In vitro assessment of the toxicity of metal compounds

A review has been compiled illustrating the directions taken in examining the genotoxic effects of metals and their compounds centering only on those studies pertaining to effects of metals and their compounds on DNA structure and function, such as the induction of DNA strand breaks, production of DNA-protein crosslinks, induction of chromosomal aberrations, and sister chromatid exchanges. Although it is premature to declare a cause and effect relationship between the carcinogenic activity of metals and their ability to induce one or more lesions in DNA, strong evidence is emerging to suggest such a relationship. Low concentrations of metals induce the appearance of DNA lesions, such as strand breaks and crosslinks, or induce sister chromatid exchanges or DNA repair synthesis. Assays based upon these events constitute extremely sensitive probes for genotoxic effects of metals and their compounds. These effects of metals on DNA are consistent with the currently accepted mechanism of chemical carcinogenesis, allowing the acquisition and propagation of altered DNA function. The lack of complete information on the activity of metals in producing DNA lesions allow only preliminary conclusions to be drawn. Certain compounds containing potentially or actually carcinogenic elements, such as Ni, Be, As, Cr, Cd, and to a minor extent Pb, have yielded positive responses in one or more DNA lesion assays. At relatively nontoxic levels of Ni and Cr, considerable evidence suggests that multiple types of DNA lesions are induced.     

Prevalence of genotoxic chemicals among animal and human carcinogens evaluated in the IARC Monograph series.

To determine whether genotoxic and non-genotoxic carcinogens contribute similarly to the cancer burden in humans, an analysis was performed on agents that were evaluated in Supplements 6 and 7 to the IARC Monographs for their carcinogenic effects in humans and animals and for the activity in short-term genotoxicity tests. The prevalence of genotoxic carcinogens on four groups of agents, consisting of established human carcinogens (group 1, n = 30), probable human carcinogens (group 2A, n = 37), possible human carcinogens (group 2B, n = 113) and on agents with limited evidence of carcinogenicity in animals (a subset of group 3, n = 149) was determined. A high prevalence in the order of 80 to 90% of genotoxic carcinogens was found in each of the groups 1, 2A and 2B, which were also shown to be multi-species/multi-tissues carcinogens. The distribution of carcinogenic potency in rodents did not reveal any specific characteristic of the human carcinogens in group 1 that would differentiate them from agents in groups 2A, 2B and 3. The results of this analysis indicate that (a) an agent with unknown carcinogenic potential showing sufficient evidence of activity in in vitro/in vivo genotoxicity assays (involving as endpoints DNA damage and chromosomal/mutational damage) may represent a hazard to humans; and b) an agent showing lack of activity in this spectrum of genotoxicity assays should undergo evaluation for carcinogenicity by rodent bioassay, in view of the present lack of validated short-term tests for non-genotoxic carcinogens. Overall, this analysis implies that genotoxic carcinogens add more to the cancer burden in man than non-genotoxic carcinogens. Thus, identification of such genotoxic carcinogens and subsequent lowering of exposure will remain the main goal for primary cancer prevention in man.     

Evaluation of the DNA repair host-mediated assay. II. Presence of genotoxic factors in various organs of mice treated with chemotherapeutic agents

The DNA repair host-mediated assay was further calibrated by testing 7 chemotherapeutic agents known to possess carcinogenic activity, namely bleomycin (BLM), cis-diamminedichloroplatinum-II (cis-Pt), cyclophosphamide (CP), diethylstilboestrol (DES), isonicotinic acid hydrazide (isoniazid, INH), natulan (NAT) and mitomycin C (MMC). Differential survival of wild-type and uvrB/recA E. coli strains served as a measure of genotoxic activity. In in vitro assays, BLM, cis-Pt and MMC exhibited high genotoxic activity. The other 4 compounds had no measurable effect on the survival of the two strains, either with or without mouse liver preparations. In the host-mediated assays BLM, cis-Pt, MMC and also NAT induced strong killing of the DNA repair-deficient bacteria recovered from liver, spleen, lungs, kidneys and the blood of treated mice compared to the wild-type strain. The results are not indicative of large organ-specific differences in genotoxically active amounts of the drugs immediately after their application to the host animals. CP, INH and DES did not show geneotix activity in these assays even at very high exposure levels. To compare the genetic endpoint measured in the DNA repair assays, i.e. induction of repairable DNA damage, with the induction of gene mutations, the ability of the 7 drugs to induce valine-resistant (VALr) mutants in E. coli was measured in host-mediated assays under identical treatment conditions. INH showed considerable mutagenic activity in E. coli cells recovered from liver and spleen, while BLM and MMC induced a 3-4-fold increase in VALr mutants above spontaneous levels. The other compounds showed no mutagenic activity under these in vivo conditions. From these results it can be concluded that the type of primary DNA lesions produced by these chemotherapeutic agents (cross-links by MMC and cis-Pt, and strand breaks by BLM and possibly by NAT; base alkylation by INH) appears to determine whether a compound will be highly positive in the DNA repair assay as in the case of BLM, cis-Pt, MMC and NAT, and less effective in inducing mutations under similar conditions, or whether the opposite will occur, as in the case of INH; DES and CP probably do not interact sufficiently with bacterial DNA to show an effect in either of the genetic endpoints; and the present DNA repair host-mediated assay may represent a sensitive, rapid and economic method for monitoring genotoxic factors in various organs of experimental animals which have been treated with cytostatic drugs.     

