[Genetic toxicology].

Problems and achievements in genotoxicology are briefly discussed with special reference to genotoxicologic assays, genotoxicologic risks, modification of genotoxicologic effects, and individual sensitivity to genotoxic agents. The basic terms and concepts of genetic toxicology as well as the main lines of its further development are considered.

Effects of fullerenol C60(OH)24 on the frequency of micronuclei and chromosome aberrations in CHO-K1 cells.

Poly-hydroxylated C(60) fullerenols (C(60)(OH)(n)) have attracted much attention in biomedical research, due to a variety of biological activities. However, the studies investigating the genotoxic effects of fullerenols are still insufficient. The aim of the present study was to analyze the genotoxic and antigenotoxic potential of fullerenol C(60)(OH)(24). The investigation was carried out with mitomycin C (MMC)-treated and control Chinese hamster ovary cells (CHO-K1), using the chromosome aberration (CA) assay and the cytokinesis-block micronucleus (CBMN) test. Cells were treated with fullerenol nanoparticles, which are well known for their antioxidative properties and cytoprotective effects, both in vivo and in vitro. Our study showed the absence of genotoxicity of fullerenol in a wide range of concentrations (11-221 microM). Fullerenol mediated the decrease in the frequency of micronuclei (MN) and chromosome aberrations compared with the controls at all endpoints examined. A dose-dependent decrease of MN frequency was found 24h after treatment with fullerenol, in contrast to the outcome of the CA assay. Cell proliferation was equally influenced by fullerenol. The majority of aberrations were of the chromosome-type. Our results show that fullerenol does not induce genotoxic effects, and that it protects both non-damaged and MMC-damaged CHO-K1 cells.

Moricandia arvensis extracts protect against DNA damage, mutagenesis in bacteria system and scavenge the superoxide anion.

The mutagenic potential of total aqueous, total oligomers flavonoids (TOF), ethyl acetate (EA), chloroform (Chl), petroleum ether (PE) and methanol (MeOH) extracts from aerial parts of Moricandia arvensis was assessed using Ames Salmonella tester strains TA100 and TA1535 with and without metabolic activation (S9), and using plasmid pBluescript DNA assay. None of the different extracts produced a mutagenic effect, except aqueous extract when incubated with Salmonella typhimurium TA100 after metabolic activation. Likewise, the antimutagenicity of the same extracts was tested using the "Ames test". Our results showed that M. arvensis extracts possess antimutagenic effects against sodium azide (SA) in the two tested Salmonella assay systems, except metabolized aqueous and PE extracts when tested with S. typhimurium TA100 assay system. Different extracts were also found to be effective in protecting plasmid DNA against the strand breakage induced by hydroxyl radicals, except PE and aqueous extracts. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) (X/XOD) and the non enzymatic (NBT/Riboflavine assay) systems. TOF extract was the more effective one in inhibiting both xanthine oxidase activity and NBT reduction.

Assessment of antioxidant / anticarcinogenic activity of plant extracts by a combination of molecular methods.

Cancer chemoprevention is considered to be a promising approach for cancer control, as it has been identified by both epidemiological and molecular studies that environmental factors are the major causes of cancer. Chemoprevention can be defined as the use of agents to prevent, inhibit or reverse the process of carcinogenesis. Several epidemiological studies have shown that fruits, vegetables and common beverages, as well as herbs and plants, are rich sources of chemopreventive compounds. In the present report, a battery of in vitro methods for the identification of chemopreventive agents are presented. These methods include: i) inhibition of bleomycin-induced mutations in Salmonella typhimurium TA102 cells, ii) inhibition of bleomycin-induced sister chromatid exchanges (SCEs) in human peripheral blood lymphocytes, iii) protection from mitomycin C-induced DNA strand breakage and iv) inhibition of topoisomerase I DNA relaxation. The first three methods are also used for the identification of agents which prevent reactive oxygen species (ROS)-mediated DNA damage.

Third International Conference on Mechanisms of Antimutagenesis and Anticarcinogenesis.

This conference, attended by scientists from 27 countries, focused on the most recent advances in the field of antimutagenesis and anticarcinogenesis. Particular emphasis was given to the mechanistic approach, which is believed to be an essential prerequisite for a safer and more effective implementation of chemoprevention of cancer and of mutation-related diseases. The arrangement of the six regular sessions basically followed and updated the detailed classification of mechanisms of inhibitors of mutagenesis and carcinogenesis proposed by S. De Flora and C. Ramel (Mutat. Res., 202: 285-306, 1988), covering both extracellular and cellular mechanisms involved in the prevention of mutations and cancer initiation, as well as in the modulation of later stages of the carcinogenesis process. In addition, a workshop was devoted to methodological aspects concerning the modulation of the genotoxic and carcinogenic response. The present report covers the main themes of overview lectures or research communications presented by more than 60 speakers. Most presentations were multi-authored, as the result of collaborative studies, in several cases at the international level, but only the names of speakers will be given.

