Ulrike Bernauer

Biotransformation of trichloroethene : dose-dependent excretion of 2,2,2-trichloro-metabolites and mercapturic acids in rats and humans after inhalation

Abstract Chronic bioassays with trichloroethene (TRI) demonstrated carcinogenicity in mice (hepatocellular carcinomas) and rats (renal tubular cell adenomas and carcinomas). The chronic toxicity and carcinogenicity is due to bioactivation reactions. TRI is metabolized by cytochrome P450 and by conjugation with glutathione. Glutathione conjugation results in S-(dichlorovinyl) glutathione (DCVG) and is presumed to be the initial biotransformation step resulting in the formation of nephrotoxic metabolites. Enzymes of the mercapturic acid pathway cleave DCVG to the corresponding cysteine S-conjugate, which is, after translocation to the kidney, cleaved by renal cysteine S-conjugate β-lyase to the electrophile chlorothioketene. After N-acetylation, cysteine S-conjugates are also excreted as mercapturic acids in urine. The object of this study was the dose-dependent quantification of the two isomers of N-acetyl-S-(dichlorovinyl)-L-cysteine, trichloroethanol and trichloroacetic acid, as markers for the glutathione- and cytochrome P450-mediated metabolism, respectively, in the urine of humans and rats after exposure to TRI. Three male volunteers and four rats were exposed to 40, 80 and 160 ppm TRI for 6 h. A dose-dependent increase in the excretion of trichloroacetic acid, trichloroethanol and N-acetyl-S-(dichlorovinyl)-L-cysteine after exposure to TRI was found both in humans and rats. Amounts of 3100 μmol trichloroacetic acid+trichloroethanol and 0.45 μmol mercapturic acids were excreted in urine of humans over 48 h after exposure to 160 ppm TRI. The ratio of trichloroacetic acid+trichloroethanol/mercapturic acid excretion was comparable in rats and humans. A slow rate of elimination with urine of N-acetyl-S-(dichlorovinyl)-L-cysteine was observed both in humans and in rats. However, the ratio of the two isomers of N-acetyl-S-(dichlorovinyl)-L-cysteine was different in man and rat. The results confirm the finding of the urinary excretion of mercapturic acids in humans after TRI exposure and suggest the formation of reactive intermediates in the metabolism of TRI after bioactivation by glutathione also in humans.

Human CYP2E1 mediates the formation of glycidamide from acrylamide

Regarding the cancer risk assessment of acrylamide (AA) it is of basic interest to know, as to what amount of the absorbed AA is metabolized to glycidamide (GA) in humans, compared to what has been observed in laboratory animals. GA is suspected of being the ultimate carcinogenic metabolite of AA. From experiments with CYP2E1-deficient mice it can be concluded that AA is metabolized to GA primarily by CYP2E1. We therefore examined whether CYP2E1 is involved in GA formation in non-rodent species with the focus on humans by using human CYP2E1 supersomes™, marmoset and human liver microsomes and in addition, genetically engineered V79 cells expressing human CYP2E1 (V79h2E1 cells). Special emphasis was placed on the analytical detection of GA, which was performed by gas chromatography/mass spectrometry. The results show that AA is metabolized to GA in human CYP2E1 supersomes™, in marmoset and human liver microsomes as well as in V79h2E1 cells. The activity of GA formation is highest in supersomes™; in human liver it is somewhat higher than in marmoset liver. A monoclonal CYP2E1 human selective antibody (MAB-2E1) and diethyldithiocarbamate (DDC) were used as specific inhibitors of CYP2E1. The generation of GA could be inhibited by MAB-2E1 to about 80% in V79h2E1 cells and to about 90% in human and marmoset liver microsomes. Also DDC led to an inhibition of about 95%. In conclusion, AA is metabolized to GA by human CYP2E1. Overall, the present work describes (1) the application and refinement of a sensitive methodology in order to determine low amounts of GA, (2) the applicability of genetically modified V79 cell lines in order to investigate specific questions concerning metabolism and (3) the involvement, for the first time, of human CYP2E1 in the formation of GA from AA. Further studies will compare the activities of GA formation in genetically engineered V79 cells expressing CYP2E1 from different species.

