New aspects in deriving health-based guidance values for bromate in swimming pool water.

Bromate, classified as a EU CLP 1B carcinogen, is a typical by-product of the disinfection of drinking and swimming pool water. The aim of this study was (a) to provide data on the occurrence of bromate in pool water, (b) to re-evaluate the carcinogenic MOA of bromate in the light of existing data, (c) to assess the possible exposure to bromate via swimming pool water and (d) to inform the derivation of cancer risk-related bromate concentrations in swimming pool water. Measurements from monitoring analysis of 229 samples showed bromate concentrations in seawater pools up to 34 mg/L. A comprehensive non-systematic literature search was done and the quality of the studies on genotoxicity and carcinogenicity was assessed by Klimisch criteria (Klimisch et al., Regul Toxicol Pharmacol 25:1-5, 1997) and SciRAP tool (Beronius et al., J Appl Toxicol, 38:1460-1470, 2018) respectively. Benchmark dose (BMD) modeling was performed using the modeling average mode in BMDS 3.1 and PROAST 66.40, 67 and 69 (human cancer BMDL10; EFSA 2017). For exposure assessment, data from a wide range of sources were evaluated for their reliability. Different target groups (infants/toddlers, children and adults) and exposure scenarios (recreational, sport-active swimmers, top athletes) were considered for oral, inhalation and dermal exposure. Exposure was calculated according to the frequency of swimming events and duration in water. For illustration, cancer risk-related bromate concentrations in pool water were calculated for different target groups, taking into account their exposure using the hBMDL10 and a cancer risk of 1 in 100,000. Convincing evidence was obtained from a multitude of studies that bromate induces oxidative DNA damage and acts as a clastogen in vitro and in vivo. Since statistical modeling of the available genotoxicity data is compatible with both linear as well as non-linear dose-response relationships, bromate should be conservatively considered to be a non-threshold carcinogen. BMD modeling with model averaging for renal cancer studies (Kurokawa et al., J Natl. Cancer Inst, 1983 and 1986a; DeAngelo et al., Toxicol Pathol 26:587-594, 1998) resulted in a median hBMDL10 of 0.65 mg bromate/kg body weight (bw) per day. Evaluation of different age and activity groups revealed that top athletes had the highest exposure, followed by sport-active children, sport-active adults, infants and toddlers, children and adults. The predominant route of exposure was oral (73-98%) by swallowing water, followed by the dermal route (2-27%), while the inhalation route was insignificant (< 0.5%). Accepting the same risk level for all population groups resulted in different guidance values due to the large variation in exposure. For example, for an additional risk of 1 in 100,000, the bromate concentrations would range between 0.011 for top athletes, 0.015 for sport-active children and 2.1 mg/L for adults. In conclusion, the present study shows that health risks due to bromate exposure by swimming pool water cannot be excluded and that large differences in risk exist depending on the individual swimming habits and water concentrations.

Critical evaluation of human health risks due to hydraulic fracturing in natural gas and petroleum production.

