Low-dose effects and biphasic effect profiles: is trenbolone a genotoxicant?

Over the last years, extensive research has documented endocrine-disrupting activities for a significant number of substances including, among others, hormones, pharmaceuticals, pesticides and surfactants. Nonetheless, for most endocrine disruptors, toxicological profiles are still incomplete or even lacking. A systematic review has shown that a number of endocrine disruptors with steroid-modulating effects may also exert mutagenic and carcinogenic activities. For trenbolone, an androgenic compound, there is controversy about its genotoxic properties in the literature, apparently with a strong dependence on the choice of the test system. Since fish and other aquatic animals are at risk of exposure to run-offs from cattle feedlots or sewage-discharge sites containing trenbolone, potential consequences to aquatic ecosystems need to be assessed. To this end, the potential genotoxic hazard of trenbolone was tested in vitro in the permanent rainbow trout-liver cell-line RTL-W1, as well as in primary cell cultures derived from zebrafish (Danio rerio) embryos after in vivo exposure. In either test system, a potential genotoxic hazard characterized by biphasic dose-response curves could be documented even at exposure concentrations of 30µg/L. These results thus confirm the conclusion that the steroid trenbolone may act as a genotoxic substance.

Environmental effect assessment for sexual endocrine-disrupting chemicals: Fish testing strategy.

Current standard testing and assessment tools are not designed to identify specific and biologically highly sensitive modes of action of chemicals, such as endocrine disruption. This information, however, can be important to define the relevant endpoints for an assessment and to characterize thresholds of their sublethal, population-relevant effects. Starting a decade ago, compound-specific risk assessment procedures were amended by specifically addressing endocrine-disrupting properties of substances. In 2002, the Conceptual Framework, agreed upon by OECD's Task Force on Endocrine Disrupters Testing and Assessment, did not propose specific testing strategies, and appropriate testing methods had not yet been developed and approved. In the meantime, the OECD Test Guidelines Programme has undertaken important steps to revise established and to develop new test methods, which can be used to identify and quantify effects of endocrine-disrupting chemicals on mammals, birds, amphibians, fish, and invertebrates. For fish testing of endocrine-disrupting chemicals, the first Test Guidelines have recently been adopted by the OECD and validation of further test systems is under progress. Based on these test systems and the experience gained during their validation procedures, we propose a 3-step fish testing strategy: 1) Weight-of-evidence approach for identifying potential sexual endocrine-disrupting chemicals; even after advanced specification of systematic criteria, this step of establishing initial suspicion will still require expert judgment; 2) in vivo evaluation of sexual endocrine-disrupting activity in fish by applying in vivo fish screening assays; sufficient data are available to diagnose the aromatase-inhibition and estrogen-receptor agonist mechanisms of action by indicative endpoints (biomarkers), whereas the ability of the respective biomarkers in the screening assay to identify the estrogen-receptor antagonists and androgen-receptor agonists and antagonists requires further validation; 3) characterization of sexual endocrine-mediated adverse effects including threshold concentrations; in cases when the most sensitive population-relevant endpoints and the most sensitive time window for exposure are known for the mechanisms of action, the fish full life-cycle or 2-generation test, which are the normal definitive tests, might be abbreviated to, e.g., the fish sexual development test. In the European Union, the measurement of indicative endpoints in the definitive test might be crucial for the authorization procedure under REACH and plant-protection products. The results of the definitive tests can be used in existing schemes of compound-specific environmental risk assessments.

Comparison of in vitro and in situ genotoxicity in the Danube River by means of the comet assay and the micronucleus test.

Genotoxicity can be correlated with adverse reproductive effects or may even result in elevated extinction risk for particular species of an ecosystem. It may thus be a valuable tool for screening of pollution and potential environmental harm. Since many genotoxicants tend to adsorb onto particulate matter, sediments and suspended materials are of particular interest for genotoxicity screening under field conditions. In order to correlate the genotoxic potential of sediments with genetic damage in fish, rainbow-trout liver (RTL-W1) cells were exposed in vitro to acetone extracts of sediments collected at 10 selected sites along the upper Danube River and analyzed in the comet and micronucleus assays. These in vitro results were compared with micronucleus formation in erythrocytes of the European barbel (Barbus barbus) caught in the field. The two in vitro bioassays showed excellent correlation, indicating comparability of genotoxic potentials in vitro. Sampling sites could be clearly differentiated with respect to severity of effects, with Rottenacker as the most heavily contaminated site, Ehingen and Schwarzach as moderately genotoxic, and with the weakest effects in the tributary Lauchert. All other sediment extracts showed intermediate genotoxic or clastogenic effects. In situ, micronucleus formation in barbel erythrocytes indicated severe genotoxicity at Rottenacker, moderate effects at Ehingen, but minor contamination at Riedlingen and Sigmaringen. In situ observations thus showed excellent correlation with corresponding in vitro tests and document the ecological relevance of in vitro studies with sediment extracts. With respect to the ecological status of the Danube River, the results overall indicate a moderate to severe genotoxic potential with a highly differential localization.

