Comparative study of DNA damage and repair induced by ten N-nitroso compounds in primary cultures of human and rat hepatocytes.

Ten carcinogenic N-nitroso compounds were assayed for DNA-damaging activity in primary cultures of human and rat hepatocytes. DNA fragmentation was measured by the alkaline elution technique, and unscheduled DNA synthesis by quantitative autoradiography. Positive dose-related responses in the range of subtoxic concentrations indicated were obtained in cells of both species with N-nitrosodiethylamine (10-32 mM), N-nitrosodi-n-propylamine (1.8-10 mM), N-nitrosomorpholine (1-3.2 mM), N-nitrosopiperidine (1-3.2 mM), N-nitrosopyrrolidine (3.2-18 mM), N-nitroso-N-methylurea (0.32-1.8 mM), N-nitroso-N-ethylurea (0.32-1.8 mM), and N-nitroso-N-butylurea (0.1-0.32 mM). N-nitrosodi-n-butylamine was practically inactive at the maximal soluble concentration (1 mM). The responses of human hepatocytes were qualitatively similar to those of rat hepatocytes, but statistically significant differences between the two species in the amounts of DNA damage and/or unscheduled DNA synthesis were observed with N-nitrosodimethylamine, N-nitrosomorpholine, N-nitrosopiperidine, N-nitrosopyrrolidine, and N-nitroso-N-butylurea. On the other hand, quantitative differences in the genotoxic effects induced by 5 mM N-nitrosodimethylamine in cultures derived from 20 human donors and from 20 rats were greater than average interspecies differences displayed by this nitrosamine and by other N-nitroso compounds. These results indicate that the rat hepatocyte DNA repair assay is a valid model for predicting the genotoxic potential of N-nitroso compounds in human hepatocytes.

Sequential analysis of DNA damage and repair during the development of carcinogen-induced rat liver hyperplastic lesions.

The level of DNA fragmentation, as evaluated by alkaline elution, and of unscheduled DNA synthesis (UDS), as measured by autoradiography, was determined in the parenchymal cells from the entire liver during the development of hyperplastic lesions induced in the rat by the following treatment: diethylnitrosamine (DEN) (200 mg/kg i.p.) on Day 0; CCl4 (2 ml/kg intragastrically) on Day 21; dietary administration of 0.02% 2-acetylaminofluorene during the third and the fourth wk; and of 0.05% phenobarbital from the sixth wk. Both DNA fragmentation and UDS were constantly detected, concomitantly with the presence of gamma-glutamyltransferase (gamma-GT)-positive hepatocytes, in the primary cultures derived from the liver of rats of this experimental group sacrificed at 4, 5, 6, and 7 wk after DEN injection, their amount being approximately the same at the fourth and at seventh wk. Moreover, evidence of DNA alterations was still present, albeit diminished, 22 wk after the beginning of treatment. In contrast, DNA fragmentation and UDS did not persist past the fifth wk, and gamma-GT-positive hepatocytes were very few or totally absent in hepatocyte primary cultures from control rats treated with DEN alone, 2-acetylaminofluorene alone, or 2-acetylaminofluorene:CCl4. CCl4 alone, and phenobarbital alone caused only a modest, albeit statistically significant, increase in DNA elution rate and UDS, respectively. In a comparison performed on hepatocyte primary cultures obtained from rats of the experimental group sacrificed at the fifth wk after the injection of DEN, the level of UDS was higher in gamma-GT-positive than in gamma-GT-negative hepatocytes. These results indicate that the regimen used to induce the selective proliferation of initiated hepatocytes actually produces extensive DNA lesions which can give rise to additional carcinogenic initiations.

DNA-damaging activity in vivo and bacterial mutagenicity of sixteen hydrazine derivatives as related quantitatively to their carcinogenicity.

