Stereochemical metabolism of styrene in volunteers.

OBJECTIVES: To study the stereochemistry of styrene metabolism in volunteers, and its interindividual variability. METHODS: Twenty healthy male volunteers (aged 18-37 years) were exposed to 360 mg/m3 styrene for 1 h while they performed 50 W physical exercise. Venous blood was drawn during and for up to 2 h after exposure. Urine was collected at time-intervals up to 24 h after exposure. The following parameters were determined: styrene, free and conjugated styrene glycol (SG) in blood, and conjugated SG, mandelic acid (MA) and phenylglyoxylic acid (PGA) in urine. RESULTS: Average pulmonary retention of styrene was 62%. Excretion of the acidic metabolites MA and PGA accounted for 58% of the pulmonary uptake. The average maximum concentration (Cmax) and area under the curve (AUC) of free (R)-SG in blood were 1.3 and 1.7 times higher than those of (S)-SG respectively; the half-life of (R)-SG was longer (82 vs 62 min, P < 0.005). Cmax and AUC of the conjugated SG enantiomers in blood did not differ, but again half-life for (R)-SG was longer (72 vs 64 min, P < 0.05). Cumulative excretion and renal clearance of conjugated (S)-SG in urine were three and four times higher, respectively, than that of (R)-SG. Cumulative excretion of (S)-MA was 1.6 times higher than (R)-MA. Interindividual differences in the kinetic parameters of the metabolites were two- to threefold. CONCLUSIONS: The enantiomeric excess found was different for each metabolite under study, implying different enantioselectivity and/or enantiospecificity of the enzymes and carrier-proteins involved in the biotransformation and excretion. The use of these metabolites as biological indicators for prediction of the enantiomeric excess of the toxic metabolite styrene-7,8-oxide (SO) is therefore not justified. Interindividual differences in the stereochemical metabolism of styrene are moderate.

Metabolic capacity and interindividual variation in toxicokinetics of styrene in volunteers.

The aim of the present study was to assess the interindividual variation in styrene toxicokinetics and to correlate this variation with the individual metabolic capacity for cytochrome P450 (CYP), CYP2E1, CYP1A2 and CYP2D6. Twenty male volunteers were exposed on separate occasions to 104+/-3 and 360+/-20 mg/m3 of styrene for 1 h while performing 50 W physical exercise on a bicycle ergometer. Styrene concentrations in blood and mandelic (MA) and phenylglyoxylic acid (PGA) in urine were measured. The metabolic capacity was assessed by phenotyping with chlorzoxazone (CYP2E1), caffeine (CYP1A2), dextromethorphan (CYP2D6) and antipyrine (CYP450). In addition, for the main styrene-metabolising enzyme, CYP2E1, genotyping for the genetic polymorphisms of the gene was performed. The average pulmonary retention of styrene was 62 +/- 7% at both exposure concentrations, and the 24-h excretion of MA and PGA accounted for 58% of the dose at both concentrations. The interindividual variation in styrene kinetics ranged from 19% for the terminal half-life (t(1/2,beta)) of styrene to 41% for the cumulative excretion of MA and PGA. However, no correlation between the apparent blood clearance of styrene (CLapp), t(1/2,beta) of styrene or excretion of MA and PGA on one hand, and the individual metabolic capacity on the other hand was found. Although other explanations cannot be excluded, this lack of correlation might be due to the high apparent blood clearance (1.4 l/min) of styrene, indicating that styrene metabolism is liver-blood-flow-dependent.

Metabolism of styrene in the human liver in vitro: interindividual variation and enantioselectivity.

1. The interindividual variation and enantioselectivity of the in vitro styrene oxidation by cytochrome P450 have been investigated in 20 human microsomal liver samples. Liver samples were genotyped for the CYP2E1*6 and CYP2E1*5B alleles. 2. Kinetic analysis indicated the presence of at least two forms of styrene-metabolizing cytochrome P450. The enzyme constants for the high-affinity component were subject to appreciable interindividual variation, i.e. Vmax1 ranged from 0.39 to 3.20 nmol mg protein(-1) min(-1) (0.96+/-0.63) and Km1 ranged from 0.005 to 0.03 mM (0.011+/-0.006). Inhibition studies with chemical inhibitors of CYP2E1, CYP1A2, CYP2C8/9 and CYP3A4 demonstrated that CYP2E1 was the primary enzyme involved in the high-affinity component of styrene oxidation. No relationship between the interindividual variation in Vmax1 and Km1 and the genetic polymorphisms of the CYP2E1 gene was found. 3. Cytochrome P450-mediated oxidation of styrene demonstrated a moderate enantioselectivity, with an enantiomeric excess (ee) of (S)-styrene oxide of 15% (range 4-27%) at low styrene concentration and an ee of (R)-styrene oxide of 7% (range -11 to +22%) at high styrene concentration. This points towards the involvement of at least two cytochrome P450, with different enantioselectivities. 4. The data indicate that cytochrome P450-mediated styrene oxidation is subject to considerable interindividual variation, but only to a moderate product enantioselectivity.

