Current aspects in metal genotoxicity

While carcinogenic metal ions are mostly non-mutagenic in bacteria, different types of cellular damage have been observed in mammalian cells, which may account for their carcinogenic potential. Two modes of action seem to be predominant: the induction of oxidative DNA damage, best established for chromium compounds, and the interaction with DNA repair processes, leading to an enhancement of genotoxicity in combination with a variety of DNA damaging agents. In the case of Cd(II), Ni(II), Co(II), Pb(II) and As(III), DNA repair processes are disturbed at low, non-cytotoxic concentrations of the respective metal compounds. Even though different steps in DNA repair are affected by the diverse metals, one common mechanism might be the competition with essential metal ions.     

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.     

Impact of arsenite and its methylated metabolites on PARP-1 activity, PARP-1 gene expression and poly(ADP-ribosyl)ation in cultured human cells

The underlying mechanisms of arsenic carcinogenicity are still not fully understood. Mechanisms currently discussed include the induction of oxidative DNA damage and the interference with DNA repair pathways. Still unclear is the role of biomethylation, which has long been considered to be one major detoxification process. Methylated arsenicals have recently been shown to interfere with DNA repair in cellular and subcellular systems, but up to now no DNA repair protein has been identified being particular sensitive towards methylated arsenicals in cultured cells. Here we report that the trivalent methylated metabolites MMA(III) and DMA(III) inhibit poly(ADP-ribosyl)ation in cultured human HeLa S3 cells at concentrations as low as 1nM, thereby showing for the first time an inactivation of an enzymatic reaction related to DNA repair by the trivalent methylated arsenicals at very low environmentally relevant concentrations. In contrast the pentavalent metabolites MMA(V) and DMA(V) showed no such effects up to high micromolar concentrations. All investigated arsenicals did not alter gene expression of PARP-1. However, all trivalent arsenicals were able to inhibit the activity of isolated PARP-1, indicating that the observed decrease in poly(ADP-ribosyl)ation in cultures human cells, predominantly mediated by PARP-1, is likely due to changes in the activity of PARP-1. Since poly(ADP-ribosyl)ation plays a major role in DNA repair, cell cycle control and thus in the maintenance of genomic stability, these findings could in part explain DNA repair inhibition and the genotoxic and carcinogenic effects of arsenic.     

Indirect mechanism of lead-induced genotoxicity in cultured mammalian cells

The data concerning the mutagenic, clastogenic and carcinogenic properties of inorganic lead compounds have been conflicting. To investigate whether the genotoxicity of lead is due to indirect effects such as interference with DNA-repair processes, the induction of mutations, sister-chromatid exchanges and strand breaks by lead ions alone as well as in combination with UV light as a standard mutagen were determined. Lead acetate alone does not induce DNA-strand breaks in HeLa cells or mutations at the HPRT locus and sister-chromatid exchanges in V79 Chinese hamster cells. However, at all endpoints tested, lead ions interfere with the processing of UV-induced DNA damage. They inhibit the closing of DNA-strand breaks after UV irradiation and enhance the number of UV-induced mutations and sister-chromatid exchanges, indicating an inhibition of DNA repair. These data point out the necessity to consider such indirect effects when assessing the genotoxicity of metal compounds. As possible mechanisms of repair inhibition we suggest either the interaction with repair enzymes such as polymerase or ligase or else the interaction with calcium-regulated processes, for example with calmodulin.     

Facilitative mechanisms of lead as a carcinogen

The carcinogenicity of lead compounds has received renewed attention because of continuing environmental and occupational sources of exposure in many countries. The epidemiological evidence for an association between lead exposures and human cancer risk has been strengthened by recent studies, and new data on mechanisms of action provide biological plausibility for assessing lead as a human carcinogen. Both epidemiological and mechanistic data are consistent with a facilitative role for lead in carcinogenesis, that is, lead by itself may not be both necessary and sufficient for the induction of cancer, but at a cellular and molecular level lead may permit or enhance carcinogenic events involved in DNA damage, DNA repair, and regulation of tumor suppressor and promoter genes. Some of these events may also be relevant to understanding mechanisms of lead-induced reproductive toxicity.     

