EDC IMPACT: Reduced sperm counts in rats exposed to human relevant mixtures of endocrine disrupters.

Human semen quality is declining in many parts of the world, but the causes are ill defined. In rodents, impaired sperm production can be seen with early life exposure to certain endocrine-disrupting chemicals, but the effects of combined exposures are not properly investigated. In this study, we examined the effects of early exposure to the painkiller paracetamol and mixtures of human relevant endocrine-disrupting chemicals in rats. One mixture contained four estrogenic compounds; another contained eight anti-androgenic environmental chemicals and a third mixture contained estrogens, anti-androgens and paracetamol. All exposures were administered by oral gavage to time-mated Wistar dams rats (n = 16-20) throughout gestation and lactation. In the postnatal period, testicular histology was affected by the total mixture, and at the end of weaning, male testis weights were significantly increased by paracetamol and the high doses of the total and the anti-androgenic mixture, compared to controls. In all dose groups, epididymal sperm counts were reduced several months after end of exposure, i.e. at 10 months of age. Interestingly, the same pattern of effects was seen for paracetamol as for mixtures with diverse modes of action. Reduced sperm count was seen at a dose level reflecting human therapeutic exposure to paracetamol. Environmental chemical mixtures affected sperm count at the lowest mixture dose indicating an insufficient margin of safety for the most exposed humans. This causes concern for exposure of pregnant women to paracetamol as well as environmental endocrine disrupters.

Burden of disease and costs of exposure to endocrine disrupting chemicals in the European Union: an updated analysis.

A previous report documented that endocrine disrupting chemicals contribute substantially to certain forms of disease and disability. In the present analysis, our main objective was to update a range of health and economic costs that can be reasonably attributed to endocrine disrupting chemical exposures in the European Union, leveraging new burden and disease cost estimates of female reproductive conditions from accompanying report. Expert panels evaluated the epidemiologic evidence, using adapted criteria from the WHO Grading of Recommendations Assessment, Development and Evaluation Working Group, and evaluated laboratory and animal evidence of endocrine disruption using definitions recently promulgated by the Danish Environmental Protection Agency. The Delphi method was used to make decisions on the strength of the data. Expert panels consensus was achieved for probable (>20%) endocrine disrupting chemical causation for IQ loss and associated intellectual disability; autism; attention deficit hyperactivity disorder; endometriosis; fibroids; childhood obesity; adult obesity; adult diabetes; cryptorchidism; male infertility, and mortality associated with reduced testosterone. Accounting for probability of causation, and using the midpoint of each range for probability of causation, Monte Carlo simulations produced a median annual cost of €163 billion (1.28% of EU Gross Domestic Product) across 1000 simulations. We conclude that endocrine disrupting chemical exposures in the EU are likely to contribute substantially to disease and dysfunction across the life course with costs in the hundreds of billions of Euros per year. These estimates represent only those endocrine disrupting chemicals with the highest probability of causation; a broader analysis would have produced greater estimates of burden of disease and costs.

Anti-androgens act jointly in suppressing spiggin concentrations in androgen-primed female three-spined sticklebacks - prediction of combined effects by concentration addition.

Increasing attention is being directed at the role played by anti-androgenic chemicals in endocrine disruption of wildlife within the aquatic environment. The co-occurrence of multiple contaminants with anti-androgenic activity highlights a need for the predictive assessment of combined effects, but information about anti-androgen mixture effects on wildlife is lacking. This study evaluated the suitability of the androgenised female stickleback screen (AFSS), in which inhibition of androgen-induced spiggin production provides a quantitative assessment of anti-androgenic activity, for predicting the effect of a four component mixture of anti-androgens. The anti-androgenic activity of four known anti-androgens (vinclozolin, fenitrothion, flutamide, linuron) was evaluated from individual concentration-response data and used to design a mixture containing each chemical at equipotent concentrations. Across a 100-fold concentration range, a concentration addition approach was used to predict the response of fish to the mixture. Two studies were conducted independently at each of two laboratories. By using a novel method to adjust for differences between nominal and measured concentrations, good agreement was obtained between the actual outcome of the mixture exposure and the predicted outcome. This demonstrated for the first time that androgen receptor antagonists act in concert in an additive fashion in fish and that existing mixture methodology is effective in predicting the outcome, based on concentration-response data for individual chemicals. The sensitivity range of the AFSS assay lies within the range of anti-androgenicity reported in rivers across many locations internationally. The approach taken in our study lays the foundations for understanding how androgen receptor antagonists work together in fish and is essential in informing risk assessment methods for complex anti-androgenic mixtures in the aquatic environment.

