Carlo Bennicelli

Genotoxic activity and potency of 135 compounds in the Ames reversion test and in a bacterial DNA-repair test

Compounds of various chemical classes were comparatively assayed in the Ames reversion test with his- S. typhimurium strains TA1535, TA157 , TA1538, TA98, TA100, and, in part, TA97 , and in a DNA-repair test with trp- E. coli strains WP2 (repair-proficient), WP67 (uvrA- polA-) and CM871 (uvrA- recA- lexA-). A liquid micromethod procedure for the assessment of the minimal inhibitory concentration (MIC) of test compounds, using the same reagents as the Ames test, was set up and calibrated in its technical details. Other techniques (spot test and treat-and-plate method) were applied to a number of compounds in order to obtain more complete information on their DNA-damaging activity in E. coli. From a qualitative standpoint, the results obtained in the reversion test and in the DNA-repair test (liquid micromethod) were overlapping for 96 (59 positive and 37 negative) out of 135 compounds (71.1%). There was disagreement for 39 compounds (28.9%), 9 of which were positive only in the reversion test (8 requiring metabolic activation and 5 genotoxic in the treat-and-plate method). 30 compounds were positive only in the lethality test, showing a direct DNA-damaging activity, which in half of the cases was completely eliminated by S9 mix. Although the experimental protocol intentionally included several compounds already reported as nonmutagenic carcinogens or as noncarcinogenic mutagens, the overall accuracy was 64.5% for the reversion test and 72.4% for the DNA-repair test, as evaluated for 75 compounds classified according to their carcinogenic activity. Quantitation of results was obtained in the Ames test by relating the net number of revertants to nmoles of compound and in the DNA-repair test by means of a formula relating the difference and ratio of MICs in repair-proficient and -deficient bacteria to nmoles of compound. Following these criteria, the genotoxic potency varied over a 4.5 X 10(7)-fold range among compounds positive in the reversion test and over a 6 X 10(9)-fold range among compounds damaging E. coli DNA. The genotoxic potencies in the two bacterial systems were correlated within the majority of the chemical classes under scrutiny.

Genotoxicity of mercury compounds. A review

This article reviews literature data concerning the genotoxicity of 29 mercury-containing agents, including laboratory compounds as well as ingredients of preparations used as fungicides, dyes, disinfectants and drugs. A variety of genetic end-points were investigated in bacteria, yeasts, moulds, plants, insects, cultured cells from fishes, rodents or humans, aquatic organisms, amphibians, mammalia and exposed humans. The overall evaluation is quite complex. Mercury compounds failed to induce point mutations in bacteria but often exerted clastogenic effects in eukaryotes, especially by binding SH groups and acting as spindle inhibitors, thereby causing c-mitosis and consequently aneuploidy and/or polyploidy. Inorganic mercury compounds were also found to induce the generation of reactive oxygen species and glutathione depletion in cultured mammalian cells. Although different mercury compounds tended to produce qualitatively comparable genetic effects, which suggests the involvement of a common toxic entity, methylmercury derivatives and other ionizable organomercury compounds were more active in short-term tests than either non-ionizable mercury compounds (e.g., dimethylmercury) or inorganic mercury salts (e.g., mercuric chloride). The results of cytogenetic monitoring in peripheral blood lymphocytes of individuals exposed to elemental mercury or mercury compounds from accidental, occupational or alimentary sources were either negative or borderline or uncertain as to the actual role played by mercury in some positive findings. Both genotoxic and non-genotoxic mechanisms may contribute to the renal carcinogenicity of mercury, which so far has been convincingly demonstrated only in male rodents treated with methylmercury chloride.

Inhibition of urethan-induced lung tumors in mice by dietary N-acetylcysteine

The thiol N-acetylcysteine (NAC), a precursor of intracellular glutathione (GSH), efficiently prevented the induction of lung tumors in Swiss albino mice, when supplemented to the diet (0.2%) both before and after an i.p. injection of the carcinogen urethan (ethyl carbamate). Irrespective of urethan administration, NAC also significantly enhanced GSH S-transferase activity in liver preparations of the same animals. These data show that, under certain conditions, it is possible to prevent chemically induced cancer by increasing the levels of physiological trapping agents.

