P. Zanacchi

Genotoxicity of chromium compounds. A review

This article reviews approximately 700 results reported in the literature with 32 chromium compounds assayed in 130 short-term tests, using different targets and/or genetic end-points. The large majority of the results obtained with Cr(VI) compounds were positive, as a function of Cr(VI) solubility and bioavailability to target cells. On the other hand, Cr(III) compounds, although even more reactive than Cr(VI) with purified nucleic acids, did not induce genotoxic effects in the majority of studies using intact cells. Coupled with the findings of metabolic studies, the large data-base generated in short-term test systems provides useful information for predicting and interpreting the peculiar patterns of Cr(VI) carcinogenicity.

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.

Genotoxic, carcinogenic, and teratogenic hazards in the marine environment, with special reference to the Mediterranean Sea

Genotoxic, carcinogenic, and teratogenic hazards arising out of pollution in the marine environment are discussed in this article, with special reference to the situation in the Mediterranean area. A number of chemical compounds or complex mixtures relevant to marine pollution, either natural or of anthropogenic origin, are tentatively listed, along with protective factors which may play a counteracting role in the same environment. Harmful substances tend to undergo interactions and transformations in seawater, sediments, and marine biota, due to physical, chemical, microbial, or light-mediated mechanisms. Bioaccumulation phenomena in marine organisms may result from food-chain biomagnification processes or from concentration of pollutants by filter feeders. A variety of sources can account for marine pollution by genotoxic, carcinogenic, and teratogenic compounds, but there is a relative paucity of analytical data concerning the Mediterranean. Metabolic transformations of xenobiotics occur in all marine organisms, the biochemical mechanisms in fish being comparable to those which have been extensively investigated in mammals. Induction of metabolic pathways, and especially of the mixed-function oxygenase system, represents the earliest warning signal of exposure to pollutants. Occurrence of neoplastic diseases is documented by experimental and field studies in marine vertebrates as well as in invertebrates. The association with local pollution phenomena has been recognized in several studies, but other etiopathogenetic factors may be also involved, and in some cases tumors have been reported to be unrelated to chemical pollution. Genotoxic agents have been detected by means of suitable techniques in seawater, sediments, and marine organisms. Several studies have investigated the presence of carcinogen-DNA adducts, DNA damage and repair processes, and cytogenetic alterations, such as chromosomal aberrations, sister-chromatid exchanges, and micronuclei, in tissues of marine organisms. However, monitoring of these end-points under field conditions encounters some limitations and problems. Even more fragmentary is the information on teratogenic effects in marine organisms, although interesting test systems have been set up. On the whole, a quite extensive database on all these toxicological issues is already available in the literature, but further studies are warranted for an adequate assessment of genotoxic, carcinogenic, and teratogenic hazards, and possibly counteracting factors in the marine environment, and specifically in the Mediterranean Sea.

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.

Protection by N-acetylcysteine of the histopathological and cytogenetical damage produced by exposure of rats to cigarette smoke

Adult male Sprague-Dawley rats were exposed whole-body to mainstream cigarette smoke (CS) once daily for 40 consecutive days. Such a treatment resulted in a significant decrease of body weight growth and in intense histopathological changes of terminal airways, including a severe inflammation of bronchial and bronchiolar mucosae, with multiple hyperplastic and metaplastic lesions and foci of micropapillomatous growth as well as emphysema, with extensive disruption of alveolar walls. All histopathological changes were efficiently prevented by the daily administration of the thiol N-acetyl-L-cysteine (NAC) by gavage. Cytological and cytogenetical changes were monitored in bronchoalveolar lavage (BAL) fluid and bone marrow cells of groups of rats killed after 1, 3, 8, 28, or 40 days of treatment. From the first day of exposure, CS significantly enhanced the proportion of polymorphonucleates among BAL cells and the frequency of micronucleated (MN) bone marrow polychromatic erythrocytes. After 8 days, a reduction was observed in the polychromatic/normochromatic erythrocytes ratio and an increase in the frequency of MN pulmonary alveolar macrophages (PAM) was also recorded, followed, after 28 days, by an increase of binucleated PAM. All these alterations immediately reached a plateau and persisted unchanged until the end of the experiment. NAC administration exhibited a significant and considerable protective effect towards the CS-induced alterations of BAL cellularity, the increase of MN PAM and bone marrow cytotoxicity.

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.

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.

Metabolic reduction of chromium, as related to its carcinogenic properties

At variance with Cr(III), Cr(VI) compounds easily cross cell membranes and exert genotoxic effects. No metabolic oxidation of Cr(III) could be detected, whereas Cr(VI) reduction was observed in the presence of body fluids and subcellular fractions of various tissues from several animal species. The differential efficiency of this process may account for the selection of target tissues in Cr(VI) carcinogenesis. For instance, reduction by saliva and gastric juice may explain a lack of carcinogenicity by the oral route; reduction inside erythrocytes may explain a lack of carcinogenicity at a distance from administration sites; reduction by the epithelial-lining fluid of terminal airways and by alveolar macrophages may be consistent with the occurrence of thresholds in lung carcinogenesis. Liver preparations displayed the top efficiency in reducing Cr(VI), whereas skeletal muscle, i.e., a typical target in experimental Cr(VI) carcinogenesis, had no detectable activity. Bronchial tree and peripheral lung parenchyma preparations from almost 100 individuals reduced Cr(VI) to a variable extent. The efficiency of lung parenchyma and of isolated alveolar macrophages was enhanced in cigarette smokers. In rats, Cr(VI) reduction by lung preparations was significantly stimulated by the repeated i.t. instillation of Cr(VI) itself. Among the electron donors (chiefly GSH) and enzymatic mechanisms responsible for the intracellular Cr(VI) reduction, such as cytochrome P-450 reductase, glutathione redactase, and aldehyde oxidase, an important role can be ascribed to cytosolic DT diaphorase activity, usually catalyzing a 2-electron reduction.

Antimutagenic and Anticarcinogenic Mechanisms of Aminothiols

Glutathione (GSH) and synthetic aminothiols, such as N-acetylcysteine (NAC), play an important role as inhibitors of mutagenesis and carcinogenesis (1). The present paper will update the studies carried out in our laboratories, which will be discussed according to a mechanistic approach. In Table 1, reporting the classification of inhibitors of mutagenesis and carcinogenesis proposed by De Flora and Ramel (2), the arrows indicate the mechanisms that we have investigated so far in the case of aminothiols.

Mechanistic aspects of chromium carcinogenicity.

There is no doubt that chromium has been the most extensively investigated metal with respect to the assessment of long-term effects and to the evaluation of the underlying mechanisms. Notwithstanding the huge data-base available in the literature, many aspects, bearing both scientific and practical interest, are still unclear or controversial. This paper will briefly discuss some relevant problems, including carcinogenicity and genotoxicity of chromium compounds as related to their valency and solubility, interactions with other compounds, bioavailability to target cells and molecules, interconversion processes between different oxidation states in various body areas and cell compartments, mechanisms of chromium metabolism and carcinogenicity, and existence of thresholds in chromium carcinogenesis.