M. Cavanna

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.

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.

Studies on the in vivo activity of E. coli L-tryptophanase

Summary Suitable amounts of a purified E. coli tryptophanase for in vivo studies were obtained from the mutant strain B/1t7A, a very rich source of enzyme. The activity in vivo has been assayed in mice. Three-four hours after a single dose, ranging from 3,400 to 10,200 I.U./Kg, a reduction of L-tryptophan plasmatic levels of about 35% was observed. Moreover, the following parameters were evaluated: a) the plasma clearance rate of administered enzyme, resulting in a half life of 115 minutes (0.05 confidence limits : 73–272 minutes); b) the plasma clearance rate of administered pyridoral-5-phosphate as coenzyme, resulting in a half life of 17 minutes (0.05 confidence limits : 14–23 minutes); c) the V max and the substrate affinity of the enzyme in rat plasma (K m = 5.9 × 10 −4 M).

DNA Alkaline Elution: Physical Basis of the Elution Process and Validation of this Method as a Screening Procedure to Identify Chemical Carcinogens

The alkaline elution technique, developed by Kohn and coworkers (Kohn and Ewig, 1973; Kohn et al., 1976; Kohn, 1979), provides a sensitive measurement of DNA single-strand size distribution in mammalian cells and represents an advanced alternative to the technique of alkaline sucrose gradient sedimentation. Alkaline elution of DNA from cell lysates allows size measurement of DNA single strands longer than the sizes that can be measured by sedimentation in alkaline sucrose gradients. The elution technique requires relatively simple and not expensive apparatuses, and is less time-consuming in comparison with the latter. Moreover, it gives more repeatable results and is more suitable to assess DNA damage from various tissues following in vivo exposure to DNA interacting agents. In the last few years, this technique has been widely applied by different groups of researchers to the study of various DNA lesions induced by several physical or chemical agents (ranging from carcinogens to antineoplastic agents), and a large series of results have been collected. The aims of this review are: to discuss the physical basis that governs DNA elution, to summarize the possible application of this method, to revise the use of alkaline elution and its reliability in the assessment of DNA fragmentation in various organs of mice and rats following in vivo exposure to chemical agents, and to evaluate critically its role as a screening procedure for carcinogenic compounds.

Evaluation of DNA damage and repair in mammalian cells exposed to chemical carcinogens. Methods potentially useful as short term prescreening tests.

Summary Methods for detecting damage and repair of DNA, induced in mammalian cells by chemical carcinogens, are examined and discussed in the perspective of their capability to predict the oncogenic potential of chemical agents. Technical aspects and possibilities of the autoradiographic method of evaluation of unscheduled DNA synthesis are considered. The limits of the experimental models employing mammalian cells cultured “in vitro” are underlined, and the advantages of the techniques able to detect DNA damage caused “in vivo” are evidenced. Results up to now obtained with the new method — “in vivo” DNA damage/alkaline elution assay — are presented. The possible advantages of such a test in comparison with the alkaline sucrose gradient sedimentation technique are discussed.

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.

Detection by alkaline elution of rat liver DNA damage induced by simultaneous subacute administration of nitrite and aminopyrine

DNA fragmentation induced in the livers of rats by oral treatment with NaNO2 and aminopyrine was evaluated by the alkaline elution technique. Whereas simultaneous administration of the two compounds in a single dose produced only a minimal increase of the DNA elution rate, their intake with drinking water for 20 successive days caused DNA fragmentation comparable to that observed after a single ip injection of 10-20 mg/kg N-nitrosodimethylamine. Either NaNO2 or aminopyrine alone induced borderline DNA damaging effects, if any, in both rats receiving a single dose and those treated for 20 successive days.

Genotoxic activity of five antidepressant hydrazines in a battery of in vivo and in vitro short-term tests.

Five antidepressant agents (monoamine oxidase inhibitors) having a hydrazino group--phenelzine, nialamide, mebanazine, isocarboxazid, and iproniazid--were assayed in four in vivo or in vitro short-term tests predictive of the potential carcinogenicity of chemicals. (1) All the compounds tested except iproniazid, produced DNA fragmentation, as evaluated by the alkaline elution technique, in liver and/or lung cells of mice treated ip or po. (2) All the compounds except mebanazine (which was no longer available for testing) were weak inducers of sister chromatid exchanges in bone marrow cells of mice treated ip. (3) Phenelzine and nialamide elicited base-pair substitutions and mebanazine elicited frameshift errors in his- Salmonella typhimurium. S9 mix containing rat liver, mouse liver, or mouse lung S9 fractions had variable effects on mutagenicity. (4) The same three compounds were positive in a DNA repair bacterial test with five trp- Escherichia coli strains lacking a variety of repair mechanisms (uvrA, polA, recA, lexA) or incorporating plasmids (R391).

DNA damage and repair by alkaline elution in N-diazo-acetylglycine amide-treated cells

Abstract In mouse fibroblast-like cells treated in vitro with N-diazoacetylglycine amide (DGA), the DNA damage and repair were evaluated by means of the alkaline elution technique. The results show that DGA induces a dose-dependent damage of DNA which is repaired only slowly. The DGA-induced damage increases for the first 2 h allowed for repair, regresses up to that displayed at the end of exposure period after 4–24 h of repair, and is evidently lower after a 48-h repair period. These results agree with the demonstrated mutagenic and carcinogenic activity of DGA.

Induction of tumors in newborn and adult Swiss mice by N-diazoacetylglycine amide

Abstract Short treatments with relatively high doses of N-diazoacetylglycine amide induced, in newborn and in adult Swiss mice, pulmonary tumors and lymphatic leukemia. The carcinogenic effect of this compound, which also exerts antineoplastic and immunodepressive activity, may be related to its alkylating action.