Molecular mechanisms of arsenic carcinogenesis

Arsenic is a metalloid compound that is widely distributed in the environment. Human exposure of this compound has been associated with increased cancer incidence. Although the exact mechanisms remain to be investigated, numerous carcinogenic pathways have been proposed. Potential carcinogenic actions for arsenic include oxidative stress, genotoxic damage, DNA repair inhibition, epigenetic events, and activation of certain signal transduction pathways leading to abberrant gene expression. In this article, we summarize current knowledge on the molecular mechanisms of arsenic carcinogenesis with an emphasis on ROS and signal transduction pathways.     

A newly adapted pulsed-field gel electrophoresis technique allows to detect distinct types of DNA damage at low frequencies in human dermal fibroblasts upon exposure to non-toxic H2O2 concentrations

Reactive oxygen species (ROS) comprise several oxygen containing compounds, among them hydrogen peroxide (H₂O₂), which are generated by internal and external sources and play pleiotropic roles in physiological and pathological states. Skin cells as well as cells from other tissues have developed antioxidant defense mechanisms to protect themselves from high concentrations of ROS. Although biological and pathological roles of ROS have previously been elucidated, so far only limited knowledge exists regarding ROS-mediated generation of DNA breaks and base lesions occurring at low frequency in intact skin cells. This study was therefore designed to probe a newly adapted pulsed-field gel electrophoresis technique for the adequate measurement of high molecular weight DNA fragments as well as to investigate the protective role of the antioxidant enzyme catalase against H₂O₂-mediated damage in human dermal fibroblasts. We stably transfected and overexpressed the full-length catalase cDNA in the human dermal fibroblast cell line 1306 in culture and found that these cells are significantly more protected from cytotoxicity, overall DNA strand breaks, and 8-oxodeoxyguanine base lesions resulting from H₂O₂-triggered oxidative stress compared to vector-transfected 1306 cells or secondary dermal fibroblasts. This work has outlined the importance of catalase in the protection from H₂O₂-mediated cytotoxicity and DNA damage which — if unbalanced — even when occurring at low frequency are known to lead to genomic instability, a hallmark in carcinogenesis and premature aging.     

Experimental study of oxidative DNA damage

Animal experiments allow the study of oxidative DNA damage in target organs and the elucidation of dose-response relationships of carcinogenic and other harmful chemicals and conditions as well as the study of interactions of several factors. So far the effects of more than 50 different chemical compounds have been studied in animal experiments mainly in rats and mice, and generally with measurement of 8-oxodG with HPLC-EC. A large number of well-known carcinogens induce 8-oxodG formation in liver and/or kidneys. Moreover several animal studies have shown a close relationship between induction of dative DNA damage and tumour formation.

In principle the level of oxidative DNA damage in an organ or cell may be studied by measurement of modified bases in extracted DNA by immunohistochemical visualisation, and from assays of strand breakage before and after treatment with repair enzymes. However, this level is a balance between the rates of damage and repair. Until the repair rates and capacity can be adequately assessed the rate of damage can only be estimated from the urinary excretion of repair products albeit only as an average of the entire body.

A number of model compounds have been used to induce oxidative DNA damage in experimental animals. The hepatocarcinogen 2-nitropropane induces up to 10-fold increases in 8-oxodG levels in rat liver DNA. The level of 8-oxodG is also increased in kidneys and bone marrow but not in the testis. By means of 2-nitropropane we have shown correspondence between the increases in 8-oxodG in target organs and the urinary excretion of 8-oxodG and between 8-oxodG formation and the comet assay in bone marrow as well potent preventive effects of extracts of Brussels sprouts. Others have shown similar effects of green tea extracts and its components. Drawbacks of the use of 2-nitropropane as a model for oxidative DNA damage relate particularly to formation of 8-aminoguanine derivatives that may interfere with HPLC-EC assays and have unknown consequences. Other model compounds for induction of oxidative DNA damage, such as ferric nitriloacetate, iron dextran, potassium bromate and paraquat, are less potent and/or more organ specific.

Inflammation and activation of an inflammatory response by phorbol esters or E. coli lipopolysaccharide (LPS) induce oxidative DNA damage in many target cells and enhance benzene-induced DNA damage in mouse bone marrow.

Experimental studies provide powerful tools to investigate agents inducing and preventing oxidative damage to DNA and its role in carcinogenesis. So far, most animal experiments have concerned 8-oxodG and determination of additional damaged bases should be employed. An ideal animal model for prevention of oxidative DNA damage has yet to he developed.     