CYP2E1 expression in bone marrow and its intra- and interspecies variability : approaches for a more reliable extrapolation from one species to another in the risk assessment of chemicals

Abstract When characterizing the health risks for man by exposure to chemicals, species-specific differences have to be taken into consideration, otherwise extrapolation from animal data to the human situation would be inadequate. The site-specific toxicity of chemicals may be explained by the following alternatives: (1) reactive metabolites are generated in the liver and subsequently transported to the target tissue(s); (2) metabolism of the parent compound occurs in the target tissue, a pathway by which the enzymes necessary for activation must be expressed in the target tissue. Cytochrome P450 2E1 (CYP2E1) is an important phase-I enzyme activating several chemicals. In the study described in this paper, myeloid intra- and interspecies variability in the expression of CYP2E1 has been investigated in rats, rabbits and man, because the bone marrow represents an important target organ for toxic effects of several chemicals, e.g. benzene. CYP2E1 at the protein level was detected by Western blotting and enzyme activities were determined by CYP2E1-dependent hydroxylation of chlorzoxazone (CLX). In the bone marrow of Wistar rats, the CLX hydroxylase activities were within the same order of magnitude (range: 0.1–0.4 pmol/mg protein per min) as previously described for mice (range 0.2–0.8 pmol/mg protein per min), whereas the CYP2E1 activities in two strains of rabbits were significantly higher (range: 1.7–4.7 pmol/mg protein per min) than in the rodents (P < 0.05). In human CD34+ bone marrow stem cells, CYP2E1 could also be detected on the protein level by Western blotting. The data demonstrate a presence of CYP2E1 in the bone marrow of all species investigated, thus supporting the hypothesis of CYP2E1-dependent local metabolism of several chemicals as a factor possibly contributing to their myelotoxicity and haematotoxicity. The data show that intraspecies/intrastrain variability of CYP2E1 activity in rodents is small. However, CYP2E1 activity between rodents and a non-rodent species was quite different indicating considerable interspecies variability.

Extrahepatic metabolism at the body's internal–external interfaces

Abstract In general, xenobiotic metabolizing enzymes (XMEs) are expressed in lower levels in the extrahepatic tissues than in the liver, making the former less relevant for the clearance of xenobiotics. Local metabolism, however, may lead to tissue-specific adverse responses, e.g. organ toxicities, allergies or cancer. This review summarizes the knowledge on the expression of phase I and phase II XMEs and transporters in extrahepatic tissues at the bodys internal–external interfaces. In the lung, CYPs of families 1, 2, 3 and 4 and epoxide hydrolases are important phase I enzymes, while conjugation is less relevant. In skin, phase I-related enzymatic reactions are considered less relevant. Predominant skin XMEs are phase II enzymes, whereby glucuronosyltransferases (UGT) 1, glutathione-S-transferase (GST) and N-acetyltransferase (NAT) 1 are important for detoxification. The intestinal epithelium expresses many transporters and phase I XME with high levels of CYP3A4 and CYP3A5 and phase II metabolism is mainly related to UGT, NAT and Sulfotransferases (SULT). In the kidney, conjugation reactions and transporters play a major role for excretion processes. In the bladder, CYPs are relevant and among the phase II enzymes, NAT1 is involved in the activation of bladder carcinogens. Expression of XMEs is regulated by several mechanisms (nuclear receptors, epigenetic mechanisms, microRNAs). However, the understanding why XMEs are differently expressed in the various tissues is fragmentary. In contrast to the liver – where for most XMEs lower expression is demonstrated in early life – the XME ontogeny in the extrahepatic tissues remains to be investigated.

CYP2E1-dependent benzene toxicity: the role of extrahepatic benzene metabolism

Abstract Benzene, a ubiquitous environmental pollutant, is haematotoxic and myelotoxic. As has been shown earlier, cytochrome P450 2E1 (CYP2E1)-dependent metabolism is a prerequisite for the cytotoxic and genotoxic effects of benzene, but which of the benzene metabolites produces toxicity is still unknown. The observed differences between the toxicity of benzene and that of phenol, a major metabolite of benzene, could be explained by alternative hypotheses. That is, whether (1) toxic benzene effects are caused by metabolites not derived from phenol (e.g. benzene epoxide, muconaldehyde), which are formed in the liver and are able to reach the target organ(s); or (2) benzene penetrates into the bone marrow, where local metabolism takes place, whereas phenol does not reach the target tissue because of its polarity. To further investigate hypothesis 2, we used various strains of mice (AKR, B6C3F1, CBA/Ca, CD-1 and C57Bl/6), for which different toxic responses have been reported in the haematopoietic system after chronic benzene exposure. In these strains, CYP2E1 expression in bone marrow was investigated and compared with CYP2E1 expression in liver by means of two independent methods. Quantification of CYP2E1-dependent hydroxylation of chlorzoxazone (CLX) by high-performance liquid chromatography (HPLC; functional analysis) was used to characterize specific enzymatic activities. Protein identification was performed by Western blotting using CYP2E1-specific antibodies. In liver microsomes of all strains investigated, considerable amounts of CYP2E1-specific protein and correspondingly high CYP2E1 hydroxylase activities could be detected. No significant differences in CYP2E1-dependent enzyme activities were found between the five strains (range of medians, 4.6–12.0 nmol 6-OH-CLX/[mg protein × min]) in hepatic tissue. In the bone marrow, CYP2E1 could also be detected in all strains investigated. However, chlorzoxazone hydroxylase activities were considerably lower (range of medians, 0.2–0.8 × 10−3 nmol 6-OH-CLX/[mg protein × min]) compared with those obtained from liver microsomes. No significant (P > 0.05) interstrain differences in CYP2E1 expression in liver and/or bone marrow could be observed in the mouse strains investigated. The data obtained thus far from our investigations suggest that strain-specific differences in the tumour response of the haematopoietic system of mice chronically exposed to benzene cannot be explained by differences in either hepatic or in myeloid CYP2E1-dependent metabolism of benzene.