Mixtures of endocrine disrupting contaminants modelled on human high end exposures: an exploratory study in rats.

By diminishing the action of androgens during gestation, certain chemicals can induce irreversible demasculinization and malformations of sex organs in the male rat after gestational exposure. Studies with mixtures of such anti-androgens have shown that substantial combined effects occur even though each individual chemical is present at low, ineffective doses, but the effects of mixtures modelled based on human intakes have not previously been investigated. To address this issue for the first time, we selected 13 chemicals for a developmental mixture toxicity study in rats where data about in vivo endocrine disrupting effects and information about human exposures was available, including phthalates, pesticides, UV-filters, bisphenol A, parabens and the drug paracetamol. The mixture ratio was chosen to reflect high end human intakes. To make decisions about the dose levels for studies in the rat, we employed the point of departure index (PODI) approach, which sums up ratios between estimated exposure levels and no-observed-adverse-effect-level (NOAEL) values of individual substances. For high end human exposures to the 13 selected chemicals, we calculated a PODI of 0.016. As only a PODI exceeding 1 is expected to lead to effects in the rat, a total dose more than 62 times higher than human exposures should lead to responses. Considering the high uncertainty of this estimate, experience on lowest-observed-adverse-effect-level (LOAEL)/NOAEL ratios and statistical power of rat studies, we expected that combined doses 150 times higher than high end human intake estimates should give no, or only borderline effects, whereas doses 450 times higher should produce significant responses. Experiments indeed showed clear developmental toxicity of the 450-fold dose in terms of increased nipple retention (NR) and reduced ventral prostate weight. The 150-fold dose group exhibited significantly increased NR. These observations suggest that highly exposed population groups, especially women of reproductive age, may not be protected sufficiently against the combined effects of chemicals that affect the hormonal milieu required for normal male sexual differentiation.

Combined exposures to anti-androgenic chemicals: steps towards cumulative risk assessment.

There is widespread exposure to anti-androgens, a group of chemicals able to disrupt androgen action in foetal life, with irreversible de-masculinizing consequences. Substances of concern include certain phthalates, pesticides and chemicals used in cosmetics and personal care products. Although people come into contact with several anti-androgens, chemicals risk assessment normally does not take account of the effects of combined exposures. However, a disregard for combination effects may lead to underestimations of risks and for this reason, we have assessed the feasibility of conducting cumulative risk assessment, where the focus is on considering the effects of exposure to multiple chemicals, via multiple routes and pathways. Following recent recommendations by the US National Research Council, we have, for the first time, included phthalates and other anti-androgenic chemicals, a total of 15 substances. On the basis of exposure estimates for the individual chemicals and reference doses for anti-androgenicity, we have used the hazard index approach. We show that the cumulative risks from anti-androgen exposures exceed acceptable levels for people on the upper end of exposure levels. The value obtained for median exposures to the 15 substances can be judged tolerable. However, significant knowledge gaps exist that prevent us from arriving at definitive conclusions. Of greatest concern is an absence of appropriate in vivo toxicity data about large numbers of in vitro androgen receptor antagonists. Knowledge about the effect profiles of these chemicals will lead to higher risk estimates. Our analysis suggests that risk reductions can be achieved by limiting exposures to the plasticizer diethyl hexyl phthalate, the cosmetic ingredients butyl- and propyl paraben, the pesticides vinclozolin, prochloraz and procymidone and bisphenol A.