Genotoxic effects of allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC)

Two isothiocyanates (ITCs) commonly found in human diet, allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC), were tested for genotoxic effects in a battery of assays: Salmonella/microsome assay with TA 98 and TA 100, differential DNA repair assay with E. coli and micronucleus (MN) induction assay with human derived Hep G2 cells. Albeit to a different degree, both ITCs induced genotoxic effects in all test systems. AITC was more genotoxic in bacterial test systems than in Hep G2 cells; in contrast, the effect of PEITC was stronger in Hep G2 cells. In in vivo assays with E. coli indicators in which mice were exposed to relatively high doses of the compounds (90 and 270 mg/kg), AITC induced moderate but significant effects; PEITC failed to induce significant effects in any of the organs. To find out the reason for the weak genotoxicity of AITC and PEITC under in vivo test conditions, we exposed E. coli indicator cells to the test substances in the absence or presence of rat liver homogenate (with and without cofactors), bovine serum albumin (BSA) and human saliva. All of them markedly attenuated the genotoxicity of AITC and PEITC, implying that the test substances are detoxified by direct non-enzymatic binding to proteins. Additional experiments carried out on the mechanistic aspects of AITC and PEITC-induced genotoxicity showed that the compounds induce the formation of thiobarbituric acid reactive substances (TBARS) in Hep G2 cells. Furthermore, in in vitro assays with E. coli, radical scavengers reduced the differential DNA damage induced by AITC and PEITC. The latter two findings give a clue that reactive oxygen species might be involved in the genotoxic effect of the ITCs. Although ITCs have been repeatedly advocated as very promising anticancer agents, the data presented here indicate that the compounds are genotoxic, and probably carcinogenic, in their own right.     

Potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive and anticancer properties

Flavonoids, including isoflavones, are natural components in our diet and, with the burgeoning interest in alternative medicine, are increasingly being ingested by the general population. Plant phenolics, which form moieties on flavonoid rings, such as gallic acid, are also widely consumed. Several beneficial properties have been attributed to these dietary compounds, including antioxidant, anti-inflammatory, and anticarcinogenic effects. Flavonoid preparations are marketed as herbal medicines or dietary supplements for a variety of alleged nontoxic therapeutic effects. However, they have yet to pass controlled clinical trials for efficacy, and their potential for toxicity is an understudied field of research. This review summarizes the current knowledge regarding potential dietary flavonoid/phenolic-induced toxicity concerns, including their pro-oxidant activity, mitochondrial toxicity (potential apoptosis-inducing properties), and interactions with drug-metabolizing enzymes. Their chemopreventive activity in animal in vivo experiments may result from their ability to inhibit phase I and induce phase II carcinogen metabolizing enzymes that initiate carcinogenesis. They also inhibit the promotion stage of carcinogenesis by inhibiting oxygen radical-forming enzymes or enzymes that contribute to DNA synthesis or act as ATP mimics and inhibit protein kinases that contribute to proliferative signal transduction. Finally, they may prevent tumor development by inducing tumor cell apoptosis by inhibiting DNA topoisomerase II and p53 downregulation or by causing mitochondrial toxicity, which initiates mitochondrial apoptosis. While most flavonoids/phenolics are considered safe, flavonoid/phenolic therapy or chemopreventive use needs to be assessed as there have been reports of toxic flavonoid-drug interactions, liver failure, contact dermatitis, hemolytic anemia, and estrogenic-related concerns such as male reproductive health and breast cancer associated with dietary flavonoid/phenolic consumption or exposures.     