The use of hydraulic fracturing (HF) to extract oil and natural gas has increased, along with intensive discussions on the associated risks to human health. Three technical processes should be differentiated when evaluating human health risks, namely (1) drilling of the borehole, (2) hydraulic stimulation, and (3) gas or oil production. During the drilling phase, emissions such as NOx, NMVOCs (non-methane volatile organic compounds) as precursors for tropospheric ozone formation, and SOx have been shown to be higher compared to the subsequent phases. In relation to hydraulic stimulation, the toxicity of frac fluids is of relevance. More than 1100 compounds have been identified as components. A trend is to use fewer, less hazardous and more biodegradable substances; however, the use of hydrocarbons, such as kerosene and diesel, is still allowed in the USA. Methane in drinking water is of low toxicological relevance but may indicate inadequate integrity of the gas well. There is a great concern regarding the contamination of ground- and surface water during the production phase. Water that flows to the surface from oil and gas wells, so-called 'produced water', represents a mixture of flow-back, the injected frac fluid returning to the surface, and the reservoir water present in natural oil and gas deposits. Among numerous hazardous compounds, produced water may contain bromide, arsenic, strontium, mercury, barium, radioactive isotopes and organic compounds, particularly benzene, toluene, ethylbenzene and xylenes (BTEX). The sewage outflow, even from specialized treatment plants, may still contain critical concentrations of barium, strontium and arsenic. Evidence suggests that the quality of groundwater and surface water may be compromised by disposal of produced water. Particularly critical is the use of produced water for watering of agricultural areas, where persistent compounds may accumulate. Air contamination can occur as a result of several HF-associated activities. In addition to BTEX, 20 HF-associated air contaminants are group 1A or 1B carcinogens according to the IARC. In the U.S., oil and gas production (including conventional production) represents the second largest source of anthropogenic methane emissions. High-quality epidemiological studies are required, especially in light of recent observations of an association between childhood leukemia and multiple myeloma in the neighborhood of oil and gas production sites. In conclusion, (1) strong evidence supports the conclusion that frac fluids can lead to local environmental contamination; (2) while changes in the chemical composition of soil, water and air are likely to occur, the increased levels are still often below threshold values for safety; (3) point source pollution due to poor maintenance of wells and pipelines can be monitored and remedied; (4) risk assessment should be based on both hazard and exposure evaluation; (5) while the concentrations of frac fluid chemicals are low, some are known carcinogens; therefore, thorough, well-designed studies are needed to assess the risk to human health with high certainty; (6) HF can represent a health risk via long-lasting contamination of soil and water, when strict safety measures are not rigorously applied.

Critical evaluation of key evidence on the human health hazards of exposure to bisphenol A.

Despite the fact that more than 5000 safety-related studies have been published on bisphenol A (BPA), there seems to be no resolution of the apparently deadlocked controversy as to whether exposure of the general population to BPA causes adverse effects due to its estrogenicity. Therefore, the Advisory Committee of the German Society of Toxicology reviewed the background and cutting-edge topics of this BPA controversy. The current tolerable daily intake value (TDI) of 0.05 mg/kg body weight [bw]/day, derived by the European Food Safety Authority (EFSA), is mainly based on body weight changes in two- and three-generation studies in mice and rats. Recently, these studies and the derivation of the TDI have been criticized. After having carefully considered all arguments, the Committee had to conclude that the criticism was scientifically not justified; moreover, recently published additional data further support the reliability of the two- and three-generation studies demonstrating a lack of estrogen-dependent effects at and below doses on which the current TDI is based. A frequently discussed topic is whether doses below 5 mg/kg bw/day may cause adverse health effects in laboratory animals. Meanwhile, it has become clear that positive results from some explorative studies have not been confirmed in subsequent studies with higher numbers of animals or a priori defined hypotheses. Particularly relevant are some recent studies with negative outcomes that addressed effects of BPA on the brain, behavior, and the prostate in rodents for extrapolation to the human situation. The Committee came to the conclusion that rodent data can well be used as a basis for human risk evaluation. Currently published conjectures that rats are insensitive to estrogens compared to humans can be refuted. Data from toxicokinetics studies show that the half-life of BPA in adult human subjects is less than 2 hours and BPA is completely recovered in urine as BPA-conjugates. Tissue deconjugation of BPA-glucuronide and -sulfate may occur. Because of the extremely low quantities, it is only of minor relevance for BPA toxicity. Biomonitoring studies have been used to estimate human BPA exposure and show that the daily intake of BPA is far below the TDI for the general population. Further topics addressed in this article include reasons why some studies on BPA are not reproducible; the relevance of oral versus non-oral exposure routes; the degree to which newborns are at higher systemic BPA exposure; increased BPA exposure by infusions in intensive care units; mechanisms of action other than estrogen receptor activation; and the current regulatory status in Europe, as well as in the USA, Canada, Japan, New Zealand, and Australia. Overall, the Committee concluded that the current TDI for BPA is adequately justified and that the available evidence indicates that BPA exposure represents no noteworthy risk to the health of the human population, including newborns and babies.

Influence of antimonite, selenite, and mercury on the toxicity of arsenite in primary rat hepatocytes.