Metabolism of styrene-7,8-oxide in human liver in vitro: interindividual variation and stereochemistry.

Styrene is an industrial solvent which is mainly oxidized by cytochrome P450 to an electrophilic, chiral epoxide metabolite: styrene-7,8-oxide (SO). SO has cytotoxic and genotoxic properties; the (R)-enantiomer is more mutagenic to Salmonella typhimurium TA 100 in the Ames test than the (S)-enantiomer. Detoxication proceeds via microsomal epoxide hydrolase (mEH). Interindividual differences in mEH activity as well as differences in mEH enantioselectivity are important factors for toxic effects of SO. To study the extent of the interindividual variation, microsomal preparations of 20 human livers were incubated with (R)- and (S)-SO separately (1-2000 microM) and Michaelis-Menten kinetics were determined. In addition, samples were genotyped for two genetic polymorphisms of the mEH gene. V(max), K(m) and V(max)/K(m) values of both enantiomers differed three- to fivefold between the livers. No association of the enzyme constants with the genetic polymorphisms of the epoxide hydrolase gene was found. Hydrolysis of the styrene oxide enantiomers proceeded in an enantioselective manner, with the (S)-enantiomer having an approximately six times higher K(m) and five times higher V(max) than the (R)-enantiomer. In vivo, both SO enantiomers are formed; therefore, time course incubations with racemic SO were carried out in vitro to investigate possible interactions between the enantiomers. When racemic SO was used as a substrate, the (R)-enantiomer acted as an inhibitor on the hydrolysis of the (S)-enantiomer. These results indicate that mEH-mediated hydrolysis of SO is subject to appreciable interindividual variation and that hydrolysis of the more toxic enantiomer is favored.

With medium-chain triglycerides, higher and faster oxygen radical production by stimulated polymorphonuclear leukocytes occurs.

BACKGROUND: Parenteral lipid emulsions are suspected of suppressing the immune function. However, study results are contradictory and mainly concern the conventional long-chain triglyceride emulsions. METHODS: Polymorphonuclear leukocytes were preincubated with parenteral lipid emulsions. The influence of the lipid emulsions on the production of oxygen radicals by these stimulated leukocytes was studied by measuring chemiluminescence. Three different parenteral lipid emulsions were tested: long-chain triglycerides, a physical mixture of medium- and long-chain triglycerides, and structured triglycerides. Structured triglycerides consist of triglycerides where the medium- and long-chain fatty acids are attached to the same glycerol molecule. RESULTS: Stimulated polymorphonuclear leukocytes preincubated with the physical mixture of medium- and long-chain triglycerides showed higher levels of oxygen radicals (p < .005) and faster production of oxygen radicals (p < .005) compared with polymorphonuclear leukocytes preincubated with long-chain triglycerides or structured triglycerides. Additional studies indicated that differences in results of various lipid emulsions were not caused by differences in emulsifier. The overall production of oxygen radicals was significantly lower after preincubation with the three lipid emulsions compared with controls without lipid emulsion. CONCLUSIONS: A physical mixture of medium- and long-chain triglycerides induced faster production of oxygen radicals, resulting in higher levels of oxygen radicals, compared with long-chain triglycerides or structured triglycerides. This can be detrimental in cases where oxygen radicals play either a pathogenic role or a beneficial one, such as when rapid phagocytosis and killing of bacteria is needed. The observed lower production of oxygen radicals by polymorphonuclear leukocytes in the presence of parenteral lipid emulsions may result in immunosuppression by these lipids.

Gas chromatography-electron capture determination of styrene-7,8-oxide enantiomers.

The enantiomers of styrene-7,8-oxide (phenyloxirane, SO) were determined using a method based on base catalysed hydrolysis with sodium methoxide. The oxirane ring opening resulted in formation, without racemisation, of the enantiomeric pairs of the two regional isomers, 2-methoxy-1-phenylethanol and 2-methoxy-2-phenylethanol. The structure of these regional isomers was confirmed by gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance (1H-NMR). To improve sensitivity of determination, the formed methoxy alcohols were subsequently derivatised with pentafluoropropionic anhydride enabling electron capture detection. This derivatization proceeded also without racemisation and the formed pentafluoropropionyl derivatives were separated on two serially coupled columns, a non-chiral AT 1705 and a chiral CP Chirasil-Dex-CB. As internal standard 2S,3S-(-)-2-methyl-3-phenyloxirane was used. The limit of quantitation of the method was 0.2 microM. The repeatability of the method was assessed at two concentration levels (2.5 and 25 microM) and ranged from 6 to 9% for both enantiomers. The method was applied to the determination of the rate and enantioselectivity of the cytochrome P-450 dependent oxidation of styrene to SO enantiomers in human liver microsomes.