Sixteen hydrazine derivatives (hydrazine, 1,1-dimethylhydrazine, 1,2-dimethylhydrazine, phenylhydrazine, procarbazine, isoniazid, isocarboxazid, nialamide, 2,4-dinitrophenylhydrazine, phenelzine, hydralazine, dihydralazine, carbamylhydrazine, mebanazine, iproniazid, and 1-carbamyl-2-phenylhydrazine) were tested for DNA-damaging activity by the alkaline elution technique and for mutagenic activity in the Salmonella-microsome (Ames) test. The first nine compounds listed (56%) were found to induce a significant DNA fragmentation in the liver and/or in the lung of i.p.-treated male Swiss mice. The DNA-damaging potency varied over an approximately 30-fold range. Thirteen of the first 14 compounds listed (81% of the total), isocarboxazid being inactive, were positive in the Ames test, with a broad range of activity towards the five bacterial strains of Salmonella typhimurium used (TA1535, TA100, TA1537. TA1538, and TA98) and of metabolic behavior in the presence of S-9 mix containing rat liver, mouse liver, or mouse lung postmitochondrial preparations from Aroclor-treated animals. The mutagenic potency varied over an almost 7000-fold range. For 11 of the 16 hydrazine derivatives tested, homogeneous carcinogenicity data (induction of pulmonary tumors in mice chronically treated p.o.) were available from literature. Elaboration of these data showed that carcinogenic potency varied over an approximately 1900-fold range. The five most potent carcinogens were all positive in the DNA damage test. Their carcinogenic potency varied over a 130-fold rage and their DNA-damaging potency varied over a 22-fold range. DNA-damaging potency seemed to vary on a more compressed scale, but regression analysis indicated the existence of a strong positive correlation between in vivo DNA-damaging and carcinogenic potencies, while a lack of correlation was found between mutagenic and carcinogenic potencies. There was no correlation between DNA-damaging and mutagenic potencies.

Formation of the N-nitroso derivatives of six beta-adrenergic-blocking agents and their genotoxic effects in rat and human hepatocytes.

Six beta-adrenergic-blocking drugs, atenolol, metoprolol, nadolol, oxprenolol, propranolol and sotalol, were found to react with sodium nitrite in HCl solution, yielding the corresponding N-nitrosamines. The genotoxic activity of the six nitrosamines was evaluated in primary cultures of both rat and human hepatocytes; DNA fragmentation was measured by the alkaline elution technique, and DNA repair synthesis by quantitative autoradiography. Positive dose-related responses were produced in cells of both species after 20 h of exposure to the following subtoxic concentrations: NO-propranolol, 0.01-0.1 mM; NO-oxprenolol, 0.03-1 mM; NO-atenolol and NO-metoprolol, 0.1-1 mM; and NO-nadolol and NO-sotalol, 0.3-3 mM. Modest but statistically significant differences between the DNA-damaging potencies for the two species were observed with NO-atenolol and NO-oxprenolol, which were both more active against rat hepatocytes, and with NO-propranolol, which was more active against human hepatocytes. At equal or higher concentrations, the six N-nitrosamines did not produce DNA fragmentation in Chinese hamster lung V79 cells; this indicates that they behave as indirectly acting compounds, which need to be transformed into reactive metabolites in order to exert a genotoxic effect.

DNA fragmentation by 2-nitropropane in rat tissues, and effects of the modulation of biotransformation processes.

The occurrence and persistence of DNA fragmentation, as detected by the alkaline elution technique, have been studied in rats treated with single oral doses of the hepatocarcinogen 2-nitropropane (2-NP). A progressive increase of liver DNA fragmentation was observed at doses ranging from 0.5 to 8 mmol/kg; single strand breaks reached the maximum frequency 6 h after administration, and were partially reduced after 36 h. In contrast, DNA fragmentation was absent in lung, kidney, bone marrow and brain of rats given 8 mmol/kg. The role of cytochrome P-450 in the activation of 2-NP is indicated by the increase of liver DNA damage in rats pretreated with phenobarbital or beta-naphtoflavone, and by its reduction produced by methoxsalen. Both administration of GSH and GSH depletion did not result in clearcut modifications of the genotoxic effect of 2-NP for the liver.

