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.

Genotoxicity of N-nitrosochlordiazepoxide in cultured mammalian cells.

Chlordiazepoxide, a benzodiazepine derivative commonly used for the treatment of anxiety, was found to react with sodium nitrite in HCl aqueous solution yielding, at pH ranging from 0.5 to 5,N-nitrosochlordiazepoxide (NO-CDE). In the absence of a metabolic activation system, a dose-dependent frequency of DNA single-strand breaks was revealed by the alkaline elution technique in V79 cells exposed to subtoxic NO-CDE concentrations ranging from 33 to 330 microM. DNA lesions were only partially repaired within 48 hr, and their promutagenic character was demonstrated by the induction of 6-thioguanine resistance in the same cells. The genotoxicity of NO-CDE was confirmed by results obtained in metabolically competent primary cultures of both rat and human hepatocytes, which displayed similar dose-related amounts of DNA fragmentation and of DNA repair synthesis after treatment with concentrations ranging from 33 to 1000 microM. In conclusions similar to those which might occur in the stomach of a patient taking chlordiazepoxide the concentration of NO-CDE in the reaction mixture (50 microM) was of the same order as the concentrations found to induce a genotoxic effect in cultured mammalian cells.

Cytotoxic, DNA-damaging and mutagenic properties of 2,6-dimethoxy-1,4-benzoquinone, formed by dimethophrine-nitrite interaction.

In conditions similar to those occurring in the stomach, the sympathomimetic drug dimethophrine was found to react with nitrite yielding 2,6-dimethoxy-1,4-benzoquinone (DMBQ). The in vitro and in vivo studies carried out to evaluate the capability of DMBQ to produce cytotoxic and genotoxic effects provided the following results. A dose-related reduction of V79 cells plating efficiency was observed for DMBQ concentrations ranging from 10 to 80 microM; a similar reduction in the fraction of viable cells excluding trypan blue occurred after exposure to 4-fold higher concentrations. A dose-dependent amount of DNA fragmentation was revealed by the alkaline elution technique either in V79 cells exposed to DMBQ concentrations ranging from 10 to 80 microM or in kidney, gastric mucosa and brain of rats treated with single p.o. doses ranging from 33 to 300 mg/kg. Both in vitro and in vivo DNA lesions were largely repaired within 24 hr, but their promutagenic character was demonstrated by the induction of 6-thioguanine-resistance in V79 cells. Primary cultures of rat hepatocytes displayed a greater resistance to the cytotoxic and DNA-damaging activities of DMBQ, and did not exhibit a clear evidence of DNA repair synthesis. Similarly, DNA fragmentation was practically undetectable in the rat liver. Therefore, DMBQ should be considered as a direct-acting genotoxic chemical which is metabolized to less, or nonreactive, species. These findings suggest that DMBQ could produce genotoxic effects in patients taking dimethophrine.

Mutagenicity of 4-hydroxynonenal in V79 Chinese hamster cells.

4-Hydroxynonenal (HNE), a major product of the peroxidation of liver microsomal lipids, was examined for mutagenic activity at the hypoxanthine-guanine phosphoribosyltransferase locus in V79 Chinese hamster lung cells. At concentrations ranging from 10 to 45 microM, HNE induced a dose-dependent increase in the number of mutations to 6-thioguanine resistance, which reached the level of 4.7X baseline at the highest concentration tested.

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.

Scintillometric determination of unscheduled DNA synthesis in primary cultures of rat-liver cells. A hydroxylapatite batch assay.

A new procedure has been examined for measuring unscheduled DNA synthesis (UDS) in hepatocyte primary cultures by liquid-scintillation counting. DNA of the hepatocyte lysates was eluted with K-phosphate buffers after absorption on hydroxylapatite in order to reduce the background produced by cytoplasmic radioactivity. To inhibit hepatocyte replicative synthesis, hydroxyurea (10 mM) and cytosine arabinoside (80 microM) were added to the cultures. This procedure was found capable of detecting UDS elicited by 0.3 - 10 mM N-nitrosodimethylamine.

Formation of DNA-damaging nitroso compounds by interaction of drugs with nitrite. A preliminary screening for detecting potentially hazardous drugs.