Evaluation of the DNA-repair host-mediated assay. I. Induction of repairable DNA damage in E. coli cells recovered from liver, spleen, lungs, kidneys, and the blood stream of mice treated with methylating carcinogens

The DNA-repair host-mediated assay was further calibrated by determining the genotoxic activities of 4 methylating carcinogens, namely, dimethylnitrosamine (DMNA), 1,2-dimethylhydrazine (SDMH), methyl nitrosourea (MNU) and methyl methanesulphonate (MMS) in various organs of treated mice. The ranking of the animal-mediated genotoxic activities of the compounds was compared with that obtained in DNA repair assays performed in vitro. The differential survival of strain E. coli K-12/343/113 and of its DNA-repair-deficient derivatives recA, polA and uvrB/recA, served as a measure of genotoxic potency. In the in vitro assays and at equimolar exposure concentrations, MMS and MNU are the most active chemicals, followed by DMNA, which shows a slight genotoxic effect only in the presence of mouse liver homogenate; SDMH has no activity under these conditions. In the host-mediated assays, the order of genotoxic potency of the compounds was quite different: those carcinogens which require mammalian metabolic activation, namely, DMNA and SDMH, show strong effects in liver and blood, a lesser effect in the lungs and kidneys and the least effect in the spleen. The activity of MNU, a directly acting compound, is similar in all organs investigated, but it is clearly lower than that of DMNA and SDMH. MMS, also a directly acting carcinogen, causes some (barely significant) effect at the highest dose tested. A similar order of potency was observed when the compounds were tested in intrasanguineous host-mediated assays with gene mutation as an endpoint. DMNA and SDMH induce comparable frequencies of L-valine-resistant mutants in E. coli K-12/343/113 recovered from liver and spleen of treated mice, the effect in the liver being the strongest. MNU is mutagenic only at a higher dose, while MMS shows no effect. The results are discussed with respect to the literature data on organ-specific DNA adduct formation induced by the compounds. It is concluded that qualitatively there is a good correlation between the degree of genotoxic activity found in the DNA repair host-mediated assay and DNA adduct formation in the animal's own cells. This is exemplified by the finding that the relative order of genotoxic activity of the 4 methylating agents in bacteria recovered from various organs (DMNA approximately equal to SDMH greater than MNU greater than MMS) is reflected by the same order of magnitude in DNA alkylation in corresponding mammalian organs. Quantitatively, the indirectly acting agents DMNA and SDMH seem to induce fewer genotoxic effects in bacteria present in the liver than would be expected on the basis of DNA-adduct formation data.     

Evaluation of the DNA repair host-mediated assay: II. Presence of genotoxic factors in various organs of mice treated with chemotherapeutic agents

The DNA repair host-mediated assay was further calibrated by testing 7 chemotherapeutic agents known to possess carcinogenic activity, namely bleomycin (BLM), cis-diamminedichloroplatinum-II (cis-Pt), cyclophosphamide (CP), diethylstilboestrol (DES), isonicotinic acid hydrazide (isoniazid, INH), natulan (NAT) and mitomycin C (MMC). Differential survival of wild-type and uvrB/recA E. coli strains served as a measure of genotoxic activity. In in vitro assays, BLM, cis-Pt and MMC exhibited high genotoxic activity. The other 4 compounds had no measurable effect on the survival of the two strains, either with or without mouse liver preparations. In the host-mediated assays BLM, cis-Pt, MMC and also NAT induced strong killing of the DNA repair-deficient bacteria recovered from liver, spleen, lungs, kidneys and the blood of treated mice compared to the wild-type strain. The results are not indicative of large organ-specific differences in genotoxically active amounts of the drugs immediately after their application to the host animals. CP, INH and DES did not show geneotix activity in these assays even at very high exposure levels. To compare the genetic endpoint measured in the DNA repair assays, i.e. induction of repairable DNA damage, with the induction of gene mutations, the ability of the 7 drugs to induce valine-resistant (VAL^r) mutants in E. coli was measured in host-mediated assays under identical treatment conditions. INH showed considerable mutagenic activity in E. coli cells recovered from liver and spleen, while BLM and MMC induced a 3–4-fold increase in VAL^r mutants above spontaneous levels. The other compounds showed no mutagenic activity under these in vivo conditions. From these results it can be concluded that (i) the type of primary DNA lesions produced by these chemotherapeutic agents (cross-links by MMC and cis-Pt, and strand breaks by BLM and possibly by NAT; base alkylation by INH) appears to determine whether a compound will be highly positive in the DNA repair assay as in the case of BLM, cis-Pt, MMC and NAT, and less effective in inducing mutations under similar conditions, or whether the opposite will occur, as in the case of INH; (ii) DES and CP probably do not interact sufficiently with bacterial DNA to show an effect in either of the genetic endpoints; and (iii) the present DNA repair host-mediated assay may represent a sensitive, rapid and economic method for monitoring genotoxic factors in various organs of experimental animals which have been treated with cytostatic drugs.     

