Genotoxicity and cell proliferative activity of a nitrosated Oroxylum indicum Vent fraction in the pyloric mucosa of rat stomach.

In vivo genotoxic activity and cell proliferative activity were examined in the stomach mucosa of male F344 rats by in vivo short-term methods after oral administration of a nitrosated Oroxylum indicum Vent (OiV) fraction, which had been found to be mutagenic without S9 mix to Salmonella typhimurium TA98 and TA100. Administration of the nitrosated OiV fraction at doses of 1 and 2 g/kg body weight induced dose-dependent DNA single-strand scission (p less than 0.02), determined by the alkaline elution method, in the stomach pyloric mucosa 2 h after its administration: a dose of 2 g/kg body weight induced an 18-fold increase in the DNA elution rate constant. Administration of the nitrosated OiV fraction at doses of 0.7-2.8 g/kg body weight also induced dose-dependent increases, up to 11-fold (p less than 0.05), in replicative DNA synthesis in the stomach pyloric mucosa 16 h after its administration. Moreover administration of the nitrosated OiV fraction at doses of 0.25-2.0 g/kg body weight induced dose-dependent increases, up to 100-fold, in ornithine decarboxylase activity in the stomach pyloric mucosa with a maximum 4 h after its administration. These results demonstrate that the nitrosated OiV fraction has genotoxic and cell proliferative activity in the pyloric mucosa of rat stomach in vivo.     

Mutagenicities of nitrosated carboline derivatives

Food-borne amines have been considered as the potential precursors of endogenous carcinogenic N-nitroso compounds in humans. A compound which yields a direct mutagen after nitrite treatment was isolated from soy sauce and was identified as 1-methyl-1,2,3,4-tetrahydro-2-carboline-3-carboxylic acid (MTCA) (Wakabayashi, et al., 1983). The mutagenicities of other carboline derivatives such as harman, norharman, harmaline, harmalol, harmine, and harmol were studied. Like MTCA, the nitrosated carboline derivatives showed higher mutagenic activity as compared to their corresponding parent compounds. The demethylated analogue of MTCA, 1,2,3,4-tetrahydro-2-carboline-3-carboxylic acid was synthesized and its nitrosated products were shown to be mutagenic to Salmonella typhimurium TA 100 and TA 98. The potent mutagen Trp-P-2 is a typical 3-carboline derivative. The mutagenicity of Trp-P-2 was suppressed remarkably after nitrosation. Several 3-carboline derivatives also showed the similar property. Nitrosation of MTCA gave several derivatives which were isolated and showed direct mutagenicity to Salmonella typhimurium TA 98. Further characterization of these new carboline derivatives is in progress.     

Mutagenicity of nitrosated food items.

Several food items, commonly consumed in South India, after nitrite treatment under simulated gastric conditions were found to be mutagenic in Salmonella typhimurium tester strain TA 100. Dichloromethane extracts containing the volatile nitroso compounds and ethyl-acetate extracts with the non-volatile nitroso compounds of some of the food items exhibited mutagenicity.     

A nitrosated arginine derivative, a powerful mutagen

An arginine derivative, benzoyl-L-arginineamide, when nitrosated, showed a powerful mutagenic action on $\text{E. coli}$ and $\text{Salmonella typhimurium}$. The active principle was identified to be 4-benzoylamido-4-carboxamidon(N-nitroso)butylcyanamide. The mutagenic activity of the new compound was more than 30 times higher than that of N-methyl-N′-nitro-N-nitrosoguanidine at neutral pH.     