Modulation of heterocyclic amine-induced mutagenicity and carcinogenicity: an 'A-to-Z' guide to chemopreventive agents, promoters, and transgenic models.

A landmark report by Widmark in 1939 describing "cancer-producing substances in roasted food", and the seminal work of Sugimura and colleagues in the 1970s on the isolation of potent mutagens from cooked meat and fish stimulated a major international effort on the study of heterocyclic amines and their modulators. The latter term is used in its broadest context to mean agents or conditions that positively or negatively influence the mutagenic or carcinogenic activities of heterocyclic amines in vitro or in vivo. An 'A-to-Z' list of these modulators includes well over 150 natural or synthetic phytochemicals, micronutrients and antioxidants, as well as several large chemical classes (polyphenols, flavones, retinoids, porphyrins), food fractions, and food preparation methods. In many cases, the findings reported in the literature can be regarded as descriptive, but for a number of specific agents there is sufficient evidence to glean some understanding of the inhibitory or promotional mechanisms of action. These mechanisms can be divided into 11 separate sub-categories, arranged within a general classification scheme that encompasses such terms as 'blocking agents', 'suppressing agents', 'desmutagens', 'bioantimutagens', 'interceptor molecules' and 'tumor promoters'. In addition, new research directions, most notably during the past 2-3 years or so, have led to the use of novel dosing protocols and unique animal models (including transgenic species) that provide insight into exposure conditions and genetic background as modulators of heterocyclic amine activity in vitro and in vivo. Overall, the more than 250 citations on the subject give ample evidence of the growing interest in modulators of heterocyclic amine carcinogenesis and mutagenesis, and their possible importance in determining human cancer risk in defined populations.

Potent antimutagenic activity of white tea in comparison with green tea in the Salmonella assay.

There is growing interest in the potential health benefits of tea, including the antimutagenic properties. Four varieties of white tea, which represent the least processed form of tea, were shown to have marked antimutagenic activity in the Salmonella assay, particularly in the presence of S9. The most active of these teas, Exotica China white tea, was significantly more effective than Premium green tea (Dragonwell special grade) against 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and four other heterocyclic amine mutagens, namely 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). Mechanism studies were performed using rat liver S9 in assays for methoxyresorufin O-demethylase (MROD), a marker for the enzyme cytochrome P4501A2 that activates heterocyclic amines, as well as Salmonella assays with the direct-acting mutagen 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline (N-hydroxy-IQ). White tea at low concentrations in the assay inhibited MROD activity, and attenuated the mutagenic activity of N-hydroxy-IQ in the absence of S9. Nine of the major constituents found in green tea also were detected in white tea, including high levels of epigallocatechin-3-gallate (EGCG) and several other polyphenols. When these major constituents were mixed to produce "artificial" teas, according to their relative levels in white and green teas, the complete tea exhibited higher antimutagenic potency compared with the corresponding artificial tea. The results suggest that the greater inhibitory potency of white versus green tea in the Salmonella assay might be related to the relative levels of the nine major constituents, perhaps acting synergistically with other (minor) constituents, to inhibit mutagen activation as well as "scavenging" the reactive intermediate(s).

Development of test systems for the detection of compounds that prevent the genotoxic effects of heterocyclic aromatic amines: preliminary results with constituents of cruciferous vegetables and other dietary constituents.

Over the past decades, strong efforts have been made to identify dietary constituents that protect against the genotoxic effects of heterocyclic aromatic amines (HAAs). However, most of the methods that have been used, in particular in vitro assays that require the addition of exogenous enzyme homogenates, have only a limited predictive value because important protective mechanisms are not adequately represented and may give misleading results. Therefore, we attempted to develop improved test systems, namely assays, with human hepatoma cells and single-cell gel electrophoresis (SCGE) tests with rats. Genotoxicity tests with human derived Hep G2 cells reflect the genotoxic effects of HAAs better than other in vitro systems. They also enable the detection of protective effects since the human derived hepatoma cells possess phase I and phase II enzymes that are involved in the activation/ detoxification of the amines. The most appropriate endpoint for experiments with Hep G2 cells appears to be micronucleus induction, but protocols for other endpoints are available as well. The second promising model is the SCGE ("comet") assay with rats that was used successfully to measure protective effects of constituents of cruciferous vegetables against 2-amino-3-methylimidazo[4,5-flquinoline (IQ) in the liver and in the colon mucosa. The present study describes the experimental design of the new approaches, as well as results obtained with various dietary constituents.