Quantitative comparative mutagenesis in bacteria, mammalian cells, and animal-mediated assays A convenient way of estimating genotoxic activity in vivo?

The accumulation of environmental compounds which exhibit genotoxic properties in short-term assays and the increasing lag of time for obtaining confirmation or not in long-term animal mutagenicity and carcinogenicity tests, makes it necessary to develop alternative, rapid methodologies for estimating genotoxic activity in vivo. In the experimental approach used here, it was assumed that the genotoxic activity of foreign compounds in animals, and ultimately humans, is determined among others by exposure level, organ distribution of (DNA) dose, and genotoxic potency per unit of dose, and that knowledge about these 3 parameters may allow to rapidly determine the expected degree of genotoxicity in various organs of exposed animals. In view of the high degree of qualitative correlation between mutagenic activity of chemicals in bacteria and in cultured mammalian cells, and their mutagenic and carcinogenic properties in animals, and in order to be able to distinguish whether mutagenic potency differences were due to differences in (DNA) dose rather than other physiological factors, the results of mutagenicity tests obtained in the present experiments using bacteria and mammalian cells were compared on the basis of DNA dose rather than exposure concentrations, with the following questions in mind: Is there an absolute or a relative correlation between the mutagenic potencies of various ethylating agents in bacteria (E. coli K12) and in mammalian cells (V79 Chinese hamster) after treatment in standardized experiments, and can specific DNA adducts be made responsible for mutagenicity? Is the order of mutagenic potency of various ethylating agents observed in bacteria in vitro representative of the ranking of mutagenic potency found in vivo? Since the answer to this last question was negative, a further question addressed to was whether short-term in vivo assays could be developed for a rapid determination of the presence (and persistence) of genotoxic factors in various organs of mice treated with chemicals. In quantitative comparative mutagenesis experiments using E. coli K12 and Chinese hamster cells treated under standardized conditions in vitro with 5 ethylating agents, there was no indication of an absolute correlation between the number of induced mutants per unit of dose in the bacteria and the mammalian cells. The ranking of mutagenic potency was, however, identical in bacteria and mammalian cells, namely, ENNG greater than ENU greater than or equal to DES greater than DEN congruent to EMS, the mutagenic activity of DEN being dependent on the presence of mammalian liver preparations.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Induction by mycotoxins of somatic mosaicism in Drosophila and DNA repair in mammalian liver cell cultures

The genotoxic activity of four mycotoxins has been studied. High level of somatic mutagenesis in imaginal discs of Drosophila melanogaster larvae and DNA repair synthesis in human embryo and adult rat liver cell cultures were inducible only by highly carcinogenic aflatoxin B1. Patulin, a weak direct-action carcinogenic substance, slightly elevated the mutagenesis in somatic cells of Drosophila but did not induce DNA repair synthesis in liver cell cultures. Citrinin that did not exhibit any carcinogenic properties when used alone and stachybotrotoxin with non-reported carcinogenic activity appeared inactive in the test-systems applied. The possibilities of rapid recognition of carcinogenic mycotoxins by detecting their genotoxic properties are discussed.     

Heritable and cancer risks of exposures to anticancer drugs: inter-species comparisons of covalent deoxyribonucleic acid-binding agents