Fifty-seven theoretically nitrosatable widely used drugs that are commonly administered orally have been screened to determine the formation of nitroso compounds by drug-nitrite interaction and to evaluate the genotoxicity of their nitrosation products against Chinese hamster ovary (CHO) cells, measured as DNA-damaging potency by the alkaline elution technique. The drug (0.1 mmol) was reacted with NaNO2 (0.4 mmol) at pH 3-3.5 for 1 h. Nitroso compounds were present in varying yield in the nitrosation mixture of 47 drugs. Twenty-two drugs formed direct-acting nitroso compounds capable of producing DNA fragmentation, i.e., a statistically significant (p less than 0.01) increase in the elution rate of CHO cell DNA. On a molar basis, their DNA-damaging potency varied over a 570-fold range, with 12 exhibiting greater potency than that of N-nitroso-N-methylurea.

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.

Viscometric detection of liver DNA fragmentation in rats treated with minimal doses of chemical carcinogens.

A new technique, using an oscillating viscometer capable of measuring changes of DNA reduced viscosity (eta red), has been used to detect DNA damage in liver of rats treated with various chemical carcinogens. In denaturing conditions (pH 12.5), the eta red of liver DNA from control rats increased slowly with time, reaching a maximum, (eta red)max, after 10 to 13 hr. Single i.p. doses of N-nitrosodimethylamine (0.07 mg/kg), N-nitrosodiethylamine (0.2 mg/kg), N-nitroso-N-methylurea (0.5 mg/kg), 1,2-dimethylhydrazine (0.06 mg/kg), procarbazine (1 mg/kg), methyl methanesulfonate (8 mg/kg), and N-diazoacetylglycine amide (3.7 mg/kg) induced a statistically significant reduction of the time (t95) required for eta red to reach its maximal value. A dose-dependent decrease of t95 was observed for dosages markedly lower than those found to be effective in eliciting DNA fragmentation by the use of alkaline elution or alkaline sucrose gradient sedimentation. 2-Acetylaminofluorene (12.5 mg/kg) and 4-nitroquinoline 1-oxide (10 mg/kg) caused a clear-cut increase of (eta red)max. 7,12-Dimethylbenz(a)anthracene (10 mg/kg) markedly prolonged t95. This viscometric assay of in vivo DNA damage allows a reliable assessment of DNA lesions induced by doses of chemical carcinogens sufficiently small not to produce significant alterations in the pharmacokinetic behavior of these compounds.

In vivo and in vitro genotoxicity of three antihypertensive hydrazine derivatives (hydralazine, dihydralazine, and endralazine).

Three antihypertensive hydrazine derivatives (hydralazine, dihydralazine, and endralazine) were found to be genotoxic in four in vivo or in vitro short-term test systems. a) In mice, a single ip administration of the LD50 of the three drugs caused a small but statistically significant increase over controls in DNA elution rate, ie, a modest amount of DNA fragmentation, in three of the four organs (liver, lung, kidney, and spleen) tested, DNA damage being absent in lung for hydralazine and endralazine and in liver for dihydralazine. Only for hydralazine DNA lesions were always repaired within 12 hr, in agreement with the constant lack of cumulative effects in mice given five successive daily doses. The rank of potencies was hydralazine greater than dihydralazine greater than endralazine. b) In mice bone marrow cells, all three hydrazine derivatives induced a modest but statistically significant increase over controls in the frequency of sister chromatid exchanges, the rank of potencies being in this case dihydralazine greater than endralazine greater than hydralazine. c) In the Ames reversion test all three drugs behaved as direct-acting mutagens of low potency, whose activity was not influenced by rat liver nor by mouse liver or lung S-9 fractions. Hydralazine and dihydralazine elicited mixed genetic mechanisms of mutations, while endralazine exclusively induced frameshift errors in Salmonella DNA. The recently developed strain TA97 was the most efficient in revealing frameshift errors with all three drugs. d) The selective lethality assays in a battery of two S typhimurium and five E coli strains confirmed the direct genotoxicity of hydralazine, dihydralazine, and endralazine, in order of potency. Potency was evaluated by means of a sensitive and reliable micromethod procedure. Among those investigated, the recA recombination repair and the lexA post-replication repair ("SOS functions") and, to a lesser extent, also the polymerase I mechanism, appeared to contribute to the specific DNA repair with all three drugs, while excision repair systems (uvrA and uvrB) did not appear to be involved.