The safety of medical devices containing DEHP plasticized PVC or other plasticizers on neonates and other groups possibly at risk (2015 update)

...................................................................................................................... 4 EXECUTIVE SUMMARY ................................................................................................ 5 1. BACKGROUND...................................................................................................... 11 2. TERMS OF REFERENCE ..................................................................................... 11 3. SCIENTIFIC RATIONALE .................................................................................. 13 3.

Immunochemical analysis of cytochrome P450 variability in human leukapheresed samples and its consequences for the risk assessment process.

Xenobiotic metabolizing cytochrome P450 (P450) enzymes were investigated in leukapheresed samples from 50 human individuals. It was the aim of the study (a). to get insight into the extent of extrahepatic P450 variability, (b). to investigate whether and to which extent P450 expression and variability as it is seen in the liver corresponds to P450 expression at extrahepatic sites, and (c). to contribute to the replacement of traditionally used default factors (usually 10 for interindividual variability) by data-derived factors in the risk assessment process. P450 enzymes were determined by Western Blotting. Immunoquantification was performed for P450 1A, 1B1, 2C, 2D6, 2E1, and 3A which were-with the exception of the polymorphically expressed CYP2D6-detectable in all samples investigated. Amounts of P450 enzymes in leukapheresed samples were (except CYP1B1) lower compared to those reported for the liver. The P450 variabilities were expressed by the ratios between the 95th and the 5th percentiles. They displayed 7-(CYP1A), 4-(CYP1B1), 6-(CYP2C), 30-(CYP2D6), 3-(CYP2E1), and 4-(CYP3A) fold variability in specific protein content. The results show (a). qualitative and quantitative differences in the expression of P450 proteins in leukapheresed samples from 50 individuals compared to liver, (b). a different extent of variability depending on the P450 enzyme, and (c). in cases where polymorphically distributed P450 enzymes are involved, the traditionally used factor of 10 might be too low to account for interindividual variability in both toxicokinetics and toxicodynamics.

Commentary: dermal penetration of bisphenol A--consequences for risk assessment.

With this comment we would raise awareness for applying appropriate procedures in route-to-route extrapolation. The paper of Demierre et al. (2012) prompted us to comment on the simple approach for route-to-route extrapolation and to explain some short comings. For the risk assessment of exposures resulting from a non-oral route, route-to-route extrapolation is often done by correcting the non-oral route exposure by the route specific absorption into the systemic circulation and comparing the result with the (oral) threshold value. Making use of this procedure means that an internal dose obtained from the non-oral route is compared with an external dose of the oral route. This procedure would be appropriate only if the absorption on the oral route is 100%. If the absorption on the oral route is less than 100% the procedure may underestimate the risk of the exposure of the non-oral route. For some chemicals with a high first pass metabolism in the liver, e.g. BPA, the situation is even more complex and in addition, the target organ for toxicity has to be taken into consideration.

Letter to the Editor: A regulatory view on the discussion on the role of alternative methods in the risk assessment of chemicals in the context of REACH