Combined exposure to anti-androgens causes markedly increased frequencies of hypospadias in the rat.

The incidence of hypospadias is increasing in young boys, but it remains unclear whether human exposure to endocrine disrupting chemicals plays a role. Risk assessment is based on estimation of no-observed-adverse-effect levels for single compounds, although humans are exposed to combinations of several anti-androgenic chemicals. In a mixture (MIX) study with three androgen receptor antagonists, vinclozolin, flutamide and procymidone, rats were gavaged during gestation and lactation with several doses of a MIX of the three chemicals or the chemicals alone. External malformations of the male reproductive organs were assessed on PND 47 using a score from 0 to 3 (normal to marked) for hypospadias. Markedly increased frequencies were observed after exposure to a MIX of the three chemicals compared to administration of the three chemicals alone. Anogenital distance at PND 1, nipple retention at PND 13, and dysgenesis score at PND 16 were highly correlated with the occurrence of hypospadias, and MIX effects were seen at doses where each of the individual chemicals caused no observable effects. Therefore, the results indicate that doses of anti-androgens, which appear to induce no hypospadias when judged on their own, may induce a very high frequency of hypospadias when they interact in concert with other anti-androgens.

Cytochrome P450 inhibitory action of Echinacea preparations differs widely and co-varies with alkylamide content.

Echinacea preparations are one of the best selling herbal medicinal products with a well established therapeutic use in the prophylaxis of upper respiratory tract infections. Their consumption is increasing, but information about their ability to inhibit cytochrome P450 enzymes (CYP) is fragmentary. The picture is further complicated by a lack of phytochemical characterization of previously tested preparations. Due to its well characterized immunomodulatory activity, the standardized Swiss registered Echinacea purpurea (L.) Moench Echinaforce extract was selected for detailed study. With the single baculovirus-expressed CYP isoforms 1A2, 2C19, 2D9 and 3A4, inhibitory actions were measured by monitoring fluorescent metabolites derived from enzyme substrates (supersome assay). The Echinaforce extract induced mild inhibition of all these isoforms, with CYP 3A4 being the most, and CYP 2D6 the least sensitive enzyme. To assess whether CYP inhibition might be a general feature of Echinacea preparations, an additional nine commercially available preparations were screened using CYP 3A4. All tested preparations were able to inhibit CYP 3A4, but inhibitory potencies (expressed as median inhibitory concentration, IC50) varied by a factor of 150. The alkylamides are thought to be responsible for the immunomodulatory activity of Echinacea, and so the concentration of 2E,4E,8Z,10E/Z-tetranoic acid isobutylamide (1) and total alkylamide content were determined in all preparations, and the latter was found to be associated with their CYP 3A4 inhibitory potency. The chemically pure alkylamides dodeca-2E,4E,8Z,10E/Z-tetranoic acid isobutylamide (1) and dodeca-2E,4E-dieonoic acid isobutylamide (2) showed inhibitory activity on CYP 2C19, 2D6 and 3A4. However, unlike the Echinaforce extract, the alkylamides did not induce CYP 1A2 inhibition. Thus, other, as yet unidentified constituents also contribute to the overall weak inhibitory effects seen with Echinacea preparations in-vitro.

Mixtures of four organochlorines enhance human breast cancer cell proliferation.

