Elsevier

Toxicology Letters

Volume 147, Issue 3, 7 March 2004, Pages 253-260
Toxicology Letters

Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP)

https://doi.org/10.1016/j.toxlet.2003.11.010Get rights and content

Abstract

The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs.

Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km=8 μM; Vmax=689 pmol/min/mg protein; n=6 livers) and a low affinity, high Km, component (Km=391 μM; Vmax=3039 pmol/min/mg protein; n=6). The high-affinity component was inhibited 79%–95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 μM), but also low Vmax (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high Vmax (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 μM, respectively), suggesting their involvement in catalysing the low-affinity component.

This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.

Introduction

Ethylbenzene is a commonly used chemical with several industrial applications. It is a solvent (often mixed with other aromatic solvents such as xylene and toluene), an intermediate in the synthesis of styrene, a raw material for the production of rubber and plastics, and an additive to some fuels (Cavender, 1994, WHO, 1996). The absorption of ethylbenzene in exposed workers arises mainly via inhalation, and to a much lesser extent through the dermal route (Gromiec and Piotrowski, 1984, Fishbein, 1985). Ethylbenzene has low acute and chronic toxicity, but it acts as a central nervous system depressant at high doses and can cause mild irritation of the mucous membranes and eyes (WHO, 1996). Therefore, it is important to control and minimise worker exposure to ethylbenzene. Its metabolites can be detected in urine, and the major product, mandelic acid, is recommended for biological monitoring of ethylbenzene (ACGIH, 2000).

Human in vivo studies have shown that mandelic acid and phenylglyoxylic acid are the major urinary metabolites following inhalation exposure to ethylbenzene (Bardodej and Bardodejova, 1970, Engstrom et al., 1984). These metabolites result from initial hydroxylation of the side chain of ethylbenzene, followed by further oxidation. Some minor ring oxidation metabolites have also been detected in urine, but these account for less than 5% of total metabolites (Engstrom et al., 1984).

Knowledge of the toxicokinetics of a chemical and the individual enzymes involved in its metabolism can improve interpretation of biological monitoring results and risk assessment by predicting the range of biotransformation rates that might be expected in the general population. In vitro metabolic systems, such as liver microsomes, have been extensively employed to study rates of biotransformation and data can be scaled to predict in vivo clearances (Houston, 1994, MacGregor et al., 2001). As part of ongoing studies of industrial solvents, the In vitro metabolism of ethylbenzene has been investigated in human liver microsomes obtained from individual donors. The aims of this study were to determine the kinetic parameters for the initial oxidation of ethylbenzene to 1-phenylethanol and to characterise the forms of cytochrome P450 (CYP) involved in this reaction.

Section snippets

Chemicals

Ethylbenzene and its metabolite, 1-phenylethanol, were obtained from Sigma–Aldrich (Gillingham, UK). All other chemicals used were of analytical grade or better.

Human liver microsomes and recombinant human cytochrome P450 isoforms

Human liver microsomes were obtained from TCS Cellworks (Botolph Claydon, UK; distributors for BioPredic, Rennes, France). Collection and processing of human tissue was conducted in compliance with all current regulatory and ethical requirements. Microsomes were characterised for activity towards a range of model CYP substrates. Donor

Analytical and experimental precision

The limit of detection for 1-phenylethanol was 50 pmol (0.5 μM). At 500 pmol (5 μM), the coefficient of variation of the assay was 4.5% within batch (n=6) and 14% between batches (n=14). Experimental precision for duplicate microsomal incubations from separate experiments analysed independently was 7% at 1 mM (n=3) and 20% at 50 μM ethylbenzene (n=5).

Ethylbenzene metabolism by human liver microsomes

The microsomal protein concentration and incubation time used in this study were both within linear ranges determined in preliminary experiments. No

Discussion

We present human liver microsomal data for the initial step of ethylbenzene metabolism, namely side chain oxidation to form 1-phenylethanol. Our findings suggest strongly that, like many other low molecular weight chemicals, ethylbenzene is predominantly metabolised by CYP2E1 (Guengerich et al., 1991). However, in common with other structurally related compounds including xylene and toluene, ethylbenzene metabolism exhibited biphasic kinetics characterised by a high- and a low-affinity

Acknowledgements

This work was funded by the UK Health and Safety Executive.

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