Luigi Robbiano

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.

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.

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.

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.

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.

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.

Update of carcinogenicity studies in animals and humans of 535 marketed pharmaceuticals.

This survey is a compendium of information retrieved on carcinogenicity in animals and humans of 535 marketed pharmaceuticals whose expected clinical use is continuous for at least 6 months or intermittent over an extended period of time. Of the 535 drugs, 530 have the result of at least one carcinogenicity assay in animals, and 279 (52.1%) of them gave a positive response in at least one assay. Only 186 drugs (34.8%) have retrievable information on carcinogenicity in humans, and 104 of them gave to a variable extent evidence of a potential carcinogenic activity. Concerning the correlation between results obtained in animals and epidemiological findings, 58 drugs gave at least one positive result in carcinogenicity assays performed in animals and to a variable extent displayed evidence of carcinogenicity in humans, but 97 drugs tested positive in animals and were noncarcinogenic in humans or vice versa. Our findings, which are in agreement with previous studies, indicate that the evaluation of the benefit/carcinogenic risk ratio should be always made in prescribing a drug.

Physico-chemical model for DNA alkaline elution: New experimental evidence and differential role of DNA length, chain flexibility and superpacking

For a better understanding of data provided by DNA alkaline elution technique, a new analytical model has been developed which takes into consideration both the physicochemical properties of in situ DNA strand (length and flexibility/superpacking) and the geometric and hydrodynamic configuration of the elution apparatus (flow and filter conditions). Simulation by this model of experimental data previously obtained before and after carcinogens administration, has shown that for constant flow and filter conditions elution profiles are dependent, not only from DNA molecular weight, but also from a parameter critically related to modifications in chain flexibility/superpacking. This has been confirmed by several independent observations, including the time-dependent changes in non-denaturing lysing solution monitored by hydroxylapatite and alkaline elution techniques.

Correlation between induction of DNA fragmentation in urinary bladder cells from rats and humans and tissue-specific carcinogenic activity.

Seven chemicals, six of which are known to induce epithelial neoplasms of the urinary bladder in rats, were assayed for their ability to induce DNA damage in primary cultures of rat and human cells from urinary bladder mucosa, and in urinary bladder, liver and kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, were obtained in cells from both rats and humans with the following concentrations of five test compounds: 2-naphthylamine and N-nitrosodi-n-butylamine 0.5 and 1 mM, phenacetin 2 and 4 mM, cyclophosphamide from 2 to 8 mM, and o-toluidine 16 and 32 mM. Nitrilotriacetic acid (1-4 mM), a rat bladder carcinogen, and 4-aminobiphenyl (0.125-0.5 mM), a bladder carcinogen in humans but not in rats, gave a weak positive response in rats cells and a more marked response in humans cells. In terms of DNA-damaging potency, 4-aminobiphenyl, cyclophosphamide, phenacetin and 4 nitrilotriacetic acid were more active in human than in rat cells, whereas the converse occurred with 2-naphthylamine. Consistently with the results observed in vitro statistically significant dose-dependent increases in the average frequency of DNA breaks were detected in the urinary bladder mucosa of rats given p.o. single doses corresponding to 14 and 12 LD50 of six of the seven test compounds; the only one which gave a substantially negative response was 4-aminobiphenyl. With the exception of N-nitrosodi-n-butylamine which caused DNA damage in liver and of phenacetin and nitrilotriacetic acid which caused damage in kidney in agreement with their tumorigenic activity, any substantial evidence of DNA lesions in these two organs was absent in rats treated with 12 LD50 of the other 4 test compounds. These findings give evidence that urinary bladder genotoxic carcinogens may be identified by the DNA damage/Comet assay using as targets cells of urinary bladder mucosa, and show that the effect may be quantitatively different in cells from rats and from human donors.

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.