In view of the large differences between the concentrations of estrogenic chemicals needed to elicit effects in in vitro assays and their levels in human tissues, it is hard to explain possible health risks in terms of exposure to individual compounds. Human populations, however, are exposed to mixtures of estrogenic and estrogen-like agents and it is necessary to consider the impact of combined effects. We assessed the combined effects of 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p'-DDT), 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE), beta-hexachlorocyclohexane (beta-HCH), and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (p,p'-DDT) on the induction of cell proliferation in MCF-7 cells. All four compounds are persistent organochlorines that can be found in human tissues. We performed extensive concentration-response analyses with the single agents to predict the effects of two mixtures of all four compounds with different mixture ratios. We calculated the predictions by using the pharmacologically well-founded models of concentration addition and independent action and then tested them experimentally. o,p'-DDT, p,p'-DDE, beta-HCH, and p,p'-DDT acted together to produce proliferative effects in MCF-7 cells. The combined effect of the four agents could be predicted on the basis of data about single agent concentration-response relationships. Regression analysis demonstrated that there were combination effects even when each mixture component was present at levels at or below its individual no-observed-effect-concentration. We assessed combination effects in two ways: First, evaluations in relation to the proliferative responses induced by single mixture components revealed that the combination effects were stronger than the effects of the most potent constituent. Thus, according to this method of evaluation, the combined effects may be termed synergistic. Second, comparisons with the expected effects, as predicted by concentration addition and independent action, showed excellent agreement between prediction and observation. With this approach, the combined effect of all four compounds can be termed additive.

Defining the impact of weakly estrogenic chemicals on the action of steroidal estrogens.

We tested whether bisphenol A (BPA) or o,p'-DDT, when combined with 17beta-estradiol (E2), would contribute to the overall mixture effect using a yeast reporter gene assay, the yeast estrogen screen. Following comprehensive concentration-response analyses of the single agents, the pharmacologically well-founded models of concentration addition and independent action were used to predict entire concentration-response relationships for mixtures of the agents with a variety of fixed mixture ratios, assuming additivity. For molar mixture ratios proportional to the levels normally found in human tissues (i.e., below 1:5000, E2:BPA or o,p'-DDT), these predictions suggest that the effects of individual xenoestrogens are too weak to create an impact on the actions of steroidal hormones. However, at mixture ratios more in favor of the xenoestrogens, a significant contribution to the overall mixture effect was predicted. The predictions were tested experimentally. The observed combined effects of mixtures of E2 with either BPA or o,p'-DDT did not deviate from the additivity expectation. On combining E2 with either BPA or o,p'-DDT at approximately equieffective concentrations corresponding to molar mixture ratios between 1:20,000 and 1:100,000 (E2:BPA or o,p'-DDT), substantial modulations of the effects of E2 became discernible. The assumption that weak xenoestrogens are generally unable to create an impact upon the already strong effects of endogenous steroidal estrogens is not supported by our observations. Our studies indicate that the potential health implication of additive combination effects between xenoestrogens and steroidal estrogens deserve serious consideration.

Prediction and assessment of the effects of mixtures of four xenoestrogens.

The assessment of mixture effects of estrogenic agents is regarded as an issue of high priority by many governmental agencies and expert decision-making bodies all over the world. However, the few mixture studies published so far have suffered from conceptual and experimental problems and are considered to be inconclusive. Here, we report the results of assessments of two-, three- and four-component mixtures of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol, all compounds with well-documented estrogenic activity. Extensive concentration-response analyses with the single agents were carried out using a recombinant yeast screen (yeast estrogen screen, YES). Based on the activity of the single agents in the YES assay we calculated predictions of entire concentration-response curves for mixtures of our chosen test agents assuming additive combination effects. For this purpose we employed the models of concentration addition and independent action, both well-established models for the calculation of mixture effects. Experimental concentration-response analyses revealed good agreement between predicted and observed mixture effects in all cases. Our results show that the combined effect of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol in the YES assay does not deviate from expected additivity. We consider both reference models as useful tools for the assessment of combination effects of multiple mixtures of xenoestrogens.

Improving the reproducibility of the MCF-7 cell proliferation assay for the detection of xenoestrogens.