Mechanisms of cadmium induced genomic instability

Cadmium is an ubiquitous environmental contaminant that represents hazard to humans and wildlife. It is found in the air, soil and water and, due to its extremely long half-life, accumulates in plants and animals. The main source of cadmium exposure for non-smoking human population is food. Cadmium is primarily toxic to the kidney, but has been also classified as carcinogenic to humans by several regulatory agencies. Current evidence suggests that exposure to cadmium induces genomic instability through complex and multifactorial mechanisms. Cadmium dose not induce direct DNA damage, however it induces increase in reactive oxygen species (ROS) formation, which in turn induce DNA damage and can also interfere with cell signalling. More important seems to be cadmium interaction with DNA repair mechanisms, cell cycle checkpoints and apoptosis as well as with epigenetic mechanisms of gene expression control. Cadmium mediated inhibition of DNA repair mechanisms and apoptosis leads to accumulation of cells with unrepaired DNA damage, which in turn increases the mutation rate and thus genomic instability. This increases the probability of developing not only cancer but also other diseases associated with genomic instability. In the in vitro experiments cadmium induced effects leading to genomic instability have been observed at low concentrations that were comparable to those observed in target organs and tissues of humans that were non-occupationally exposed to cadmium. Therefore, further studies aiming to clarify the relevance of these observations for human health risks due to cadmium exposure are needed.     

Low doses of selenium specifically stimulate the repair of oxidative DNA damage in LNCaP prostate cancer cells

Epidemiological studies have demonstrated an inverse relationship between selenium (Se) intake and cancer incidence and/or mortality. However, the molecular mechanisms underlying the cancer chemopreventive activity of Se compounds remain largely unknown. The objective of this study was to investigate the effect of low doses of Se on the stimulation of DNA repair systems in response to four different qualities of DNA damage. P53-proficient LNCaP human prostate adenocarcinoma cells were grown either untreated or in the presence of low concentrations of two Se compounds (30° nM sodium selenite, or 10 μM selenomethionine) and exposed to UVA, H2O2, methylmethane sulfonate (MMS) or UVC. Cell viability as well as DNA damage induction and repair were evaluated by the alkaline Comet assay. Overall, Se was shown to be a very potent protector against cell toxicity and genotoxicity induced by oxidative stress (UVA or H2O2) but not from the agents that induce other types of deleterious lesions (MMS or UVC). Furthermore, Se-treated cells exhibited increased oxidative DNA repair activity, indicating a novel mechanism of Se action. Therefore, the benefits of Se could be explained by a combination of antioxidant activity, the reduction in DNA damage and the enhancement of oxidative DNA repair capacity.     

Modulation by Co(II) of UV-induced DNA repair, mutagenesis and sister-chromatid exchanges in mammalian cells

In bacterial test systems, Co(II) has been shown to be antimutagenic in combination with several chemical and physical agents. To investigate whether such modulations also apply to mammalian cells, the effect of Co(II) on UV-induced mutagenesis, sister-chromatid exchanges as well as DNA damage and its removal was determined. Co(II) itself is weakly mutagenic at the HPRT locus and increases the frequency of sister-chromatid exchanges. Additionally, at both endpoints the metal ions enhance the genotoxicity of UV light. To discriminate between an enhancement of DNA damage and an interference with repair processes, the number of pyrimidine cyclobutane dimers was determined by HPLC. While the induction of these DNA lesions is not affected by Co(II), their removal is inhibited at concentrations of 75 microM Co(II) and higher. Analysis of the kinetics of strand-break induction and closure after UV irradiation by nucleoid sedimentation reveals an accumulation of strand breaks in the presence of Co(II). This indicates that either the polymerization or the ligation step in excision repair is affected. Since similar interactions with the processing of UV-induced DNA damage have been observed with other carcinogenic and/or mutagenic metal ions, this appears to be a common mechanism of metal genotoxicity.     

Oxidative stress in Lactococcus lactis

Lactococcus lactis, the most extensively characterized lactic acid bacterium, is a mesophilic- and microaerophilic-fermenting microorganism widely used for the production of fermented food products. During industrial processes, L. lactis is often exposed to multiple environmental stresses (low and high temperature, low pH, high osmotic pressure, nutrient starvation and oxidation) that can cause loss or reduction of bacterial viability, reproducibility, as well as organoleptic and/or fermentative qualities. Among these stress factors, oxidation can be considered one of the most deleterious to the cell, causing cellular damage at both molecular and metabolic levels. During the last two decades, considerable efforts have been made to improve our knowledge of oxidative stress in L. lactis. Many genes involved with both oxidative stress resistance and control mechanisms have been identified; functionally they seem to overlap. The finding of new genes, and a better understanding of the molecular mechanisms of stress resistance in L. lactis and other lactic acid bacterium, will lead to the construction and isolation of stress-resistant strains. Such strains could be exploited for both traditional and probiotic uses.     