Induction of DNA fragmentation and DNA repair synthesis in human and rat hepatocytes by diethylstilbestrol.

The synthetic estrogen diethylstilbestrol (DES), a known human carcinogen, was examined for cytotoxicity, and the induction of DNA damage and repair in primary cultures of human and rat hepatocytes. In both species concentrations of DES ranging from 5.6 to 18 micrograms/ml constantly produced reduction of cell viability and DNA fragmentation in dose-related amounts. However, large individual quantitative differences in the sensitivity to the cytotoxic and DNA-damaging activities of DES were observed among cultures derived from the 5 human donors. DES capability of eliciting DNA-excision repair was weak but statistically significant in both human and rat hepatocytes. Taken as a whole these results contribute to support the hypothesis of a genotoxic mechanism in DES-induced carcinogenesis.

A study of the potential genotoxicity of cimetidine using human hepatocyte primary cultures: discrepancy from results obtained in rat hepatocytes.

The genotoxicity of cimetidine, a drug widely used in the treatment of peptic ulcer, was examined in human hepatocyte primary cultures. No induction of unscheduled DNA synthesis, as detected by autoradiography, or of DNA fragmentation, as measured by alkaline elution, was seen in metabolically competent human hepatocytes exposed for 20 h to cimetidine concentrations ranging from 0.33 to 9 mM. These findings, which are in contrast with the previously observed capability of cimetidine to induce DNA damage and repair in rat hepatocyte primary cultures, suggest that for some chemicals the rat hepatocyte model might be an inappropriate predictor of potential genotoxic effects in the analogous human cells.

DNA damage induced by 3,3'-dimethoxybenzidine in liver and urinary bladder cells of rats and humans.

3,3'-Dimethoxybenzidine (DMB), a congener of benzidine used in the dye industry and previously found to be carcinogenic in rats, was evaluated for its genotoxic activity in primary cultures of rat and human hepatocytes and of cells from human urinary bladder mucosa, as well as in liver and bladder mucosa of intact rats. A similar modest 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 DMB concentrations ranging from 56 to 180 microM. Replicating rat hepatocytes displayed a modest increase in the frequency of micronucleated cells after a 48-h exposure to 100 and 180 microM concentrations. In primary cultures of human urinary bladder mucosa cells exposed for 20 h to 100 and 180 microM DMB, the Comet assay revealed a clear-cut increase of DNA fragmentation. In rats given one-half LD50 of DMB as a single oral dose, the GSH level was reduced in both the liver and urinary bladder mucosa, whereas DNA fragmentation was detected only in the bladder mucosa. Taken as a whole, these results suggest that DMB should be considered a potentially genotoxic chemical in both rats and humans; the selective effect on the rat urinary bladder might be the consequence of pharmacokinetic behavior.

Toxicity of jellyfish and sea-anemone venoms on cultured V79 cells.

Cnidarian toxins exert an influence on human activities and public health. The cytotoxicity of crude toxins (nematocyst and surrounding tissue venom) of Aequorea aequorea, Rhizostoma pulmo and Anemonia sulcata was assessed on V79 cells. Rhizostoma pulmo and Anemonia sulcata crude venoms showed remarkable cytotoxicity and killed all treated cells at highest tested concentration within 2 and 3 hr, respectively. Aequorea aequorea crude venom greatly affected growth rate during long-term experiments. No genotoxic effect was observed.

Cytotoxicity of the venom of Pelagia noctiluca forskål (Cnidaria: Scyphozoa).