In the past years, several methodologies were developed for potency ranking of genotoxic carcinogens and germ cell mutagens. In this paper, we analyzed six sub-classes of covalent deoxyribonucleic acid (DNA) binding antineoplastic drugs comprising a total of 37 chemicals and, in addition, four alkyl-epoxides, using four approaches for the ranking of genotoxic agents on a potency scale: the EPA/IARC genetic activity profile (GAP) database, the ICPEMC agent score system, and the analysis of qualitative and quantitative structure-activity and activity-activity relationships (SARs, AARs) between types of DNA modifications and genotoxic endpoints. Considerations of SARs and AARs focused entirely on in vivo data for mutagenicity in male germ cells (mouse, Drosophila), carcinogenicity (TD₅₀s) and acute toxicity (LD₅₀s) in rodents, whereas the former two approaches combined the entire database on in vivo and in vitro mutagenicity tests. The analysis shows that the understanding and prediction of rank positions of individual genotoxic agents requires information on their mechanism of action. Based on SARs and AARs, the covalent DNA binding antineoplastic drugs can be divided into three categories. Category 1 comprises mono-functional alkylating agents that primarily react with N7 and N3 moieties of purines in DNA. Efficient DNA repair is the major protective mechanism for their low and often not measurable genotoxic effects in repair-competent germ cells, and the need of high exposure doses for tumor induction in rodents. Due to cell type related differences in the efficiency of DNA repair, a strong target cell specificity in various species regarding the potency of these agents for adverse effects is found. Three of the four evaluation systems rank category 1 agents lower than those of the other two categories. Category 2 type mutagens produce O-alkyl adducts in DNA in addition to N-alkyl adducts. In general, certain O-alkyl DNA adducts appear to be slowly repaired, or even not at all, which make this kind of agents potent carcinogens and germ cell mutagens. Especially the inefficient repair of O-alkyl—pyrimidines causes the high mutational response of cells to these agents. Agents of this category give high potency scores in all four expert systems. The major determinant for the high rank positions on any scale of genotoxic of category 3 agents is their ability to induce primarily structural chromosomal changes. These agents are able to cross-link DNA. Their high intrinsic genotoxic potency appears to be related to the number of DNA cross-links per target dose unit they can induce. A confounding factor among category 3 agents is that often the genotoxic endpoints occur closed to or toxic levels, and that the width of the mutagenic dose range, i.e., the dose area between the lowest observed effect level and the LD₅₀, is smaller (usually no more than 1 logarithmic unit) than for chemicals of the other two categories. For all three categories of genotoxic agents, strong correlations are observed between their carcinogenic potency, acute toxicity and germ cell specificity.     

IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. International Programme on Chemical Safety

The purpose of these guidelines is to provide concise guidance on the planning, performing and interpretation of studies to monitor groups or individuals exposed to genotoxic agents. Most human carcinogens are genotoxic but not all genotoxic agents have been shown to be carcinogenic in humans. Although the main interest in these studies is due to the association of genotoxicity with carcinogenicity, there is also an inherent interest in monitoring human genotoxicity independently of cancer as an endpoint.The most often studied genotoxicity endpoints have been selected for inclusion in this document and they are structural and numerical chromosomal aberrations assessed using cytogenetic methods (classical chromosomal aberration analysis (CA), fluorescence in situ hybridisation (FISH), micronuclei (MN)); DNA damage (adducts, strand breaks, crosslinking, alkali-labile sites) assessed using bio-chemical/electrophoretic assays or sister chromatid exchanges (SCE); protein adducts; and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations. The document does not consider germ cells or gene mutation assays other than HPRT or markers of oxidative stress, which have been applied on a more limited scale.     

Evaluation of a genotoxicity test measuring DNA-strand breaks in mouse lymphoma cells by alkaline unwinding and hydroxyapatite elution

A rapid genotoxicity test, based on the measurement of the proportion of single- to double-stranded DNA by alkaline unwinding and hydroxyapatite elution in mouse lymphoma cells treated in vitro with various chemicals, was evaluated. Seventy-eight compounds from diverse chemical groups, including commonly tested mutagens, toxic compounds not usually tested for genotoxicity and non-toxic compounds not thought to be genotoxic were tested. The results obtained were compared with those from the mouse lymphoma TK locus forward-mutation assay, providing a basis for assessing the relative sensitivity of the 2 assays using the same cells exposed to chemicals under similar conditions. Clear evidence of DNA-damaging activity was obtained with 43 of the compounds, while 4 gave equivocal results. Of the remaining 31 compounds, 14 were toxic without inducing DNA damage while the rest were non-toxic and did not induce any DNA damage. Results were available from both the alkaline unwinding assay and the mouse lymphoma assay for 61 compounds; they showed a concordance between the 2 assays of 77%. Of the 47 compounds that were positive or equivocal in the alkaline unwinding assay, only carbon tetrachloride and prednisolone were negative in the mouse lymphoma assay, while 12 of the 19 compounds that were negative in the alkaline unwinding assay were positive in the mouse lymphoma assay. These included 3 compounds that interfere with nucleic acid metabolism, and 3 crosslinking agents, which would be expected to produce mutations to a greater extent than strand breaks. The other 6 compounds were anthranilic acid, benzoquinone, p-chloroaniline, diethylmaleate, glucose and procarbazine HCl. Of these only the last is a known carcinogen. It is concluded from the present study that there was good overall agreement between the results of the DNA alkaline unwinding and mouse lymphoma TK locus assays, but that the sensitivity of the alkaline unwinding assay is lower for some classes of compounds. Bearing this in mind, the alkaline unwinding assay is considered suitable as a rapid screen for genotoxic activity in eukaryotic cells.     