The moose,purine degradation,and environmental adaptation

It is accepted that allantoin is the end-product of purine degradation in mammals, except that uricase activity has been lost during the evolution of humans in which uric acid protects the brain from oxidative damage. However, we have found that the moose Alces americanus excretes extremely low urinary concentrations of allantoin and high concentrations of uric acid very similar to those of humans. Exposure to extreme cold is known to cause oxidative damage, and we suggest that the retention of uric acid by the moose represents an adaptation enabling the species to survive at high latitudes.     

Total body irradiation causes residual bone marrow injury by induction of persistent oxidative stress in murine hematopoietic stem cells

Ionizing radiation (IR) and/or chemotherapy causes not only acute tissue damage but also late effects including long-term (or residual) bone marrow (BM) injury. The induction of residual BM injury is primarily attributable to the induction of hematopoietic stem cell (HSC) senescence. However, the molecular mechanisms by which IR and/or chemotherapy induces HSC senescence have not been clearly defined, nor has an effective treatment been developed to ameliorate the injury. Thus, we investigated these mechanisms in this study. The results from this study show that exposure of mice to a sublethal dose of total body irradiation (TBI) induced a persistent increase in reactive oxygen species (ROS) production in HSCs only. The induction of chronic oxidative stress in HSCs was associated with sustained increases in oxidative DNA damage, DNA double-strand breaks (DSBs), inhibition of HSC clonogenic function, and induction of HSC senescence but not apoptosis. Treatment of the irradiated mice with N-acetylcysteine after TBI significantly attenuated IR-induced inhibition of HSC clonogenic function and reduction of HSC long-term engraftment after transplantation. The induction of chronic oxidative stress in HSCs by TBI is probably attributable to the up-regulation of NADPH oxidase 4 (NOX4), because irradiated HSCs expressed an increased level of NOX4, and inhibition of NOX activity with diphenylene iodonium but not apocynin significantly reduced TBI-induced increases in ROS production, oxidative DNA damage, and DNA DSBs in HSCs and dramatically improved HSC clonogenic function. These findings provide the foremost direct evidence demonstrating that TBI selectively induces chronic oxidative stress in HSCs at least in part via up-regulation of NOX4, which leads to the induction of HSC senescence and residual BM injury.     

Titanium dioxide particles phosphorylate histone H2AX independent of ROS production

Zinc oxide (ZnO) nanoparticles are finding applications in a wide range of products including cosmetics, food packaging, imaging, etc. This increases the likelihood of human exposure to these nanoparticles through dermal, inhalation and oral routes. Presently, the majority of the studies concerning ZnO nanoparticle toxicity have been conducted using in vitro systems which lack the complex cell-cell, cell-matrix interactions and hormonal effects found in the in vivo scenario. The present in vivo study in mice was aimed at investigating the oral toxicity of ZnO nanoparticles. Our results showed a significant accumulation of nanoparticles in the liver leading to cellular injury after sub-acute oral exposure of ZnO nanoparticles (300 mg/kg) for 14 consecutive days. This was evident by the elevated alanine aminotransferase (ALT) and alkaline phosphatase (ALP) serum levels and pathological lesions in the liver. ZnO nanoparticles were also found to induce oxidative stress indicated by an increase in lipid peroxidation. The DNA damage in the liver and kidney cells of mice was evaluated by the Fpg-modified Comet assay which revealed a significant (p<0.05) increase in the Fpg-specific DNA lesions in liver indicating oxidative stress as the cause of DNA damage. The TUNEL assay revealed an induction of apoptosis in the liver of mice exposed to ZnO nanoparticles compared to the control. Our results conclusively demonstrate that sub-acute oral exposure to ZnO nanoparticles in mice leads to an accumulation of nanoparticles in the liver causing oxidative stress mediated DNA damage and apoptosis. These results also suggest the need for a complete risk assessment of any new engineered nanoparticle before its arrival into the consumer market.     

