Gas chromatography–negative ion chemical ionization mass spectrometry as a powerful tool for the detection of mercapturic acids and DNA and protein adducts as biomarkers of exposure to halogenated olefins

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Abstract

The studies on metabolism of halogenated olefins presented here outline the advantages of modern mass spectrometry. The perchloroethene (PER) metabolite N-acetyl-S-(trichlorovinyl)-l-cysteine (N-ac-TCVC) is an important biomarker for the glutathione dependent biotransformation of PER. In urine of rats and humans exposed to PER, N-ac-TCVC was quantified as methyl ester after BF3–MeOH derivatization by gas chromatography with chemical ionization and negative ion detection mass spectrometry (GC–NCI-MS). The detection limit was 10 fmol/μl injected solution using [2H3]N-ac-TCVC methyl ester as the stable isotope internal standard. Cleavage of S-(trichlorovinyl)-l-cysteine by β-lyase enzymes results in an electrophilic and highly reactive thioketene which reacts with nucleophilic groups in DNA and proteins. Protein adduct formation was shown in kidney mitochondria by identification of dichloroacetylated lysine after derivatization with 1,1,3,3-tetrafluoro-1,3-dichloroacetone by GC–NCI-MS. In addition, chlorothioketene was generated in organic solvents and reacted with cytosine to give N4-chlorothioacetyl cytosine. After derivatization with pentafluorobenzyl bromide this compound exhibited good gas chromatographic properties and was detectable with a limit of detection of 50 fmol/injected volume. The detection of chemically induced protein modifications in the target organ of toxic metabolite formation and the study of DNA modifications with chemically generated metabolites provide important information on organ toxicity and possible tumorigenicity of halogenated olefins.

Introduction

The determination of biomarkers such as urinary metabolites of particular xenobiotics or DNA and protein adducts are very important for the classification of chemicals in human risk assessment [1]. A main emphasis for these studies is therefore the development of simple and sensitive methods for the detection and quantitation of these biomarkers [2], [3].

Halogenated olefins (tetrachloroethene, PER; trichloroethene, TRI) which are extensively used in industry as metal degreasing solvents and as dry cleaning agents are good examples to demonstrate applications of modern mass spectrometry (MS) for such problems. Long-term exposure of rodents to both PER and TRI has been shown to increase the incidence of liver tumors in male mice and of renal tumors in male rats [4], [5]. The chronic toxicity of TRI and PER is most likely mediated by bioactivation reactions. Halogenated olefins such as PER and TRI are metabolized by both cytochrome P450 and glutathione dependent biotransformation pathways leading to the generation of reactive metabolites which may covalently bind to cellular macromolecules (Fig. 1). Cytochrome P450 oxidation of both PER and TRI results in formation of the corresponding chlorinated acetyl chlorides which react with amino groups in macromolecules or with water to give dichloro- or trichloroacetate [6], [7]. In addition, the bioactivation by glutathione conjugation of PER and TRI and the cleavage of the gluathione conjugates to the cysteine conjugates S-(trichlorovinyl)-l-cysteine (TCVC) and S-(1,2-dichlorovinyl)-l-cysteine (1,2-DCVC) by transpeptidases is likely responsible for the nephrotoxicity and possible renal tumorgenicity of these halogenated olefins [8]. The corresponding thioketenes formed by the β-lyase mediated cleavage of TCVC respectively 1,2-DCVC are presumed to be the ultimate metabolites responsible for the mutagenic and nephrotoxic effects [9], [10], [11].

The cysteine conjugates are transformed by N-acetyltransferases to mercapturic acids which are excreted with urine. Therefore, mercapturic acids and the thioketene adducts of proteins and DNA may represent important biomarkers of exposure, which have to be detected and quantified in biological samples. Nevertheless, both mercapturic acids and thioketene adducts of DNA and protein represent only minor metabolites and after exposure to workplace relevant concentrations of PER or TRI very low amounts of these biomarkers have to be detected.

Several methods may be used to quantify biomarkers of exposure ranging from high-performance liquid chromatography–UV absorbance detection (HPLC–UV) for urine metabolites to 32P-postlabelling methods determining DNA adducts. However, these methods often have several disadvantages. HPLC–UV is not sufficiently sensitive for mercapturic acids without an efficient chromophore. 32P-postlabelling methods to quantify DNA adducts are difficult to standardize because of many possible variations for optimal use [1].

For protein adduct detection immunochemical methods are often used, however, problems with cross-reactivity and quantitation of adduct concentrations are limiting their usefulness [12], [13].

The work presented here shows that three similar analytical methods using only one tool, gas chromatography–negative chemical ionization mass spectrometry (GC–NCI-MS), may be applied for detection and quantitation of the biomarkers.

Section snippets

Reagents

Chlorodifluoroacetic acid was obtained from Fluorochem, Old Glossop, UK. Chloroacetic chloride, diazabicyclo[2.2.2.]octane, DNA bases, lysine, methanolic BF3 (14% in MeOH), pentafluorobenzyl bromide, PER, TRI and all other chemicals were obtained from Sigma–Aldrich (Deisenhofen, Germany) in the highest purity available.

Synthesis

N6-Chloroacetyl adenine, N4-chloroacetyl cytosine and N2-chloroacetyl guanine, potential DNA adducts formed from chloroketene were synthesized by the method of Müller et al. [14]

Quantitation of mercapturic acids by electron-capture MS

After derivatization of mercapturic acids to corresponding volatile esters these esters may be to quantified by GC–MS. A method based on GC–NCI-MS has been described for the quantitation of N-ac-TCVC in the urine of PER or N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine [N-ac-S-(1,2-DCVC)] in the urine of TRI-exposed rodents and humans [21], [22]. The method used in this study is based on these procedures, but uses a simplified clean-up, a derivatization procedure with higher yields and a GC column

Acknowledgments

This work was supported by the US Environmental Protection Agency (EPA) (contract No. CR824456-01-0) and the Biomed Program of the European Union (contract No. BN4-CT96-0184).

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