Isolation and characterization of structurally novel antimutagenic flavonoids from spinach (Spinacia oleracea).

Thirteen compounds, isolated from spinach (Spinacia oleracea), acted as antimutagens against the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline in Salmonella typhimurium TA 98. The antimutagens were purified by preparative and micropreparative HPLC from a methanol/water (70:30, v/v) extract of dry spinach (commercial product) after removal of lipophilic compounds such as chlorophylls and carotenoids by solid-phase extraction (SPE). Pure active compounds were identified by instrumental analysis including FT-IR, (1)H and (13)C NMR, UV-vis spectroscopy, and mass spectrometry. All of these compounds were flavonoids and related compounds that could be attributed to five groups: (A, methylenedioxyflavonol glucuronides) 5,3'-dihydroxy-4'-methoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 1), 5,2',3'-trihydroxy-4'-methoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 2), 5-hydroxy-3',4'-dimethoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 3); (B, flavonol glucuronides) 5,6,3'-trihydroxy-7,4'-dimethoxyflavonol 3-O-beta-glucuronide (compound 4), 5,6-dihydroxy-7,3',4'-trimethoxyflavonol 3-O-beta-glucuronide (compound 5); (C, flavonol disaccharides) 5,6,4'-trihydroxy-7,3'-dimethoxyflavonol 3-O-disaccharide (compound 6), 5,6,3',4'-tetrahydroxy-7-methoxyflavonol 3-O-disaccharide (compounds 7 and 8); (D, flavanones) 5,8,4'-trihydroxyflavanone (compound 9), 7,8,4'-trihydroxyflavanone (compound 10); (E, flavonoid-related compounds) compounds 11, 12, and 13 with incompletely elucidated structures. The yield of compound 1 was 0.3%, related to dry weight, whereas the yields of compounds 2-13 ranged between 0.017 and 0.069%. IC(50) values (antimutagenic potencies) of the flavonol glucuronides ranged between 24.2 and 58.2 microM, whereas the flavonol disaccharides (compounds 7 and 8), the flavanones (compounds 9 and 10), and the flavonoid-related glycosidic compounds 11-13 were only weakly active. The aglycons of compounds 7 and 8, however, were potent antimutagens (IC(50) = 10.4 and 13.0 microM, respectively).

[Mutagenesis, antimutagenesis, DNA repair]. / Mutagenez, antimutagenez, reparatsiia DNK.

The paper presents a classification of antimutagens and the mechanism of their action. The mutagens included interferons and vitamins. The paper also provides evidence for the unique protective action of interferons against various mutagens and reveals their activity depending on the genotype of cells, in some DNA repair-deficiency cells, interferons failed to act. Surveys of workers contacting with heavy metals who were on ascorbic acid, retinol or their combination have indicated a reduction in chromosomal aberrations, sister chromatid exchanges and stimulation of DNA repair, which are induced by reference mutagens in the lymphocytes in vitro.

Structural basis of antimutagenicity of chemicals towards 4-nitroquinoline 1-oxide in Salmonella typhimurium.

Eighty-eight organic compounds representing a variety of chemical classes, a proportion of which possess antimutagenic and/or anticarcinogenic properties, were compared for their ability to inhibit the direct-acting mutagenicity of 4-nitroquinoline 1-oxide (4NQO) in strain TA100 of Salmonella typhimurium. Twelve compounds were found to decrease 4NQO-induced mutagenicity by at least 50%, with an antimutagenic potency varying over a 1225-fold range, corresponding to a 4-4900 molar excess of inhibitor over that of 4NQO. The resulting antimutagenicity data base was subjected to structural analysis by the computer automated structure evaluation (CASE) methodology, which identified one structural determinant (biophore) and four biophobes. The biophore contained the sulfhydryl group, present in three thiols and five aminothiols, all of which inhibited 4NQO mutagenicity. Conversely, the corresponding disulphides or thioethers as well as dithiocarbamates, isothiocyanates and other sulfur-containing compounds were devoid of antimutagenicity towards 4NQO. A distinctive dichotomy in the ability to inhibit 4NQO mutagenicity was observed with pairs of structurally and metabolically related compounds, such as the polyamines spermidine (antimutagenic) versus spermine and the tetrapyrroles bilirubin (antimutagenic) versus biliverdine. Curcumin and myricetin were the only inhibitors of 4NQO-induced mutagenicity out of a group of 12 phenols and three bioflavonoids, respectively. One biophobe was an oxidized sulfhydryl moiety, while the other three were derived from benzene or its derivatives. Some of these phenyl compounds are known antioxidants. Thus, nucleophilicity rather than antioxidant properties appears to play a role in antimutagenicity to 4NQO.(ABSTRACT TRUNCATED AT 250 WORDS)