The MCF-7 cell proliferation assay is potentially a simple and highly reproducible tool for the identification of estrogenic compounds. However, its widespread use has been complicated by the lack of a standardised protocol, resulting in considerable inter-laboratory variability. We have explored the sources of variability both in relation to cell lines and test regimens and report on optimised procedures for the identification of estrogenic agents. Two supposedly identical MCF-7 parent cell lines (designated UCL and SOP), and the BUS subline were cultured according to an existing protocol, and responses to 17-estradiol (E2) assessed. Despite yielding almost identical EC50 values, the proliferative response varied widely between cell lines from 0.98-fold over controls (UCL) to 8.9-fold (BUS) indicating major differences between them. The underlying causes may be genetic, and to assess this we used comparative genomic hybridisation (CGH), a technique which allows the detection of DNA sequence copy number changes on a genome-wide scale. Although numerous similarities existed between the different cell lines, the least oestrogen-responsive line (MCF-7/UCL) exhibited the greatest number of cytogenetic changes, many of which were not seen in MCF-7/SOP cells. We suggest that care must be taken, therefore, when choosing a cell line for MCF-7 cell-based experiments. Selecting the MCF-7/SOP line for further work, we carried out a thorough and systematic optimisation of the MCF-7 cell proliferation assay, finding that a 72-h period in oestrogen-free medium before treatment strongly influenced the cells response to E2. With 1 nM E2, proliferation increased from 1.5-fold to 6.5-fold relative to vehicle-treated controls, a response similar to that seen with MCF-7/BUS cells in the E-SCREEN protocol devised by Soto et al. With parent MCF-7 cells, other laboratories have reported only 4.5-fold increases as maximal. Here we present evidence that the choice of cell line and culture conditions are crucial in determining test outcomes, and once chosen and adhered to the assay yields reproducible results.

Comparative genomic hybridization reveals extensive variation among different MCF-7 cell stocks.

Comparative genomic hybridization (CGH) allows the detection of DNA sequence copy number changes on a genome-wide scale in a single hybridization reaction. The ability of CGH to be applied to formalin-fixed, paraffin-embedded tumor samples has lead to its widespread application in the cytogenetic analysis of archival material. When setting up CGH in the laboratory, rigorous control experiments must be carried out to ensure that the losses and gains are scored correctly. Groups interested in breast cancer frequently use the MCF-7 cell line as a positive control in these experiments, comparing the results to previously described genetic alterations. Here we present the results of CGH carried out with three stocks of MCF-7 cells. The cells differ widely in their proliferative response to 17-beta estradiol and show extensive variation in copy number changes affecting specific chromosomal regions. We suggest that care must be taken, therefore, when choosing a cell line as a positive control for CGH experiments.

RAPD library fingerprinting of bacterial and human DNA: applications in mutation detection.

Random amplified polymorphic DNA (RAPD) fingerprinting is a modification of the polymerase chain reaction (PCR), which utilises a single, arbitrarily-chosen primer to amplify a number of fragments from a given template DNA to generate a discrete "fingerprint" when resolved by gel electrophoresis. Alterations by as little as a single base in the primer sequence lead to marked alterations in the fingerprints generated with a given template under optimised conditions. By inference, single base alterations in the genomic template DNA may also lead to changes in the RAPD fingerprints. We have examined this potential application to detect mutations in bacteria and cultured human cells. We have utilised Escherichia coli and human lymphoblastoid cell lines exposed to UV radiation, selected for by cellular mutation assays, and compared RAPD fingerprints of mutant and non-mutant samples. Polymorphisms became evident as the presence and/or absence of DNA fragments between the two samples. A dose-dependent increase in the number of polymorphic bands was seen with UV irradiation of E. coli. To a lesser degree, polymorphisms were also evident for human lymphoblastoid DNA. The possible underlying mechanisms for these alterations in fingerprints as a result of mutation(s) in the primer binding site(s) are discussed. The ability of RAPD fingerprinting to detect a mutant in a population of non-mutants is evaluated, and whilst the lack of sensitivity inherent in the technique precludes its use as a mutation screening assay, its potential for generation of mutant and non-mutant DNA probes for other mutation detection techniques may prove to be of great merit. Teratogenesis Carcinog. Mutagen. 20:49-63, 2000.

Chromium(VI)-mediated DNA damage: oxidative pathways resulting in the formation of DNA breaks and abasic sites.