N-Acetyl-S-(1-carbamoyl-2-hydroxy-ethyl)-L-cysteine (iso-GAMA) a further product of human metabolism of acrylamide: comparison with the simultaneously excreted other mercaptuic acids.

The N-acetyl-S-(1-carbamoyl-2-hydroxy-ethyl)-L: -cysteine (iso-GAMA) could be identified as a further human metabolite of acrylamide. In this study, we report the excretion of d(3)-iso-GAMA in human urine after single oral administration of deuterium labelled acrylamide (d(3)-AA). One healthy male volunteer ingested a dose of about 1 mg d(3)-AA which is equivalent to a dose of 13 microg/kg bodyweight. Over a period of 46 h the urine was collected and the d(3)-iso-GAMA levels analysed by LC-ESI-MS/MS. The excretion of iso-GAMA begins five hours after application. It rises to a maximum concentration (c (max)) of 43 microg/l which was quantified in the urine excreted after 22 h (t (max)). The excretion pattern is parallel to that of the major oxidative metabolite N-acetyl-S-(2-carbamoyl-2-hydroxy-ethyl)-L-cysteine (GAMA). Total recovery of iso-GAMA was about 1% of the applied dose. Together with N-acetyl-S-(2-carbamoylethyl)-L: -cysteine (AAMA) and GAMA, 57% of the applied dose is eliminated as mercapturic acids. The elimination kinetics of the three mercapturic acids of AA are compared. We show that dietary doses of acrylamide (AA) cause an overload of detoxification via AAMA and lead to the formation of carcinogenic glycidamide (GA) in the human body.

Hemoglobin adducts and mercapturic acid excretion of acrylamide and glycidamide in one study population.

The aim of this study was to determine the relationship between the oxidative and reductive metabolic pathways of acrylamide (AA) in the nonsmoking general population. For the first time both the blood protein adducts and the urinary metabolites of AA and glycidamide (GA) were quantified in an especially designed study group with even distribution of age and gender. The hemoglobin adducts N-carbamoylethylvaline (AAVal) and N-( R, S)-2-hydroxy-2-carbamoylethylvaline (GAVal) were detected by GC-MS/MS in all blood samples with median levels of 30 and 34 pmol/g of globin, respectively. Concentrations ranged from 15 to 71 pmol/g of globin for AAVal and from 14 to 66 pmol/g of globin for GAVal. The ratio GAVal/AAVal was 0.4-2.7 (median = 1.1). The urinary metabolites were determined by LC-MS/MS. Of all urine samples examined 99% of N-acetyl- S-(2-carbamoylethyl)- l-cysteine (AAMA) levels and 73% of N-( R/ S)-acetyl- S-(2-carbamoyl-2-hydroxyethyl)- l-cysteine (GAMA) levels were above the LOD (1.5 microg/L). Concentrations ranged from

Investigations on permeation of mitomycin C through double layers of natural rubber gloves.

Treating peritoneal carcinomatosis by the aggressive cytoreductive surgery with the hyperthermic intraoperative intraperitoneal chemotherapy (HIPEC) surgeons expose their gloved hands for up to 90 min to a peritoneal dialysis solution (PDS) containing mitomycin C (MMC). We investigated the permeation of MMC through the material of three different natural rubber gloves under conditions similar to the in-use during HIPEC as well as under worst-case exposure scenario. Two different methods, a two-chamber diffusion cell and a single-chamber glass chamber method, were used to demonstrate the permeation capability. The permeation of MMC dissolved in 0.9% NaCl solution and PDS through double natural rubber glove material was tested over 2 h using four concentrations (c = 0.004, 0.008, 0.016 and 0.4 mg ml(-1)) and three receptor fluids (0.9% NaCl solution, PDS and a novel artificial sweat). In none of four glass chamber experiments and in only one of 40 diffusion cell experiments was permeation through glove material detected. The permeation occurred between 15 and 30 min under worst-case exposure scenario at a approximately 100-fold higher MMC concentration than under in-use conditions during HIPEC. The double-layer natural rubber gloves tested were effective to prevent a permeation of MMC in vitro under HIPEC-similar exposure. Our results support the glove wearing procedure in our university hospital. However, occupational exposure to antineoplastic drugs should be minimized, since there is insufficient knowledge regarding harmful effects from a long-term exposure to low doses.

Urinary excretion of acrylamide and metabolites in Fischer 344 rats and B6C3F(1) mice administered a single dose of acrylamide.