The activity of Pelagia noctiluca venom was never assessed on cultured cells; therefore, we have evaluated on V79 cells the cytotoxicity, genotoxicity and ATP depletion induced after treatment. Venom did not cause alteration on cell DNA, but showed remarkable cytotoxic properties. With the highest nematocyst concentration (150,000 nematocyst/ml) 74 and 39% cells survived after 1 and 3 h, respectively, when evaluated by Trypan blue. Treated cells showed increased ATP levels during the same time. Preliminary HPLC analyses have showed the occurrence of a protein containing peak.

Reduction of DNA repair efficiency during the initial phase of rat liver carcinogenesis.

Chemically induced DNA fragmentation and unscheduled DNA synthesis (UDS) were determined in hepatocyte primary cultures (HPC) obtained from the entire liver during the development of hyperplastic lesions induced in rats by the following treatment: 200 mg/kg i.p. of N-nitrosodiethylamine (DEN) on day 0; 2 ml/kg i.g. of CCl4 on day 21; dietary administration of 0.02% 2-acetylaminofluorene (2-AAF) during the 3rd and the 4th week, and of 0.05% phenobarbital (PB) from the 6th week. At 4, 5, 6, and 7 weeks after DEN injection, the level of DNA fragmentation elicited by either the activation-independent carcinogen methyl methanesulfonate (MMS) or the activation-dependent carcinogen N-nitrosodimethylamine (DMN) was significantly lower than that in HPC from age-matched normal rats. In the same HPC, a statistically significant decrease was observed in the capability of repairing DNA damage induced by MMS, while the reduction of the DNA repair efficiency did not reach the level of significance after exposure to DMN.

DNA damage induced by nitrosated ranitidine in cultured mammalian cells.

Ranitidine, a new H-2 receptor antagonist more potent than cimetidine in inhibiting gastric secretion, reacted under acid conditions with a twofold molar amount of nitrite (a nitrite/ranitidine ratio about 1000 times that likely to occur in gastric juice of treated humans) yielding a nitroso derivative capable of inducing a dose-dependent DNA fragmentation in cultured Chinese hamster ovary cells.

DNA damage induced by seven N-nitroso compounds in primary cultures of human and rat kidney cells.

Seven N-nitroso compounds (NOC), known to induce kidney tumors in rats, were assayed for DNA-damaging activity in primary cultures of human and rat kidney cells. DNA fragmentation was measured by the alkaline elution technique. Positive responses were obtained in cells of both species with N-nitrosodimethylamine (32 mM), N-nitrosodiethylamine (32 mM), N-nitrosodi-n-propylamine (10 mM), N-ethyl-N-hydroxyethylnitrosamine (18 mM), and streptozotocin (1 mM). N-nitrosodiethanolamine and N-nitrosomorpholine were inactive at the highest concentration tested (32 mM). The responses of human kidney cells were qualitatively similar to those of rat kidney cells, but statistically significant differences between the two species in the DNA-damaging potencies were observed with N-ethyl-N-hydroxyethylnitrosamine and streptozotocin, both more genotoxic in rat cells. Taken as a whole, the results suggest on the one hand that the five active NOC might be carcinogenic for the kidney in humans, and on the other hand that the rat kidney cell/DNA damage assay is a valid model for predicting the genotoxic potential of NOC in human kidney cells.

DNA-damaging activity of tripelennamine in primary cultures of human hepatocytes.

The genotoxicity of tripelennamine, an antihistamine used in the treatment of allergic disorders, was examined in human hepatocyte primary cultures derived from 3 different donors, after exposure to non-toxic concentrations ranging from 10 to 100 microM. A modest but statistically significant and dose-related amount of autoradiographic DNA repair was present in cultures from two donors. DNA fragmentation, as measured by alkaline elution, was found to occur in dose-dependent amounts in cultures of all the 3 donors. These findings, which agree with the previously observed capability of tripelennamine to induce DNA repair and fragmentation in rat hepatocytes, strengthen the suspicion of a potential genotoxic risk of this drug to humans.