Stressed out mitochondria: The role of mitochondria in ageing and cancer focussing on strategies and opportunities in human skin

Mitochondrial DNA damage has been used as a successful and unique biomarker of tissue stress. A valuable example of this is sun damage in human skin which leads to ageing and skin cancer. The skin is constantly exposed to the harmful effects of sunlight, such as ultraviolet radiation, which causes it to age with observable characteristic features as well as clinical precancerous lesions and skin cancer. Formation of free radicals by the sun's harmful rays which contribute to oxidative stress has been linked to the induction of deletions and mutations in the mitochondrial DNA. These markers of mitochondrial DNA damage have been proposed to contribute to the mechanisms of ageing in many tissues including skin and are associated with many diseases including cancer. In this article we highlight the role of this important organelle in ageing and cancer with particular emphasis on experimental strategies in the skin.     

Role of the Alkali Labile Sites,Reactive Oxygen Species and Antioxidants in DNA Damage Induced by Methylated Trivalent Metabolites of Inorganic Arsenic

In the last decade arsenic metabolism has become an important matter of discussion. Methylation of inorganic arsenic (iAs) to monomethylarsonic acid (MMA^V) and dimethylarsinic acid (DMA^V) is considered to decrease arsenic toxicity. However, in addition to these pentavalent metabolites, the trivalent metabolites monomethylarsonous (MMA^III) and dimethylarsinous acid (DMA^III) have been identified recently as intermediates in the metabolic pathway of arsenic in cultured human cells. To examine the role of oxidative damage in the generation of DNA strand breaks by methylated trivalent arsenic metabolites, we treated human lymphocytes with both metabolites at non-cytotoxic concentrations. We further tested whether these effects are sensitive to modulation by the antioxidants ascorbate (Vitamin C) and selenomethionine (Se-Met). Both trivalent metabolites produced oxidative stress related DNA damage, consisting of single strand breaks and alkali-labile sites, with MMA^III being more potent at low concentrations than DMA^III. Neither MMA^III nor DMA^III induced DNA-double strand breaks. The oxidative stress response profiles of the metabolites were parallel as determined by lipid peroxidation induction. MMA^III induced peroxidation from the lowest concentration tested, while effects of DMA^III were apparent only at concentrations above 10 μM. The antioxidant Se-Met exhibited a more pronounced inhibition of trivalent arsenic metabolite-induced oxidative-DNA damage than did vitamin C. The present findings suggest that DNA damage by methylated trivalent metabolites at non-cytotoxic concentrations may be mediated by a mix of reactive oxygen and nitrogen oxidized species.     

Induction of retrovirus gene expression by aflatoxin B1 and 2-acetylaminofluorene

The abilities of 2-acetylaminofluorene (AAF) and aflatoxin B1 (AFB) to induce the expression of endogenous murine retrovirus in AKR mouse embryo fibroblasts were examined. AFB was able to induce virus expression at concentrations as low as 0.067 microM, although the induction efficiency was increased at higher doses. AAF gave somewhat more variable results. In some experiments induction was observed with as little as 0.1 microM while in other experiments induction was not observed at concentrations as high as 100 microM. Addition of an S9 metabolizing system enhanced the ability of AAF to induce virus expression but also increased the background induction frequency. These results are discussed in relation to the mechanism of virus induction and the potential usefulness of virus induction as an assay for carcinogenic potential.     

Arsenite and its biomethylated metabolites interfere with the formation and repair of stable BPDE-induced DNA adducts in human cells and impair XPAzf and Fpg

The underlying mechanisms of arsenic carcinogenicity are only poorly understood and especially the role of biomethylation is still a matter of debate. Besides the induction of oxidative DNA damage the interference with DNA repair processes have been proposed to contribute to arsenic-induced carcinogenicity. Within the present study the effects of arsenite and its mono- and dimethylated trivalent and pentavalent metabolites on BPDE-induced DNA adduct formation and repair has been investigated and compared in cultured human lung cells. Whereas only arsenite and MMA(III) increased BPDE-DNA adduct formation, arsenite (>/=5 microM), the trivalent (>/=2.5 microM) and the pentavalent (>/=250 microM) metabolites diminished their repair at non-cytotoxic concentrations. As potential molecular targets, interactions with the zinc finger domain of the human XPA protein (XPAzf) and the Escherichia coli zinc finger protein Fpg, involved in NER and BER, respectively, have been investigated. All trivalent arsenicals were able to release zinc from XPAzf; furthermore, MMA(III) and DMA(III) inhibited the activity of isolated Fpg. Altogether the results suggest that besides arsenite, especially the trivalent methylated metabolites may contribute to diminished NER at low concentrations.