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in mice and rats. AA is also formed during cooking in many commonly consumed starchy foods. Our previous toxicokinetic investigations of AA and its genotoxic metabolite, glycidamide (GA), in rodents showed that AA is highly bioavailable from oral routes of administration, is widely distributed to tissues, and that the dietary route, in particular, favors metabolism to GA. Formation and accumulation of mutagenic GA-DNA adducts in many tissues support the hypothesis that AA is carcinogenic in rodent bioassays through metabolism to GA. The current investigation describes the quantification of 24 h urinary metabolites, including free AA and GA and their mercapturic acid conjugates (AAMA and GAMA, respectively), using LC/MS/MS in F344 rats and B6C3F(1) mice following a dose of 0.1 mg/kg bw given by intravenous, gavage, and dietary routes of administration. Similar groups of rodents were used previously for serum/tissue toxicokinetic and adduct determinations (DNA and hemoglobin). The goal was to investigate relationships between urinary and circulating biomarkers of exposure, toxicokinetic parameters for AA and GA, and tissue GA-DNA adducts in rodents from single doses of AA. Significant linear correlations were observed between urinary levels of AA with AAMA and GA with GAMA in the current data sets for rats and mice. Concentrations of AA and AAMA correlated significantly with average AUC values determined previously for AA in groups of rats and mice similarly dosed with AA. Urinary GA and GAMA concentrations showed significant correlations with average AUC values for GA and liver GA-DNA adducts determined previously in rats and mice similarly dosed with AA. Despite statistical significance, considerable inter-animal variability was observed in all urinary measurements, which limited the degree of correlation with either average toxicokinetic or biomarker data collected from different groups of animals. These results suggest that urinary measurements of AA and its metabolites may be useful for prediction of internal exposures to AA and GA.

Acrylamide exposure via the diet: influence of fasting on urinary mercapturic acid metabolite excretion in humans.

Acrylamide (AA) is carcinogenic in animals and classified by the International Agency for Research on Cancer as probably carcinogenic in humans. Regarding the AA contents of food the diet significantly contributes to the overall AA burden of the general population. However, it is unclear to which degree the diet, apart from smoking, contributes to the internal AA exposure. Therefore the influence of an AA-free diet on the excretion of urinary mercapturic acid metabolites derived from AA in three healthy volunteers fasting for 48 h was examined. Urinary AA mercapturic acid metabolites were considerably reduced after 48 h of fasting. The levels were even well below the median level in non-smokers. This confirms that the diet is the main source of environmental AA exposure in humans, apart from smoking. Other possible AA sources could be of minor quantitative importance only.

Toxicokinetics of acrylamide in humans after ingestion of a defined dose in a test meal to improve risk assessment for acrylamide carcinogenicity.

High amounts of acrylamide in some foods result in an estimated daily mean intake of 50 microg for a western style diet. Animal studies have shown the carcinogenicity of acrylamide upon oral exposure. However, only sparse human toxicokinetic data is available for acrylamide, which is needed for the extrapolation of human cancer risk from animal data. We evaluated the toxicokinetics of acrylamide in six young healthy volunteers after the consumption of a meal containing 0.94 mg of acrylamide. Urine was collected up to 72 hours thereafter. Unchanged acrylamide, its mercapturic acid metabolite N-acetyl-S-(2-carbamoylethyl)cysteine (AAMA), its epoxy derivative glycidamide, and the respective metabolite of glycidamide, N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine (GAMA), were quantified in the urine by liquid chromatography-mass spectrometry. Toxicokinetic variables were obtained by noncompartmental methods. Overall, 60.3 +/- 11.2% of the dose was recovered in the urine. Although no glycidamide was found, unchanged acrylamide, AAMA, and GAMA accounted for urinary excretion of (mean +/- SD) 4.4 +/- 1.5%, 50.0 +/- 9.4%, and 5.9 +/- 1.2% of the dose, respectively. Apparent terminal elimination half-lives for the substances were 2.4 +/- 0.4, 17.4 +/- 3.9, and 25.1 +/- 6.4 hours. The ratio of GAMA/AAMA amounts excreted was 0.12 +/- 0.02. In conclusion, most of the acrylamide ingested with food is absorbed in humans. Conjugation with glutathione exceeds the formation of the reactive metabolite glycidamide. The data suggests an at least 2-fold and 4-fold lower relative internal exposure for glycidamide from dietary acrylamide in humans compared with rats or mice, respectively. This should be considered for quantitative cancer risk assessment.

Excretion of mercapturic acids of acrylamide and glycidamide in human urine after single oral administration of deuterium-labelled acrylamide.

