, a modest (2.1- to 2.8-fold), but significant, increase in the frequency of mutation to 6-thioguanine resistance was observed in V79 cells in the absence of a metabolic activation system. In rats, a single p.o. dose of 80 mg/kg produced a clastogenic effect in the liver, but not in the bone marrow, and the p.o. administration for 14 successive days of approximately 46 mg/kg/day increased the average diameter of liver basophilic foci initiated by diethylnitrosamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Testing of metoclopramide and procainamide for their ability to induce genotoxic effects in cultured mammalian cells.

Metoclopramide (MCA) and procainamide (PCA), two widely used benzamide drugs developed before the present regulatory climate but recently found to induce DNA breaks in human lymphocytes, were evaluated for their genotoxic effects in cultured rodent and human cells. In subtoxic concentrations neither MCA (from 0.10 to 0.32 mM) nor PCA (from 0.18 to 0.56 mM) induced DNA fragmentation and repair in primary cultures of metabolically competent rat and human hepatocytes. In the absence of metabolic activation a meaningful increase in the frequency of 6-thioguanine-resistant V79 cells was produced by the maximum tolerated concentration of MCA (3.2 mM), whereas PCA resulted nonmutagenic. Any clastogenic effect was absent in human lymphocytes exposed to MCA for 28 hr, but a statistically significant increase in the frequency of micronucleated cells was observed when the exposure was prolonged to 72 hr. In contrast, PCA was never clastogenic under the same experimental conditions. These results suggest that of the two benzamides tested only MCA should be considered potentially capable of producing mutagenic and clastogenic effects; it presumably behaves as an agent which is rapidly transformed by the liver into inactive metabolites, and the clinical relevance of its genotoxic activity remains to be ascertained.

Cytotoxic and genotoxic effects of five n-alkanals in primary cultures of rat and human hepatocytes.

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.

In vitro and in vivo evaluation of the genotoxicity of N-nitrosooxprenolol.

N-nitrosooxprenolol (NO-oxprenolol) might be formed in the stomach of patients taking the beta-adrenergic blocking drug, oxprenolol. This nitroso derivative has previously been shown to induce DNA damage and repair in both rat and human cultured hepatocytes. The results of the present study show that in the presence of co-cultured rat hepatocytes, 0.03 mM NO-oxprenolol produced a significant increase in the frequency of 6-thioguanine-resistant but not of ouabain-resistant mutants. No mutagenic activity was detected in the absence of metabolic activation. In mice, NO-oxprenolol (1 g/kg) increased the incidence of micronucleated cells in the liver but not in the bone marrow and the spleen. These results suggest that NO-oxprenolol, consistent with its chemical nature of nitrosamine, is biotransformed into short-lived reactive species.

Mutation induction in Chinese hamster lung V79 cells by five alk-2-enals produced by lipid peroxidation.

Five alk-2-enals--pent-2-enal, hex-2-enal, hept-2-enal, oct-2-enal and non-2-enal--produced by lipid peroxidation were tested for mutagenic activity in V79 Chinese hamster cells. At concentrations ranging from 0.003 to 0.3 mM all 5 alk-2-enals induced a dose-dependent increase in the frequency of 6-thioguanine-resistant mutants, and their mutagenic potency was found to increase with the length of the carbon chain. In contrast, only hept-2-enal produced a statistically significant increase in the number of mutations to ouabain resistance.

Mutagenicity in V79 Chinese hamster cells of n-alkanals produced by lipid peroxidation.

The mutagenicity for mammalian cells of five n-alkanals produced by lipid peroxidation was tested in V79 Chinese hamster lung cells either at the hypoxanthine-guanine phosphoribosyltransferase locus as resistance to 6-thioguanine or at the Na/K ATPase locus as resistance to ouabain. The results show that propanal, butanal, pentanal and hexanal induced a dose-dependent increase in the frequency over controls of both 6-thioguanine- and ouabain-resistant mutants at concentrations ranging from 3 to 30 mM. With nonanal the same effects were observed with concentrations of 0.1-0.3 mM.

Methylglyoxal-induced mutation to 6-thioguanine resistance in V79 cells.

Methylglyoxal, at concentrations ranging from 0.16 to 1.5 mM, caused a dose-dependent increase in the frequency of HGPRT-deficient mutants in V79 cells. Its mutagenic activity was reduced when V79 cells were co-cultured with freshly isolated rat hepatocytes.