Cobalt and antimony: genotoxicity and carcinogenicity

The purpose of this review is to summarise the data concerning genotoxicity and carcinogenicity of Co and Sb. Both metals have multiple industrial and/or therapeutical applications, depending on the considered species. Cobalt is used for the production of alloys and hard metal (cemented carbide), diamond polishing, drying agents, pigments and catalysts. Occupational exposure to cobalt may result in adverse health effects in different organs or tissues. Antimony trioxide is primarily used as a flame retardant in rubber, plastics, pigments, adhesives, textiles, and paper. Antimony potassium tartrate has been used worldwide as an anti-shistosomal drug. Pentavalent antimony compounds have been used for the treatment of leishmaniasis. Co(II) ions are genotoxic in vitro and in vivo, and carcinogenic in rodents. Co metal is genotoxic in vitro. Hard metal dust, of which occupational exposure is linked to an increased lung cancer risk, is proven to be genotoxic in vitro and in vivo. Possibly, production of active oxygen species and/or DNA repair inhibition are mechanisms involved. Given the recently provided proof for in vitro and in vivo genotoxic potential of hard metal dust, the mechanistic evidence of elevated production of active oxygen species and the epidemiological data on increased cancer risk, it may be advisable to consider the possibility of a new evaluation by IARC. Both trivalent and pentavalent antimony compounds are generally negative in non-mammalian genotoxicity tests, while mammalian test systems usually give positive results for Sb(III) and negative results for Sb(V) compounds. Assessment of the in vivo potential of Sb2O3 to induce chromosome aberrations (CA) gave conflicting results. Animal carcinogenicity data were concluded sufficient for Sb2O3 by IARC. Human carcinogenicity data is difficult to evaluate given the frequent co-exposure to arsenic. Possible mechanisms of action, including potential to produce active oxygen species and to interfere with DNA repair systems, still need further investigation.     

Identification of genotoxic stress in human cells by fluorescent monitoring of p53 expression.

The tumor suppressor protein p53 is induced upon DNA damage essentially by post-translational regulatory mechanisms, which lead to a substantial increase of p53 levels. To exploit this essential property of p53, we developed a novel reporter system for monitoring accumulation and subcellular translocation of p53 protein, which is able to function as a simple test for detecting mutagenic and genotoxic stress in human cells. For this purpose, we constructed a plasmid with a specific translational TP53::EGFP gene fusion and selected stable transfected clones in the human cell line HEK293, in which p53 is functionally stabilized due to the expression of the transgenic adenoviral E1A oncoproteins. HEK293-TP53::EGFP clones may be used as a living cell system for monitoring not only of the induction of p53 protein in the cell, but also of its subcellular localization. Using this human reporter cell system, we examined levels of p53 by fluorescence microscopy and by FACS analysis following treatment with several classes of genotoxic and carcinogenic compounds. All tested DNA damaging agents caused a significant increase of intracellular p53-EGFP levels in a concentration-dependent manner. On the other hand, non-genotoxic carcinogens and stress conditions that cannot damage DNA were not able to induce p53-EGFP accumulation. The induction effect caused by genotoxic stress was found to be dependent on the endogenous p53 status, because it was not observed in p53-deficient cell lines. This corroborates the notion that p53 may be used as an universal sensor for genotoxic stress and demonstrates the usefulness of HEK293-p53-EGFP cells as a reporter system for identification of mutagens and genotoxic carcinogens in human cells by means of visualizing and monitoring intracellular p53 levels and localization.     