Genotoxicity of HNE

Since previous investigations on the genotoxicity of 4-hydroxynonenal (HNE) were carried out with prokaryotic systems or eukaryotic cell lines which may not adequately reflect the response of cells in vivo due to differences in the metabolism, the genotoxic potential of HNE was further evaluated in primary cells (hepatocytes) and cell clones of cerebral endothelial cells expressing specific functions, i.e. blood-brain barrier (BBB) and capillary formation associated phenotypes. Treatment of hepatocytes with HNE induced statistically significant levels of SCE at concentrations >/=0.1 microM, micronuclei at concentrations >/=1 microM and chromosomal aberrations at a concentration of 10 microM. Treatment of cloned cerebral microvascular endothelial cells induced significantly elevated levels of chromosomal aberrations at concentrations >/=1 microM and micronuclei at concentrations >/=10 microM in both cEC phenotypes, compared to the controls. Additionally, cytotoxicity was observed at a concentration of 50 microM HNE and was significantly higher in type II cells. These results indicate that cells expressing differentiated functions representative for the in vivo situation react more sensitive to HNE than cell lines, and may reflect the sensitivity of the target cells. The different response with respect to the endpoints of genotoxicity tested most probably depends on the different metabolizing capacities and thus the action of different metabolites of HNE.     

Genotoxicity of phytoestrogens

Plant extracts containing phytohormones are very popular as 'alternative' medicine for many kinds of diseases. They are especially favored by women who enter menopause and are concerned about the side effects of hormone replacement therapy. However, adverse health effects of phytoestrogens have often been ignored. This review examines the literature on genotoxicity and apoptotic effects of phytohormones. Genistein, coumestrol, quercetin, zearalenone, and resveratrol exerted genotoxic effects in in vitro test systems. Other phytoestrogens such as lignans, the isoflavones daidzein and glycetein, anthocyanidins, and the flavonol fisetin exhibited only weak or no effects in vitro. However, some metabolites of daidzein showed a genotoxic activity in vitro. Practically all of the phytoestrogens exhibit pro-apoptotic effects in some cell systems. Further investigations regarding dose-response-relationships and other aspects relevant for extrapolation to human exposure seem necessary. Until then, care may be advised in taking concentrated phytohormones. Nevertheless, the intake of substantial amounts of plant-food in a normal diet constitutes an important, individual contribution to cancer prevention.     

Genotoxicity of streptozotocin

Streptozotocin (Streptozocin, STZ, CAS No. 18883-66-4) is a monofunctional nitrosourea derivative isolated from Streptomyces achromogenes. It has broad spectrum antibiotic activity and antineoplastic properties and is often used to induce diabetes mellitus in experimental animals through its toxic effects on pancreatic beta cells. STZ is a potent alkylating agent known to directly methylate DNA and is highly genotoxic, producing DNA strand breaks, alkali-labile sites, unscheduled DNA synthesis, DNA adducts, chromosomal aberrations, micronuclei, sister chromatid exchanges, and cell death. This antibiotic was found to be mutagenic in bacterial assays and eukaryotic cells. STZ is also carcinogenic; a single administration induces tumors in rat kidney, liver, and pancreas. Several lines of evidence indicate that free radicals are involved in the production of DNA and chromosome damage by this compound. Because of the use of STZ as an antineoplastic agent, the study of its genotoxicity has considerable practical significance. The purpose of this review is to present our current knowledge regarding the genotoxicity of STZ.     

Genotoxicity of singlet oxygen

Singlet oxygen, ¹O₂(¹Δ_g), fulfills essential prerequisites for a genotoxic substance, like hydroxyl radicals and other oxygen radicals: it can react efficiently with DNA and it can be generated inside cells, e.g. by photosensitization and enzymatic oxidation. As might be anticipated from the non-radical character of singlet oxygen, the pattern of DNA modifications it produces is very different from that caused by hydroxyl radicals. While hydroxyl radicals produce DNA strand breaks and sites of base loss (AP sites) in high yield and react with all four bases of DNA, singlet oxygen generates predominantly modified guanine residues and few strand breaks and AP sites. There is now convincing evidence that a major product of base modification caused by singlet oxygen is 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). Indeed, the recently reported miscoding properties of 8-hydroxyguanine can explain the predominant type of mutations observed when DNA modified by singlet oxygen is replicated in cells. There are also strong indications that singlet oxygen generated by photosensitization can act as an ultimate DNA modifying species inside cells. However, indirect genotoxic mechanisms involving other reactive oxygen species produced from singlet oxygen are also possible and appear to predominate in some cases. The cellular defense system against oxidants consists of effective singlet oxygen scavengers such as carotenoids. The observation that carotenoids can inhibit neoplastic cell transformation when administered not only together with but also after the application of chemical or physical carcinogens might indicate a role of singlet oxygen in tumor promotion that could be independent of the direct or indirect DNA damaging properties.     