In a paper published recently in Archives of Toxicology, Lilienblum et al. present their view on available and ready to use alternative methods for the risk assessment of chemicals (Lilienblum et al. 2008). As they refer speciWcally to the new European legislation (REACH) which came into force mid 2007 we would like to comment some of the aspects of the paper from the perspective of regulators. First of all there is general agreement with the evaluation of the current status of alternative methods of the authors (Lilienblum et al. 2008). Considering complex endpoints, we consent that emphasis should be given to reWne tests with the aim to reduce the number of animals rather than to try to replace the tests by developing in vitro alternatives to the current testing methods. However, we would more clearly express the potential for the vitro methods for some of the endpoints which are discussed in the publication. For example, the in vitro dermal resorption test and the in vitro test on phototoxicity are both validated test methods and accepted at the regulatory level. Hence, in our view no in vivo tests are necessary to cover these endpoints. In addition, we see a high value in the available prediction tool for skin irritation and corrosion (ECB 2008) which is extremely helpful in a tiered approach. For classiWcation and labelling negative results can be used as they have clearly been identiWed in the validation study (Hulzebos et al. 2005). Hence, even under the GHS rules where two levels for positive results are required (which the prediction tool is not able to distinguish) the tool is nevertheless helpful considering that negative results for skin irritation and corrosion account for more than 70% of the cases in the EU New Chemicals Database. It can be assumed that using this alternative method would reduce the need for in vivo testing under REACH in 70% of 15,500 chemicals with a production volume below 10 tons per year. We also would put more emphasis in the potential the currently available in vitro test strategy can oVer for genotoxicity and mutagenicity testing. We agree with the assessment and the critique presented in the Lilienblum paper (Lilienblum et al. 2008). However, we would like to draw the attention to the fact that the currently used regulatory tiered testing approach reduces the necessity to perform in vivo testing to a small number of cases. Based on the numbers in the New Chemicals Data Base, the current approach to start with in vitro testing and to proceed with in vivo testing only in the positive in vitro cases was required in only around 25% of the cases which reduces the number of required in vivo tests under REACH to a quite remarkable low number of cases. Furthermore, we would like to give some considerations to the topic of toxicokinetic and metabolism which is extensively discussed in the Lilienblum paper. We agree with the assessment that toxicokinetic and metabolism is of importance and is a crucial issue and that metabolism often plays a key role in intraand inter-species diVerences (to document the regulatory need we refer to work of Bernauer et al. 2000, 2002, 2003, 2006; Abraham et al. 2005; Mielke et al. 2005; Bernauer and Gundert-Remy 2008). We agree with other scientists that the interpretation of testing results need additional data for extrapolation of the results to humans (Greim 2007) and toxicokinetic information is regarded as very helpful for the development of integrated testing strategies (ITS) which are propagated in the REACH guidance documents according to REACH legislation. In addition, toxicokinetic data can be used to support waiving arguments (Annex VIII, 8.6.1 90 days study; Annex IX, 8.7 studies on reprotoxiccity; Annex X, 8.4 germ cell mutageU. Gundert-Remy (&) · U. Bernauer · S. Madle · A. Oberemm · A. Schulte · H.-B. Richter-Reichhelm Berlin, Germany e-mail: ursula.gundert-remy@bfr.bund.de

Opinion of the Scientific Committee on Consumer Safety (SCCS)--The safety of the use of formaldehyde in nail hardeners.

2015 Elsevier Inc. All rights reserved. The substance formaldehyde (CAS Number 50-00-0) is anticipated to be classified as a carcinogen category 1B under the CLP Regulation (EC) No. 1272/2008. However, such substances may be used in cosmetic products by way of exception where, subsequent to their classification as CMR substances of category 1A or 1B under Part 3 of Annex VI to Regulation (EC) No. 1272/2008, all of the conditions of Article 15.2 of the Cosmetics Regulation are fulfilled: (a) They comply with the food safety requirements as defined in Regulation (EC) No. 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matter of food safety; (b) there are no suitable alternative substances available, as documented in an analysis of alternatives; (c) the application is made for a particular use of the product category with a known exposure; and (d) they have been evaluated and found safe by the SCCS for use in cosmetic products, in particular in view of exposure to these products and taking into consideration the overall exposure from other sources, taking particular account of vulnerable population subgroups. Formaldehyde is used in nail hardeners for its specific crosslinking functionality with keratin. The use of formaldehyde in nail hardeners is currently restricted as specified in the Entry 13 of Annex III of Regulation (EC) No. 1223/2009 – i.e., a maximum concentration in the finished products of 5% (as formaldehyde); labelled as ‘contains formaldehyde’ when the finished cosmetic product contains formaldehyde in a concentration above 0.05% and with the warning ‘protect cuticles with grease or oil’. On 23 May 2013, the European Commission published a call for data on formaldehyde use in cosmetics and/or formaldehyde released by others substances used in cosmetics, seeking also information of the suitable alternatives. The Commission only received a full application from Cosmetics Europe which supports the use of formaldehyde in nail hardeners at the maximum level of 2.2% (as free formaldehyde). In view of the data that became available, the independent Scientific Committee on Consumer Safety (SCCS) was asked (i) to assess if condition d) of Article 15.2 is fulfilled, in order to confirm or not the safe use of formaldehyde in nail hardeners at the maximum level of 2.2% (as free formaldehyde) and (ii) to indicate if there are any further scientific concerns with regard to the use of formaldehyde in nail hardeners.