Alessandra Allavena

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.

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.

Low clastogenic activity in vivo of the N-nitroso derivatives of 5 β-adrenergic-blocking drugs proved to be potent genotoxins in vitro

The N-nitroso derivatives formed by the in vitro reaction of 5 beta-adrenergic-blocking drugs with sodium nitrite and previously found to induce DNA fragmentation and repair in primary cultures of both rat and human hepatocytes were tested for their ability to exert a clastogenic effect in vivo. In partially hepatectomized rats given by gavage a single dose of 1000 mg/kg, all 5 N-nitroso derivatives caused a statistically significant increase in the frequency of micronucleated hepatocytes, the clastogenic potencies of NO-propranolol, NO-metoprolol, and NO-nadolol being slightly greater than those of NO-atenolol and NO-sotalol. In contrast, none of them produced a significant increase in the frequency of micronucleated polychromatic erythrocytes in the bone marrow and the spleen. For all 5 N-nitroso derivatives the in vivo clastogenic potency, i.e. the ratio between the number of micronuclei observed in the liver and the dose administered, was markedly lower than the in vitro DNA-damaging potency calculated as the ratio between the amount of DNA fragmentation and the concentration tested. This suggests a preferential detoxification in vivo of the 5 N-nitroso derivatives.

Comparison in human and rat hepatocytes of the DNA-damaging activity of five chemicals probably carcinogenic to humans

Five chemicals-acrylonitrile, adriamycin, bischloroethyl nitrosourea, phenacetin and procarbazine-classified by the International Agency for Research on Cancer as probably carcinogenic to humans were assayed for DNA-damaging activity in primary cultures of human and rat hepatocytes in order to assess possible interspecies differences that might cast doubt on the extrapolation to humans of results obtained in rodents. DNA damage was measured by the alkaline elution technique. In the range of subtoxic concentrations indicated, dose-related increases in the frequencies of DNA single-strand breaks were induced in cells of both species by acrylonitrile (1.0-5.6 mm) and procarbazine (5.6-18 mm), whereas phenacetin was inactive up to the maximal soluble dose (3.2 mm). Adriamycin (1.8-5.6 mum) and bischloroethyl nitrosourea (18-56 mum) produced in cells of both species dose-dependent increases in the frequencies of both DNA breaks and cross-links. The responses of human hepatocytes were qualitatively similar to those of rat hepatocytes, but modest statistically significant differences between the two species in the average frequencies of DNA lesions were observed with the four active agents: the amount of DNA damage was greater in rat than in human hepatocytes with acrylonitrile (1.7-fold), adriamycin (1.4-fold), and BCNU (1.3-fold), whereas procarbazine was more genotoxic (1.4-fold) for human hepatocytes. However, as the interindividual variability of the response was greater than that occurring between the two species, the results should be interpreted as indicating that rat hepatocytes are good predictors of metabolic activation/detoxification and DNA-damaging activity in humans for the five chemicals studied.