Natural isothiocyanates: genotoxic potential versus chemoprevention

Isothiocyanates, occurring in many dietary cruciferous vegetables, show interesting chemopreventive activities against several chronic-degenerative diseases, including cancer, cardiovascular diseases, neurodegeneration, diabetes. The electrophilic carbon residue in the isothiocyanate moiety reacts with biological nucleophiles and modification of proteins is recognized as a key mechanism underlying the biological activity of isothiocyanates. The nuclear factor-erythroid-2-related factor 2 system, which orchestrates the expression of a wide array of antioxidant genes, plays a role in the protective effect of isothiocyanates against almost all the pathological conditions reported above. Recent emerging findings suggest a further common mechanism. Chronic inflammation plays a central role in many human diseases and isothiocyanates inhibit the activity of many inflammation components, suppress cyclooxygenase 2, and irreversibly inactivate the macrophage migration inhibitory factor. Due to their electrophilic reactivity, some isothiocyanates are able to form adducts with DNA and induce gene mutations and chromosomal aberrations. DNA damage has been demonstrated to be involved in the pathogenesis of various chronic-degenerative diseases of epidemiological relevance. Thus, the genotoxicity of the isothiocyanates should be carefully considered. In addition, the dose-response relationship for genotoxic compounds does not suggest evidence of a threshold. Thus, chemicals that are genotoxic pose a greater potential risk to humans than non-genotoxic compounds. Dietary consumption levels of isothiocyanates appear to be several orders of magnitude lower than the doses used in the genotoxicity studies and thus it is highly unlikely that such toxicities would occur in humans. However, the beneficial properties of isothiocyanates stimulated an increase of dietary supplements and functional foods with highly enriched isothiocyanate concentrations on the market. Whether such concentrations may exert a potential health risk cannot be excluded with certainty and an accurate evaluation of the toxicological profile of isothiocyanates should be prompted before any major increase in their consumption be recommended or their clinical use suggested.     

Genotoxicity and potential carcinogenicity of cyanobacterial toxins - a review

The occurrence of cyanobacterial blooms has increased significantly in many regions of the world in the last century due to water eutrophication. These blooms are hazardous to humans, animals, and plants due to the production of cyanotoxins, which can be classified in five different groups: hepatotoxins, neurotoxins, cytotoxins, dermatotoxins, and irritant toxins (lipopolysaccharides). There is evidence that certain cyanobacterial toxins are genotoxic and carcinogenic; however, the mechanisms of their potential carcinogenicity are not well understood. The most frequently occurring and widespread cyanotoxins in brackish and freshwater blooms are the cyclic heptapeptides, i.e., microcystins (MCs), and the pentapeptides, i.e., nodularins (NODs). The main mechanism associated with potential carcinogenic activity of MCs and NOD is the inhibition of protein phosphatases, which leads to the hyperphosphorylation of cellular proteins, which is considered to be associated with their tumor-promoting activity. Apart from this, MCs and NOD induce increased formation of reactive oxygen species and, consequently, oxidative DNA damage. There is also evidence that MCs and NOD induce micronuclei, and NOD was shown to have aneugenic activity. Both cyanotoxins interfere with DNA damage repair pathways, which, along with DNA damage, is an important factor involved in the carcinogenicity of these agents. Furthermore, these toxins increase the expression of TNF-α and early-response genes, including proto-oncogenes, genes involved in the response to DNA damage, cell cycle arrest, and apoptosis. Rodent studies indicate that MCs and NOD are tumor promotors, whereas NOD is thought to have also tumor-initiating activity. Another cyanobacterial toxin, cylindrospermopsin (CYN), which has been neglected for a long time, is lately being increasingly found in the freshwater environment. The principal mechanism of its toxicity is the irreversible inhibition of protein synthesis. It is pro-genotoxic, and metabolic activation by cytochrome P-450 enzymes is needed for its genotoxic activity. In metabolically competent cells, it induces DNA strand breaks and exerts clastogenic and aneugenic activity. In addition, CYN increased the expression of p53 regulated genes involved in cell cycle arrest, DNA damage repair, and apoptosis. It also has cell transforming potential, and limited preliminary rodent studies indicate that CYN could have tumor-initiating activity. In 2010, the International Agency for Research on Cancer (IARC) classified MCLR as possible human carcinogen (Group 2B). Although there is not enough available information for the classification of other cyanobacterial toxins, the existing data from in vitro and in vivo studies indicate that NOD and especially CYN may be even more hazardous than MCLR to human and animal health. In addition in the environment, cyanobacterial toxins occur in complex mixtures as well as together with other anthropogenic contaminants, and numerous studies showed that the toxic/genotoxic potential of the extracts from cyanobacterial scums is higher than that of purified toxins. This means that the mixtures of toxins to which humans are exposed may pose higher health risks than estimated from the toxicological data of a single toxin. Future research efforts should focus on the elucidation of the carcinogenic potential of NOD, CYN, and the mixture of cyanobacterial extracts, as well as on the identification of possible novel toxins.     