The long-term toxicity of arsenic (As) as a result of exposure to contaminated drinking water might be modified by coinciding exposures to elements like selenium, antimony, or mercury. In this study the influence of tetravalent selenite, trivalent antimonite, and divalent mercury was investigated in vitro using cultured primary rat hepatocytes. The cell vitality was assessed in the 3-[4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] (MTT), assay with concurrent exposures of the cells to up to 50 microM sodium arsenite(III) and a potential modifier [50 microM sodium(IV) selenite, 10 microM antimony(III) chloride, 25 microM mercuric(II) chloride], which indicated an additive increase in the combined cytotoxicity. Sodium arsenite was tested for genotoxicity in the micronucleus test in a concentration range of 0.25 up to 7.5 microM. In this range, the MTT conversion was at least 80%, indicating high cell viability. Adose-dependent induction of micronuclei was observed. The lowest concentration causing a significantly elevated frequency of micronuclei was 1 microM As (p < 0.05). A significant influence (i.e., reduction of the combined genotoxicity as a result of the presence of a potential modifier) was only observed for 10 and 25 microM antimony chloride (p < 0.05, Fisher's exact test). The metabolic methylation of arsenite was not affected by concurrent incubation with any of the potential modifiers.

Micronuclei assessment in buccal cells of people environmentally exposed to arsenic in northern Chile.

To determine the genotoxic risk associated to environmental arsenic exposure, the frequency of micronuclei in buccal cells (BCMN) of people drinking arsenic-contaminated water has been evaluated. A group of 105 individuals from the Antofagasta region (north Chile), and 102 individuals from the area of Concepcion, used as reference group, were included in the study. Arsenic concentration in drinking water was high (0.75 mg/L) in the Antofagasta area, 75-fold the maximum recommended level by WHO (0.01 mg/L), while the values obtained in Concepcion were significantly lower (0.002 mg/L). Individual measures of arsenic exposure were also determined in fingernails, which clearly confirm the existence of chronic exposure in the sampled populations from the Antofagasta region (10.15 microg/g versus 3.57 microg/g). The cytogenetic results indicate that, although the BCMN frequency is higher in exposed than in controls, this increase does not attain statistical significance. When the exposure biomarkers were related with the cytogenetic values, no correlations were observed between BCMN and arsenic content in water or in fingernails. In addition, the genotoxicity values do not seem to be related to the ethnic origin from people belonging to the exposed group. As a conclusion it appears that, in the studied population, the chronic ingestion of arsenic-contaminated water does not induce cytogenetic damage, measured as micronuclei, in the cells of the oral mucous in a significant extent.

Induction of the peroxisome proliferator activated receptor by fenofibrate in rat liver.

The process of peroxisome proliferation in rodent liver by hypolipidemic compounds and related substances has recently been shown to be receptor-mediated. In the present study, we have examined the effect of oral administration of the strong peroxisome proliferator fenofibrate on the hepatic expression level of the peroxisome proliferator activated receptor (PPAR) in rats. Immunoblots of rat liver cytosols and nuclear extracts using antibodies raised against recombinant PPAR/beta-galactosidase fusion proteins revealed a pronounced increase in the amount of PPAR protein in response to fenofibrate treatment. This induction could also be confirmed at the level of RNA by Northern blotting. A time-course investigation showed a delayed accumulation of mRNA in response to the treatment, starting on day 2 after a latency period of at least one day. Thus, induction of the PPAR as a response to peroxisome proliferators represents one important dimension of the pleiotropic effects of peroxisome proliferators.

The occurrence of carbonyl reduction in continuous cell lines emphasizes the essentiality of this metabolic pathway.

Using the ketone compound metyrapone (MPON) as a substrate for carbonyl reduction it has been verified for the first time that various permanent cell lines in culture express carbonyl reducing activity. This is even true for the dedifferentiated and fibroblastoid cell line V79, emphasizing the essentiality of this metabolic pathway. MPON reducing enzyme activities are located in the endoplasmic reticulum as well as in the cytoplasm of the cells. Compared to MPON-reductase in rat liver microsomes, no immunological homology to microsomal C2REV7 rat liver hepatoma cell MPON-reductase could be detected, indicating differences in antigenic determinants between the enzymes of the solid organ and respective cells in continuous culture.