We investigated the human metabolism of AA to the mercapturic acids N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and N-(R/S)-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L: -cysteine (GAMA) which are derived from AA itself and from its oxidative genotoxic metabolite glycidamide (GA), respectively. A healthy male volunteer received a single dose of about 1 mg deuterium-labelled acrylamide (d(3)-AA), representing 13 microg/kg body weight, in drinking water. Urine samples before dosing and within 46 h after the dose were analysed for d(3)-AAMA and d(3)-GAMA by LC-ESI-MS/MS. A first phase of increase in urinary concentration was found to last 18 h with a broad plateau between 8 and 18 h for AAMA, and 22 h for GAMA. Elimination half-lives of both AAMA and GAMA were estimated to be approximately 3.5 h for the first phase and more than 10 h up to few days for the second phase. Total recovery in urine after 24 h was about 51% as the sum of AAMA and GAMA and hereby well in accordance with former studies in rats. After 2 days AAMA, accounting for altogether 52% of the total AA dose, was the major metabolite of AA in humans. GAMA, accounting for 5%, appeared as a minor metabolite of AA. In humans we found a urinary ratio of 0.1 for GAMA/AAMA compared to previously reported values of 0.2 for rats and 0.5 for mice. Therefore, the metabolic fate of AA in humans was more similar to that in rats than in mice as already demonstrated in terms of the haemoglobin adducts. Consequently a genotoxic potency of AA mediated by GA could be supposed to be comparable in rats and humans.

Determination of the major mercapturic acids of acrylamide and glycidamide in human urine by LC-ESI-MS/MS.

We developed a LC-MS/MS method for the quantitative determination of the mercapturic acid (MA) metabolites of acrylamide (AA) AAMA and of its oxidative metabolite glycidamide (GA) GAMA in urine samples from the general population. The method requires 4 mL of urine which is solid phase extracted prior to LC-MS/MS analysis. The metabolites are detected by ESI-tandem mass spectrometry in negative ionisation mode and quantified by isotope dilution. Detection limits ranged down to 1.5 microg/L urine for both AAMA and GAMA. The imprecision expressed as R.S.D. lay between 2% and 6% for both analytes (intra- and inter-assay). First results on a small group of 29 persons out of the general population ranged from 5 to 338 microg/L AAMA and

Towards an alternative for the acute fish LC(50) test in chemical assessment: the fish embryo toxicity test goes multi-species -- an update.

After its standardisation at the national level in Germany (DIN 38415-6, 2001, 2001), the 48 h sewage testing assay with zebrafish (Danio rerio) embryos has been submitted for standardisation to ISO. As an alternative to the conventional acute (96 h) fish test, a modified fish embryo test will be submitted to the OECD for chemical testing in late 2005. For this, a protocol originally designed for zebrafish was adapted to fit also the requirements of other OECD species, namely medaka (Oryzias latipes) and fathead minnow (Pimephales promelas). Results document that the transfer of the protocol is possible with only minor modifications. Data obtained from embryo tests with the three species are comparable. Statistical analysis of existing zebrafish embryo toxicity data resulted in the conclusions (1) that there is a reliable correlation between the fish embryo test and the acute fish test, (2) that the confidence belt of the regression line was relatively small, but that the prediction range was relatively wide. The regression thus seems appropriate to describe the relationship between acute fish and embryo LC(50) with good confidence, but is less appropriate as a prediction model. Investigations into oxygen requirements of zebrafish embryos reveal that they adapt to a broad range of oxygen levels and survive at concentrations of 2 mg/l without malformations. Zebrafish embryos can thus be exposed in very small toxicant volumes (100 microl), which is of particular interest for the testing of metabolites. Dechorionation studies with 48 h old zebrafish embryos indicate that the barrier function of the chorion increases with the lipophilicity of the test compound. Finally, examples are given as to how additional endpoints can be incorporated into the fish embryo test protocol to extend its scope, e.g. to sediment toxicity assessment or genotoxicity and mutagenicity testing.

Mercapturic acids of acrylamide and glycidamide as biomarkers of the internal exposure to acrylamide in the general population.

Acrylamide (AA), a widely used industrial monomer which is categorised to be carcinogenic, was found to be generated in starch-containing foods during the heating process. This discovery has caused reasonable concern about possible health risks to humans due to dietary acrylamide uptake. In order to gain more information on human metabolism of acrylamide and to contribute to the assessment of the human carcinogenic risk due to AA uptake we measured the mercapturic acid of AA and its epoxide glycidamide (GA) i.e. N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and N-(R,S)-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA) in human urine. The relation between AAMA and GAMA is important in this context because GA is thought to be the ultimate carcinogenic metabolite of AA. The median levels in smokers (n=13) were found to be about four times higher than in non-smokers (n=16) with median levels of 127 microg/l versus 29 microg/l for AAMA and 19 microg/l versus 5 microg/l for GAMA. Therefore cigarette smoke proved to be an important source of acrylamide exposure. The level of AAMA in the occupationally non-exposed collective (n=29) ranged from 3 to 338 microg/l, the level of GAMA from