The Escherichia coli K-12 SOS chromotest agar spot test for simple, rapid detection of genotoxic agents

The Escherichia coli K-12 SOS chromotest is a colorimetric (beta-galactosidase induction) system for detecting genotoxic chemicals as agents which induce filamentation in response to DNA damage. The chromotest was modified from a liquid suspension assay to a simple, convenient agar spot test, which was performed in the manner of a related colorimetric prophage induction assay (BIA). Chromotest agar dishes yielded optimal results after 16-18 h incubation, presumably because of the agar growth characteristics of tester strain PQ37. Of 44 tested chemicals, nitro aromatics, cytotoxic/antitumor agents, polycyclic hydrocarbons and aflatoxins showed good activity. Alkylating agents such as MNNG and MMS were active only at high concentrations. Compounds active in both the chromotest and BIA were active at 10-100-fold lower concentrations in the chromotest. The chromotest appeared to be less effective than the Salmonella Ames mutagenicity test in the detection of diverse classes of chemical carcinogens. The chromotest may be a useful alternative to the BIA in the study of particular classes of genotoxic compounds.     

Genotoxicity and potential carcinogenicity of cyanobacterial toxins – a review

The occurrence of cyanobacterial blooms has increased significantly in many regions of the world in the last century due to water eutrophication. These blooms are hazardous to humans, animals, and plants due to the production of cyanotoxins, which can be classified in five different groups: hepatotoxins, neurotoxins, cytotoxins, dermatotoxins, and irritant toxins (lipopolysaccharides). There is evidence that certain cyanobacterial toxins are genotoxic and carcinogenic; however, the mechanisms of their potential carcinogenicity are not well understood. The most frequently occurring and widespread cyanotoxins in brackish and freshwater blooms are the cyclic heptapeptides, i.e., microcystins (MCs), and the pentapeptides, i.e., nodularins (NODs). The main mechanism associated with potential carcinogenic activity of MCs and NOD is the inhibition of protein phosphatases, which leads to the hyperphosphorylation of cellular proteins, which is considered to be associated with their tumor-promoting activity. Apart from this, MCs and NOD induce increased formation of reactive oxygen species and, consequently, oxidative DNA damage. There is also evidence that MCs and NOD induce micronuclei, and NOD was shown to have aneugenic activity. Both cyanotoxins interfere with DNA damage repair pathways, which, along with DNA damage, is an important factor involved in the carcinogenicity of these agents. Furthermore, these toxins increase the expression of TNF-α and early-response genes, including proto-oncogenes, genes involved in the response to DNA damage, cell cycle arrest, and apoptosis. Rodent studies indicate that MCs and NOD are tumor promotors, whereas NOD is thought to have also tumor-initiating activity. Another cyanobacterial toxin, cylindrospermopsin (CYN), which has been neglected for a long time, is lately being increasingly found in the freshwater environment. The principal mechanism of its toxicity is the irreversible inhibition of protein synthesis. It is pro-genotoxic, and metabolic activation by cytochrome P-450 enzymes is needed for its genotoxic activity. In metabolically competent cells, it induces DNA strand breaks and exerts clastogenic and aneugenic activity. In addition, CYN increased the expression of p53 regulated genes involved in cell cycle arrest, DNA damage repair, and apoptosis. It also has cell transforming potential, and limited preliminary rodent studies indicate that CYN could have tumor-initiating activity. In 2010, the International Agency for Research on Cancer (IARC) classified MCLR as possible human carcinogen (Group 2B). Although there is not enough available information for the classification of other cyanobacterial toxins, the existing data from in vitro and in vivo studies indicate that NOD and especially CYN may be even more hazardous than MCLR to human and animal health. In addition in the environment, cyanobacterial toxins occur in complex mixtures as well as together with other anthropogenic contaminants, and numerous studies showed that the toxic/genotoxic potential of the extracts from cyanobacterial scums is higher than that of purified toxins. This means that the mixtures of toxins to which humans are exposed may pose higher health risks than estimated from the toxicological data of a single toxin. Future research efforts should focus on the elucidation of the carcinogenic potential of NOD, CYN, and the mixture of cyanobacterial extracts, as well as on the identification of possible novel toxins.