Mutagenicity testing with TA97 and TA102 of 30 DNA-damaging compounds, negative with other Salmonella strains

In a comparative study on 135 compounds of various chemical classes, 30 agents inducing direct nonreparable DNA damage in repair-deficient E. coli failed in reverting strains TA1535, TA1537, TA1538, TA98 and TA100 of S. typhimurium (De Flora et al., 1984b). These compounds were re-assayed in the Ames test using strains TA97 and TA102. A dose-dependent mutagenic response was detected with aminoantipyrine and p-rosaniline in TA97 and with streptomycin and formaldehyde in TA102. p-Rosaniline was the only mutagen requiring metabolic activation. 5 compounds, i.e. o-aminophenol in TA97 and methanol, ethanol, cadmium chloride and cadmium sulfate in TA102, induced a reproducible increase in revertants over controls, but this was less than 2-fold. The remaining 21 chemicals--including amino compounds, aliphatics, aromatics, heterocycles, hydrazine derivatives and inorganics--confirmed their inactivity in the Ames test. Overall data for 135 compounds, comparing the Ames test (7 strains) and the DNA-repair test (3 strains), are re-assessed on the basis of these findings.

Modulation of cigarette smoke-related end-points in mutagenesis and carcinogenesis.

The epidemic of lung cancer and the increase of other tumours and chronic degenerative diseases associated with tobacco smoking have represented one of the most dramatic catastrophes of the 20th century. The control of this plague is one of the major challenges of preventive medicine for the next decades. The imperative goal is to refrain from smoking. However, chemoprevention by dietary and/or pharmacological agents provides a complementary strategy, which can be targeted not only to current smokers but also to former smokers and passive smokers. This article summarises the results of studies performed in our laboratories during the last 10 years, and provides new data generated in vitro, in experimental animals and in humans. We compared the ability of 63 putative chemopreventive agents to inhibit the bacterial mutagenicity of mainstream cigarette smoke. Modulation by ethanol and the mechanisms involved were also investigated both in vitro and in vivo. Several studies evaluated the effects of dietary chemopreventive agents towards smoke-related intermediate biomarkers in various cells, tissues and organs of rodents. The investigated end-points included metabolic parameters, adducts to haemoglobin, bulky adducts to nuclear DNA, oxidative DNA damage, adducts to mitochondrial DNA, apoptosis, cytogenetic damage in alveolar macrophages, bone marrow and peripheral blood erytrocytes, proliferation markers, and histopathological alterations. The agents tested in vivo included N-acetyl-L-cysteine, 1,2-dithiole-3-thione, oltipraz, phenethyl isothiocyanate, 5,6-benzoflavone, and sulindac. We started applying multigene expression analysis to chemoprevention research, and postulated that an optimal agent should not excessively alter per se the physiological background of gene expression but should be able to attenuate the alterations produced by cigarette smoke or other carcinogens. We are working to develop an animal model for the induction of lung tumours following exposure to cigarette smoke. The most encouraging results were so far obtained in models using A/J mice and Swiss albino mice. The same smoke-related biomarkers used in animal studies can conveniently be applied to human chemoprevention studies. We participated in trials evaluating the effects of N-acetyl-L-cysteine and oltipraz in smokers from Italy, The Netherlands, and the Peoples Republic of China. We are trying to develop a pharmacogenomic approach, e.g. based on genetic metabolic polymorphisms, aimed at predicting not only the risk of developing cancer but also the individual responsiveness to chemopreventive agents.

Metabolic reduction of chromium by alveolar macrophages and its relationships to cigarette smoke.

Pulmonary alveolar macrophages (PAM), obtained by bronchoalveolar lavage from 47 individuals, reduced hexavalent chromium [Cr(VI)] and decreased its mutagenicity. Their specific activity--mostly mediated by cytosolic, enzyme-catalyzed mechanisms--was significantly higher than in corresponding preparations of mixed-cell populations from human peripheral lung parenchyma or bronchial tree, or from rat lung or liver. At equivalent number of PAM, Cr(VI) reduction, total protein, and some oxidoreductase activities were significantly increased in smokers. No appreciable variation could be detected between lung cancer and noncancer patients. In rats, the Cr(VI)-reducing activity of PAM preparations was induced by Aroclor 1254. Thus, alveolar macrophages provide crucial defense mechanisms not only by phagocytizing metals, but also by metabolically reducing Cr(VI). The epithelial-lining fluid (ELF) also displayed some Cr(VI) reduction. Together with already investigated metabolic processes occurring inside lung cells, these mechanisms are expected to determine thresholds in the pulmonary carcinogenicity of chromium.

High sensitivity of Salmonella TA102 in detecting hexavalent chromium mutagenicity and its reversal by liver and lung preparations.