Characterization of carbonyl reducing activity in continuous cell lines of human and rodent origin.

Carbonyl reduction was investigated in the continuous cell lines V79, NCI-H322 and C2REV7 by using the ketone compound metyrapone as a substrate. Metyrapone reducing enzymes were characterized by evaluating the cosubstrate requirement and by testing the sensitivity of this reaction to specific inhibitors. All cell lines were found to produce metyrapol at a linear rate over a time course of at least 48 hr, when tested in cultured monolayers. In general, cytosolic metyrapone reduction exceeds microsomal activity several-fold in all three cell lines. Quercitrin turned out to be the strongest inhibitor in all fractions, except in NCI-H322 microsomes where it had no effect. Consequently, carbonyl reductase is suspected to be responsible for metyrapone reduction in the cytosol and microsomes of V79 and C2REV7 cells as well as in the cytosol of NCI-H322 cells. Simultaneous sensitivity towards quercitrin, dicoumarol, indomethacin and 5 alpha-dihydrotestosterone in some cases points to the existence of different isozymes of carbonyl reductase. In NCI-H322 microsomes only dicoumarol and indomethacin decrease metyrapol formation, thus pointing to an isozyme of NAD(P)H:quinone-oxidoreductase. Concerning cosubstrate requirements metyrapone reducing enzymes show a strong preference for NADPH, thus confirming the involvement of carbonyl reductase in this reaction. In conclusion, carbonyl reduction of metyrapone in continuous cell lines is mediated by carbonyl reductases due to the common sensitivity towards the diagnostic inhibitor quercitrin and due to the strong preference for NADPH as cosubstrate. According to its maintenance in permanent cell lines carbonyl reductase seems to be an essential and constitutive enzyme, which probably fills an important role in normal cell physiology.

Carbonyl reduction of metyrapone in human liver.

Carbonyl reduction was investigated in cytosolic and microsomal fractions of human liver using the ketone metyrapone as a substrate. The cytosolic enzyme has a stronger preference for NADPH over NADH than the microsomal enzyme: the former shows only 14% of the NADPH-supported activity while the latter exhibits 36% activity with NADH. Barbitone and quercitrin, the classic inhibitors of carbonyl reductases, do not affect metyrapone reduction in either fraction. Dicumarol and indomethacin, the specific inhibitors of NAD(P)H: quinone-oxidoreductase and dihydrodiol dehydrogenase, respectively, only slightly decreased metyrapol formation. In contrast, 5 alpha-dihydrotestosterone, the active form of the androgen steroid testosterone, inhibited metyrapone reduction very strongly in the microsomal fractions and is postulated to be the physiological substrate of the enzyme. This resembles the situation in mouse liver [E. Maser and K. J. Netter, Biochem Pharmacol 38: 3049-3054, 1989] where microsomal metyrapone reductase was inhibited by steroids and the purified enzyme was demonstrated to mediate androsterone oxidation. Immunoblot analysis revealed antigenic cross-reaction of antibodies against the 34 kDa metyrapone reductase from mouse liver microsomes with the homologous protein in human liver microsomes pointing to structural homologies between the respective enzymes of the two species. These results--together with previous findings, which have shown that there exist functional as well as structural relationships between microsomal mouse liver metyrapone reductase and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni [E. Maser, U. Oppermann and K. J. Netter, Eur J Pharmacol 183:1366, 1990]--suggest that metyrapone reduction in human liver microsomes might be catalysed by a microsomal hydroxysteroid dehydrogenase.

Human biomonitoring of arsenic and antimony in case of an elevated geogenic exposure.