Inside cells chromium(VI) is activated to its ultimate carcinogenic form by reducing agents including glutathione (GSH) and ascorbate (AsA). The precise mechanism by which DNA damaging species are formed is unclear. In earlier in vitro work with isolated DNA we have shown that chromium(VI) in combination with GSH or AsA is able to induce similar numbers of single strand breaks and apurinic/apyrimidinic sites (AP-sites). Moreover, the formation of both lesions followed a similar temporal pattern. It is conceivable that the two forms of DNA damage arise from a common precursor lesion (e.g. hydrogen abstraction at C4' of the DNA sugar moiety) with a partitioning along two pathways, one yielding an AP-site, the other a single strand break (SSB) and a base propenal. The present study is intended to test this hypothesis by analysing whether oxidation products of deoxyribose can be formed in the presence of chromium(VI) and GSH or AsA. It was found that mixtures of chromium(VI) and GSH or AsA were able to oxidise 2-deoxyribose to yield malondialdehyde, which was detected by reaction with thiobarbituric acid. The characteristic pink chromogen, which forms upon reaction with thiobarbituric acid, was also observed with calf thymus DNA as the substrate. In both experimental systems the addition of catalase prevented the formation of deoxyribose breakdown products. Hydroxyl radicals did not seem to be important for the generation of DNA damage as the characteristic modified DNA bases could not be detected by using gas chromatography-mass spectrometry. These results lead us to conclude that the formation of SSB during the reductive conversion of chromium(VI) proceeds primarily via hydrogen abstraction from C4'. The observation that Fenton chemistry is not involved in these processes is intriguing and necessitates further research into the ways in which chromium can activate molecular oxygen to form DNA damaging species.

Approaches to assessing combination effects of oestrogenic environmental pollutants.

Concerns about possible combination effects of environmental chemicals with estrogenic activity have motivated the search for synergisms between such agents. However, much published work has taken no account of the concepts and methods for analysing combination effects, which were developed in toxicology and pharmacology. In the present communication, we draw attention to conceptual frameworks relevant for a sound analysis of the effects of mixtures of oestrogenic compounds. A model calculation is presented demonstrating that it is conceivable that weakly oestrogenic compounds may be able to act together to produce significant effects, even when they are present at concentrations below their individual effect thresholds. Our results suggest that it may not be necessary to invoke synergisms in order to explain the discrepancy between the high concentrations of these agents required to produce effects in in vitro assay systems and their low concentrations in the environment.

Synergisms with mixtures of xenoestrogens: a reevaluation using the method of isoboles.

There are concerns about possible combination effects of environmental chemicals with oestrogenic activity and their implications for human health. Such chemicals are present in complex mixtures in our environment. A number of studies searching for possible synergistic interactions between xenoestrogens have appeared in the literature. However, in these studies no account was taken of established concepts and methods for analysing combination effects. In the present review, we highlight conceptual issues which may be useful for a sound analysis of the effects of mixtures of xenoestrogens. We find that much published work suffers from an undue focus on measuring effects of mixtures at only one dose level. Assessments of combination effects are frequently complicated by a lack of information on dose-response relationships. Some studies which purportedly show absence of synergy have in fact overlooked synergisms.

Genotypic selection of mutated DNA sequences using mismatch cleavage analysis, a possible basis for novel mutation assays.

A novel technique for the selection of mutated DNA sequences, termed mismatch cleavage-polymerase chain reaction (MC-PCR), is proposed. The method is based on hybridizing genomic DNA with a suitable probe, several 100 bp long. Mutated DNA sequences will form mismatched heteroduplexes which are cleaved by using resolvases. Cleaved heteroduplexes are detected by ligation to an oligonucleotide adaptor and then amplified by using PCR. If practical, this technique would have considerable advantages over the restriction site mutation (RSM) method. Failure to achieve cleavage efficiencies of close to 100% will not compromise success. This is because positive signals (PCR amplification) arise from cleaved mutated sites and not, as in RSM, from DNA sequences resistant to cleavage by restriction endonucleases. Furthermore, the mutational target is much larger than in RSM. It would be possible to screen stretches of DNA several 100 bp in length for mutations. Any mutation, independent of its location, could be identified. The usefulness of MC-PCR for the genotypic selection of mutants will depend on the effectiveness with which a small number of mismatched heteroduplexes can be recognized, cleaved and ligated.