The recently developed strain TA102, particularly suited to the detection of oxidative mutagens (Levin et al., 1983), was the most sensitive out of 9 strains of S. typhimurium his- in revealing the mutagenicity of Cr(VI) compounds (sodium dichromate, calcium chromate and chromium trioxide). The rank of sensitivity was the following: TA102, TA100, TA97, TA92, TA1978, TA98, TA1538 and TA1537, TA1535 being the only insensitive strain. Cr(III) compounds (chromic acetate, chromic nitrate and chromic potassium sulfate) were totally inactive with all strains. The direct mutagenicity of Cr(VI) was markedly decreased, through NADPH-requiring mechanisms, by rat-liver S9 fractions and, to a lower extent, by human lung S12 fractions, which supports the hypothesis of a metabolically regulated threshold in chromium pulmonary carcinogenicity.

Xenobiotic metabolizing enzymes in uninduced and induced rainbow trout (Oncorhynchus mykiss): Effects of diets and food deprivation

Abstract 1. Untreated and β-naphtoflavone (BNF)-treated trout ( Oncorhynchus mykiss ) were maintained for 15–21 days under 3 different feeding regimens or under starvation. 2. Possible diet effects were studied by measuring the following 13 hepatic parameters: aminopyrine demethylase (APDM), ethoxyresorufin-O-deethylase (EROD), benzo(a)pyrene hydroxylase (AHH), UDP glucuronyltransferase (UDPGT), glutathione reductase (GR), glutathione-S-transferase (GST), glutathione peroxidase (GPx), glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) enzyme activities; the content of total cytochrome P-450 (P-450); proteic and non-proteic SH groups (SHP, SHNP) and liver somatic index (LSI). 3. The results show that some feeding regimens affect a large number of these parameters in both uninduced and BNF-treated fish. 4. Hence, caution should be imposed in the use of the biochemical markers as a water-quality monitoring tool. 5. In particular, it is advisable to maintain fish without feeding during short-term experiments, especially because trout exhibit an excellent biochemical response to inducer even after a 21-day starvation period. 6. Conversely, when fish are to be necessarily fed (long-term experiments), diet must be carefully chosen and controlled by preliminary tests because some commercial fish pellets contain both inducers and inhibitors of several microsomal/cytosolic enzymes.

The epidemiological revolution of the 20th century

Until 100 years ago the epidemiological scenario of human diseases had substantially remained unchanged. The 20th century has been characterized by a fantastic advance in life expectancy and by a shift from infectious to chronic degenerative diseases as prevailing causes of death. As an example of the epidemiological revolution in a developed country, we reconstructed, year by year from 1901 to 2000, the situation in Italy. Reference to the situation in other countries is also made. Both crude and age‐adjusted mortality data were made available for males and females. A new turning point became evident in the second half of the 20th century with the decline of mortality for cardiovascular diseases and, more recently, for tumors. This review discusses the roots and rationale for these epidemiological changes. The discoveries made in the area of biomedical sciences, the progress in preventive and curative medicine, and the improvement of hygienic conditions have been so spectacular that 1 million lives are saved every year in Italy as compared with the late 19th century. De Flora, S., Quaglia, A., Bennicelli, C., Vercelli, M. The epidemiological revolution of the 20th century. FASEB J. 19, 892–897 (2005)

Mutagenicity of active oxygen species in bacteria and its enzymatic or chemical inhibition

The mono-electronic reduction of oxygen in the hypoxanthine-xanthine oxidase system led to the formation of active species eliciting an evident and highly reproducible mutagenic response in strain TA104 of S. typhimurium. Similar effects were observed by generating oxy radicals either extracellularly or inside bacterial cells. Mutagenicity was selectively detected in TA104 and not in other Salmonella strains, which points out the importance of the hisG428 mutation and of the deletion excising the uvrB gene, as far as sensitivity to oxy radicals is concerned. The mutagenicity of the system was further enhanced in the presence of superoxide dismutase. Catalase did not affect the mutagenicity of hypoxanthine plus xanthine oxidase, whereas it inhibited the mutagenicity induced by the mixture of hypoxanthine with xanthine oxidase and superoxide dismutase. This demonstrates that not only hydrogen peroxide but also the superoxide radical anion is positive in this system. Glutathione and 2 synthetic thiols, i.e., N-acetylcysteine and alpha-mercaptopropionylglycine, besides decreasing the high spontaneous mutagenicity of TA104, efficiently prevented the mutagenicity of active oxygen species.