Genotoxicity evaluation of tetramethylbenzenes

The three tetramethyl isomers of benzene (prenitene, 1,2,3,4-; izodurene, 1,2,3,5-; and durene, 1,2,4,5-tetramethylben- zene) were studied using in vitro mutagenicity and in vivo genotoxicity tests. Potency of mutate induction by these solvents was evaluated in Salmonella typhimurium cells with, and without S9-mix made from Aroclor 1254-induced rat liver S9. The potency of induction of micronuclei (MN) and sister chromatid exchanges (SCEs) by solvents was evaluated in bone marrow of mice. Izodurene displayed mutagenic potency in strains TA97a, TA98 and TA100 only in the absence of the S9-mix. In MN tests, all three tetramethylbenzenes demonstrated no clastogenic activity on the bone marrow cells. All the tested solvents were active as genotoxic compounds in the SCE tests, demonstrating a dose-response relationships.     

Genotoxicity of alpha-asarone analogues

The role of cholesterol in the formation of atherosclerotic lesions during hypercholesterolemia has been confirmed. alpha-Asarone is a substance of a potent hypolipidemic activity which is isolated from plants. We previously described the synthesis of several alpha-asarone analogues exhibiting hypolipidemic and antiplatelet activity. Genotoxic activity of four selected alpha-asarone analogues was theoretically evaluated based on quantum-mechanical method for calculation of enthalpy of carbocations formation (DeltaH(R)) according to the Testa's method. In the present paper, we evaluated the mutagenic and genotoxic activity of alpha-asarone isomers 2-5 based on the reference Ames test and micronucleus test. Results obtained in the study show that tested isomers were non-mutagenic, however, they exhibited growing cytotoxic activity. Relationship between the heat of formation of their putative metabolic intermediates and mutagenic/genotoxic activity was not confirmed.     

Genotoxicity of heat-processed foods

Gene-environment interactions include exposure to genotoxic compounds from our diet and it is no doubt, that humans are regularly exposed to e.g. food toxicants, not least from cooked foods. This paper reviews briefly four classes of cooked food toxicants, e.g. acrylamide, heterocyclic amines, nitrosamines and polyaromatic hydrocarbons. Many of these compounds have been recognised for decades also as environmental pollutants. In addition cigarette smokers and some occupational workers are exposed to them. Their occurrence, formation, metabolic activation, genotoxicity and human cancer risk are briefly presented along with figures on estimated exposure. Several lines of evidence indicate that cooking conditions and dietary habits can contribute to human cancer risk through the ingestion of genotoxic compounds from heat-processed foods. Such compounds cause different types of DNA damage: nucleotide alterations and gross chromosomal aberrations. Most genotoxic compounds begin their action at the DNA level by forming carcinogen-DNA adducts, which result from the covalent binding of a carcinogen or part of a carcinogen to a nucleotide. The genotoxic and carcinogenic potential of these cooked food toxicants have been evaluated regularly by the International Agency for Research on Cancer (IARC), which has come to the conclusion that several of these food-borne toxicants present in cooked foods are possibly (2A) or probably (2B) carcinogenic to humans, based on both high-dose, long-term animal studies and in vitro and in vivo genotoxicity tests. Yet, there is insufficient scientific evidence that these genotoxic compounds really cause human cancer, and no limits have been set for their presence in cooked foods. However, the competent authorities in most Western countries recommend minimising their occurrence, therefore this aspect is also included in this review.     