Problems in the biological monitoring of chromium(VI) exposed individuals.

The usefulness of currently available techniques for the biological monitoring of chromium(VI) exposed individuals is reviewed. Chromium levels in body fluids, such as urine and blood plasma, are reliable markers of exposure to chromium in oxidation states (VI) and (III) and provide a measure of the internalized dose of chromium. These markers are sufficiently sensitive to be useful in most occupational settings encountered today. In contrast, the majority of cytogenetic surveillance studies among chromium platers, ferrochromium workers and stainless steel welders using the manual metal arc (MMA) method have yielded negative or inconclusive results. As a marker for genotoxicity, the number of sister chromatid exchanges in blood lymphocytes proved to be relatively insensitive towards exposure to chromium(VI). There were however significant increases in rare chromosome aberrations among MMA stainless steel welders, although the reported levels of all aberrations combined were similar to those observed among control groups of many other studies. The relative lack of success of cytogenetic surveillance studies using blood lymphocytes is surprising in view of the strong genotoxicity of chromium(VI). A possible explanation comes from recent studies which showed that the differences in chromium lymphocyte levels between exposed and controls were disproportionately small. Another factor which complicates attempts to correlate genotoxic effects in lymphocytes with the processes giving rise to cancers of the respiratory system is the toxicokinetics of inhaled chromium(VI). Only small fractions of the total inhaled dose are distributed in the body while the bulk of chromium(VI) deposited in the lungs remains there for very long periods of time. The vast majority of lymphocytes will therefore come into contact with chromium(VI) not while travelling through the supporting tissues of the lungs but during their migration through the blood. There they take up chromium(VI) that has leached from the lungs. Blood lymphocytes therefore seem to be inappropriate for the monitoring of the biologically effective dose, and of early biological effects arising from exposure to chromium(VI). Thus there is an urgent need to develop techniques which would allow the non-invasive monitoring of internalized doses of chromium in the lung.

A role for molecular oxygen in the formation of DNA damage during the reduction of the carcinogen chromium (VI) by glutathione.

Although well-established as carcinogens, the way in which chromium (VI) compounds exert their carcinogenic, mutagenic, and DNA-damaging potential remains obscure. It is clear that inside cells chromium(VI) is activated to its ultimate carcinogenic form by reducing agents including glutathione (GSH). The present study is intended to clarify if Fenton mechanisms are likely to be important in the formation of DNA lesions by chromium(VI) in combination with GSH. In buffer solutions which were treated to remove Fenton-active metal ions as well as in those not further purified, chromate and GSH induced similar numbers of single-strand breaks (SSB) in isolated PM2 DNA. Molecular oxygen was found to be essential for the formation of SSB, but chromium(V) species arising from chromate/GSH, unless activated by oxygen, appeared to be unreactive toward DNA. Upon addition of Mn(II) to solutions of chromium(VI) and GSH a diminution of Mn(II) ESR signals was observed, good evidence for the presence of chromium(IV) species. Using gas chromatography/mass spectrometry in selective ion-monitoring mode and high-performance liquid chromatography with electrochemical detection, we were able to show that Cr(VI)/GSH failed to induce base modifications typical of hydroxyl radical attack on DNA. Experimental conditions which readily induced SSB gave rise to the formation of chromium-DNA adducts, clearly demonstrating that the generation of these two DNA lesions is not mutually exclusive. We conclude that models which ascribe the induction of chromium-DNA adducts to chromium(V) and the generation of oxidative DNA damage including SSB to hydrogen peroxide are oversimplistic. It is not necessary to invoke a mechanism requiring the presence of added hydrogen peroxide to account for the ability of Cr(VI)/GSH to cause SSB. Our findings suggest that the combination of GSH, molecular oxygen, and chromium(VI) can damage DNA via non-Fenton pathways.