Part of the northern Palatinate region in Germany is characterized by elevated levels of arsenic and antimony in the soil due to the presence of ore sources and former mining activities. In a biomonitoring study, 218 residents were investigated for a putative increased intake of these elements. Seventy-six nonexposed subjects in a rural region in south lower Saxony were chosen as the reference group. Urine and scalp hair samples were obtained as surrogates to determine the internal exposures to arsenic and antimony. The analyses were performed using graphite furnace atomic absorption spectrometry except for arsenic in urine, which was determined by the hydride technique. This method does not detect organoarsenicals from seafood, which are not toxicologically relevant. In the northern Palatinate subjects, slightly elevated arsenic contents in urine and scalp hair (presumably not hazardous) could be correlated with an increased arsenic content in the soil. On the other hand, the results did not show a correlation between the antimony contents in the soil of the housing area and those in urine and hair. Except for antimony in scalp hair, age tended to be associated with internal exposures to arsenic and antimony in both study groups. Consumption of seafood had a slight impact on the level of urinary arsenic, which is indicative of the presence of low quantities of inorganic arsenicals and dimethylarsinic acid in seafood. The arsenic and antimony contents in scalp hair were positively correlated with the 24-hr arsenic excretion in urine. However, antimony in scalp hair was not correlated with seafood consumption as was arsenic in scalp hair and in urine. This indicated the existence of unidentified common pathways of exposure contributing to the alimentary body burden. Short time peaks in the 24-hr excretion of arsenic in urine, which could not be assigned to a high consumption of seafood, were detected for six study participants. This suggests that additional factors relevant in the exposure to arsenic are still unidentified.

Confounding variables in the environmental toxicology of arsenic.

Arsenic is one of the most important global environmental toxicants. For example, in regions of West Bengal and Inner Mongolia, more than 100000 persons are chronically exposed to well water often strongly contaminated with As. Unfortunately, a toxicologically safe risk assessment and standard setting, especially for long-term and low-dose exposures to arsenic, is not possible. One reason is that the key mechanism of arsenic's tumorigenicity still is not elucidated. Experimental data indicate that either DNA repair inhibition or DNA methylation status alteration may be causal explanations. Moreover, when comparing epidemiological data, it cannot be ruled out that the susceptibility to arsenic's carcinogenicity may be different between Mexican and Taiwanese people. Some other studies indicate that some Andean populations do not develop skin cancer after long-term exposure to As. It is not known yet how this resistance could be mediated. Finally, the situation is even more complicated when taking into consideration that there are several compounds suspected to modulate the chronic environmental toxicity of arsenic, variables that may either enhance or suppress the in vivo genotoxicity and carcinogenicity of the metalloid. Among them are nutritional factors like selenium and zinc as well as drinking water co-contaminants like antimony. Further, yet unidentified factors influencing the body burden and/or the excretion of arsenic are possibly prevailing: preliminary data from own human biomonitoring studies showed a peaking of As in urine samples of non-exposed people which was not caused by elevated exposure to As through seafood consumption. The relevance of these putative confounding variables cannot be finally evaluated yet. Further experimental as well as epidemiological studies are needed to answer these questions. This would help to conduct a toxicologically improved risk assessment, especially for low-dose and long-term exposures to arsenic.

Comparative and environmental genotoxicity of antimony and arsenic.

Antimony and arsenic compounds are known to have a genotoxic potential. Soil contamination with these elements can be due to the presence of natural ore sources of fahlore (gray copper). As a result, human and animal populations may be highly exposed. The sister chromatid exchange (SCE) test is an adequate tool for the sensitive detection of antimony and arsenic genotoxicity. We used this assay to investigate the coergism of the two elements in vitro to gain data for the assessment of a putative risk from coexposure. The combinative effect of antimony and arsenic in the SCE test appeared subadditive. Additionally, the SCE served to determine the genotoxic potential in extracts of contaminated fahlore soil samples gained under mildly acidic conditions. The genotoxicity observed was very low because antimony and arsenic predominated in the pentavalent, non-genotoxic state, but, the partial antagonism observed in the in vitro experiments could be an additional explanation for the low genotoxicity.

Arsenic(III), but not antimony(III), induces DNA-protein crosslinks.