Genotoxicity of hyperbaric oxygen

Hyperbaric oxygen (HBO) treatment is applied as a therapy for a wide variety of diseases with symptoms caused by lack of oxygen in the target tissues. However, it is known that exposure to high concentrations of oxygen may lead to oxidative stress and cause cell and tissue damage. Oxygen toxicity and possible cancer-promoting effects of HBO therapy have been a matter of serious concern. Although a cancer-inducing effect of HBO was not found to date, recent studies clearly indicated an induction of oxidative DNA damage in blood cells of healthy subjects after HBO under therapeutic conditions. The biological significance of this finding has been investigated in a series of in vitro and in vivo tests. This review summarizes these studies and critically discusses potential adverse genetic effects of HBO therapy. Furthermore, since an induction of anti-oxidative defense mechanisms has been determined after HBO exposure, a modified treatment regimen of HBO therapy is proposed which avoids genotoxic effects.     

In vitro testing and the carcinogenic potential of several nitrosated indole compounds

4-chloro-methoxyindole is a naturally occurring compound in Vicia faba which can easily react with nitrite to form a N-nitroso compound. In this in vitro study, the potential genotoxic effects of nitrosated 4-chloro-6-methoxyindole and its structural analogue 4-chloroindole were evaluated for the first time by using both Salmonella and Chinese hamster V79 cells. Additionally, the inhibition of gap junctional intercellular communication in V79 cells by these compounds was determined; this is a validated parameter for tumor-promoting activity. Most assays were also performed with nitrosated indole-3-acetonitrile, a naturally occurring compound in brassicas. Both nitrosated chloroindoles were highly mutagenic to Salmonella typhimurium TA100 without the need of exogenous metabolic activation and were potent inducers of Sister Chromatid Exchanges. Nitrosated indole-3-acetonitrile generated the same effects, although at much higher concentrations. Equivocal results were obtained for the nitrosated chloroindoles in a forward mutation assay using the hypoxanthine guaninephosphoribosyltransferase locus. All nitrosated indole compounds significantly inhibited gap junctional intercellular communication. These results indicate that nitrosated chloroindoles and nitrosated indole-3-acetonitrile should be considered as mutagens and agents with potential tumor-promoting capacity.Abbreviations BrdU 5-bromo-2-deoxyuridine - 4Cl 4-chloroindole - 4C6MI 4-chloro-6-methoxy-indole - DMSO dimethyl sulfoxide - EBSS Earle's balanced salt solution - EMS ethyl methanesulfonate - GJIC gap junctional intercellular communication - HBSS Hanks balanced salt solution - HGPRT hypoxanthine guaninephosphoribosyl transferase - I₃A indole-3-acetonitrile - MNNG 1-methyl-1-nitroso-3-nitroguanidine - NOC N-nitroso compounds - NQO 4-nitroquinolone-N-oxide - SCE sister chromatid exchange - 6TG 6-thioguanine - TPA 12-O-tetradecanoylphorbol-13-acetate     

Genotoxicity of naturally occurring hydroxyanthraquinones

A variety of structurally related hydroxyanthraquinones (HA) were investigated in a test battery for the evaluation of mutagenicity and cell-transforming activity. The tests were: (1) the Salmonella typhimurium mutagenicity assay, (2) the V79-HGPRT mutagenicity assay, (3) the DNA-repair induction assay in primary rat hepatocytes and (4) the in vitro transformation of C3H/M2 mouse fibroblasts. In Salmonella, most of the tested compounds were mutagenic in strain TA1537, but only a few were active in other strains. Among these were HA with a hydroxymethyl group, such as lucidin and aloe-emodin. In V79 cells, only HA with 2 hydroxy groups in the 1,3 positions (1,3-DHA, purpurin, emodin) or with a hydroxymethyl sidechain (lucidin and aloe-emodin) were mutagenic. The compounds found to be active in V79 cells were also active in the DNA-repair assay and in the C3H/M2 transformation assay. Thus, it appears that the genotoxicity of HA is dependent on certain structural requirements.