Genotoxicity of cimetidine in primary cultures of rat hepatocytes.

The genotoxicity of cimetidine was examined in the hepatocyte primary culture/DNA-repair test and by the DNA-damage/alkaline-elution assay. A dose-dependent amount of unscheduled DNA synthesis was elicited by cimetidine, whereas DNA fragmentation occurred only in hepatocytes exposed to the highest (3 mM) concentration of the drug. These findings are in contrast with the negative results previously obtained in long-term and short-term carcinogenesis assays.

Increased frequency of micronucleated kidney cells in rats exposed to halogenated anaesthetics.

Six halogenated anaesthetics were tested for their ability to induce micronuclei formation in the rat kidney. A statistically significant increase in the frequency of micronucleated cells was detected in rats given a single p.o. dose of 4 mmol/kg of halothane (3.48 x baseline), chloroform (3.32 x baseline), trichloroethylene (3.24 x baseline), sevoflurane (2.98 x baseline), and isoflurane (2.95 x baseline). In contrast, the response was substantially negative in rats given the same dose of enflurane. As compared to controls, rats treated with halothane and trichloroethylene displayed a reduction in the frequency of binucleated cells presumably due to a toxicity-induced inhibition of cellular proliferation. These findings suggest a potential genotoxic activity of halogenated anaesthetics for the rat kidney.

An in vivo micronucleus assay for detecting the clastogenic effect in rat kidney cells.

A micronucleus assay in vivo has been developed that is based on the use of freshly isolated kidney cells from mononephrectomized rats. In this validation study, a statistically significant increase in the frequency of micronucleated cells was detected in rats given i.p. a single dose of four kidney carcinogens, N-nitrosodimethylamine, N-nitrosodiethylamine, N-ethyl-N-hydroxyethylnitrosamine and N-nitroso-N-methylurea. The clastogenic effect was more marked when the same dose was injected for 3 successive days. As compared to controls, treated rats displayed a reduction in the frequency of binucleated cells, presumably due to a toxicity-induced inhibition of cellular proliferation. The proposed method should be suitable for the detection of the clastogenic effect of procarcinogens biotransformed into reactive species in the kidney.

Genotoxicity testing of chloramphenicol in rodent and human cells.

The results of this work, carried out to extend the limited information at present available on the genotoxic potential of chloramphenicol (CAP), indicate that in millimolar concentrations this antibacterial agent produced a minimal amount of DNA fragmentation in both V79 cells and metabolically competent rat hepatocytes. Moreover, a level of DNA-repair synthesis indicative of a weak but positive response was detected in primary cultures of liver cells obtained from 2 of 3 human donors, and a borderline degree of repair was present in those prepared from rats. The promutagenic character of CAP-induced DNA lesions was confirmed by a low but significant increase in the frequency of 6-thioguanine-resistant clones of V79 cells, which, however, was absent when the exposure was done in the presence of co-cultured rat hepatocytes. Finally, oral administration to rats of 1/2 LD50 CAP did not increase the incidence of either micronucleated polychromatic erythrocytes or micronucleated hepatocytes. Taken as a whole these findings suggest that CAP should be considered a compound intrinsically capable of producing a very weak genotoxic effect, but only at concentrations about 25 times higher than those occurring in patients treated with maximal therapeutic dosages.

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.

DNA fragmentation by N-nitrosodimethylamine and methyl methanesulfonate in human hepatocyte primary cultures.

The ability of N-nitrosodimethylamine (DMN) and methyl methanesulfonate (MMS) to induce DNA damage in primary cultures of human hepatocytes was examined by the alkaline elution technique. Both the agents induced a dose-dependent increase in DNA elution rate, but appreciable differences in the degree of response to the procarcinogen DMN were observed among cultures obtained from the livers of four patients. A comparative analysis of DNA fragmentation indicated a substantial similarity between human and concurrently studied rat hepatocytes in their response to both DMN and MMS.