Antimony compounds are supposed to resemble to arsenicals in some toxicological features. Comparative investigations with antimony and arsenic were performed to collect data on the genotoxicity of antimony, on which the knowledge is scarce. In comparison to trivalent arsenic, trivalent antimony proved to be five times less cytotoxic in the neutral red assay and one order of magnitude less genotoxic in the cytokinesis-block micronucleus test using V79 Chinese hamster cells. The data obtained in the comet assay showed that the As(III)-mediated generation of DNA-protein crosslinks and DNA strand lesions seem to be two independent processes. In contrast, Sb(III) induced DNA strand lesions but not DNA-protein crosslinks. Further studies will have to investigate whether the genotoxicity of Sb(III) is mediated by an inhibition of DNA repair as it seems to be the case for As(III).

Arsenic and antimony: comparative approach on mechanistic toxicology.

A chemico-toxicological similarity between arsenic and antimony exists and their toxicology is often seen. Indeed, both elements possess several common properties, e.g. they are clastogenic but not mutagenic in the trivalent state and they have a carcinogenic potential: trivalent arsenicals are known to be human carcinogens and antimony(III) oxide (by inhalation) has been shown to cause lung cancer in female rats. For years, arsenic has been known to be environmentally toxic. Elevated human exposure to this element, mostly caused by the intake of contaminated tap water, is associated with increased incidences of cancer at various sites. It is still not clear how arsenic compounds exert their genotoxic effect. It may be connected with an inhibition of DNA repair or the induction of oxidative stress. Little work has been done on the toxicology of antimony as it is less widely present in the environment. There is evidence that in mammals antimony, unlike arsenic, is not detoxified via methylation but it still remains unclear what mechanism is responsible for antimony's genotoxicity. In general, there is little information known about this element to accurately determine its impact on human health. Thus, the aim of this paper is to review current knowledge for future risk assessment and further scientific work.

Genotoxicity of selected pesticides in the mouse bone-marrow micronucleus test and in the sister-chromatid exchange test with human lymphocytes in vitro.

Selected pesticides (aldicarb, 1,3-dichloropropene, methidathion, parathion, triadimefon, vinclozolin) were tested for their clastogenic and aneugenic activities in the mouse bone-marrow micronucleus (MN) test in vivo and for their sister-chromatid exchange-inducing activities in human lymphocytes in vitro in the presence and absence of an exogenous metabolizing system from rat-liver S9. 1,3-Dichloropropene significantly increased the frequencies of micronucleated polychromatic erythrocytes (PCE) in bone-marrow cells of female mice from 3.3 MN/1000 PCE to 15.3 MN/1000 PCE (187 mg per kg body weight). 1,3-Dichloropropene (100 microM) induced 16.0 SCE/metaphase after 24 h of incubation as compared with the basal rate of 11.2 SCE/metaphase (-S9) and of 15.4 SCE/metaphase as compared with 10.5 SCE/metaphase of the control (+S9). These values were statistically significantly different from each other. The other pesticides tested did neither increase the rate of micronuclei significantly in polychromatic erythrocytes in male nor in female animals. Aldicarb and methidathion induced a significant increase in SCEs in human lymphocytes in vitro only without the metabolic activating system: aldicarb, 5 microM, 24 h incubation: 15.5 SCE/metaphase; control: 12.6 SCE/metaphase; methidathion, 100 microM, 24 h incubation: 15.8 SCE/metaphase, control: 11.1 SCE/metaphase. Parathion, triadimefon and vinclozolin did not have any SCE-inducing effects.

Suppression of arsenic-induced chromosome mutagenicity by antimony.

Arsenic and antimony are two semimetals sharing some chemical as well as toxicological properties. Both elements are clastogenic but not point mutagenic in their trivalent state of valency. Environmental exposure to arsenic was proven to be associated with increased rates of various types of cancers. Antimony is suspected to be carcinogenic to humans. Arsenic and antimony can be found as environmental co-contaminants resulting in co-exposure to man. However, in most regions where arsenic was found in elevated environmental amounts, it was not investigated whether an additional exposure to antimony was predominating. In this study, the chromosome mutagenicity induced by arsenic(III) was significantly suppressed by antimony(III) in the micronucleus test with V79 cells. The results demonstrate the necessity to identify putative environmental co-contaminations of antimony in the regions contaminated with arsenic and to determine the impact of antimony co-exposure on arsenic genotoxicity and carcinogenicity in man in vivo.