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.

Estimation of the VA/(Q+VTIS) distribution from single-breath alkane uptake.

In this paper, it was investigated if the ventilation-perfusion distribution can be estimated from the uptake (U) of inert gases with different solubilities during the single-breath maneuver. A model was implemented that describes U as a function of solubility for inhomogeneously distributed alveolar volume (VA) versus blood and tissue volume (Q + VTIS). The VA/(Q + VTIS) distribution describes the relative contribution of gas-exchange units with different VA/(Q + VTIS) ratios to the expiratory volume. U was derived as the sum of uptakes corresponding to different modes in the distribution, weighted with the relative contribution to the expiratory volume. This permits an estimation of the distribution parameters by fitting U as a function of solubility. The n alkanes were used because of their different solubilities. Analysis of the sensitivity of the estimated VA/(Q + VTIS) distribution parameters to measurement errors showed that mostly two modes can be discerned. The influence of fixed model parameters appeared relatively small. The model could well explain U in normal and emphysematous subjects, with a larger contribution of high VA/(Q + VTIS) ratios in the emphysematous subjects. It was concluded that the VA/(Q + VTIS) distribution can be estimated noninvasively from single-breath alkane uptake.

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.

Study on the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene in humans.

OBJECTIVES: To investigate in humans the contribution of the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene (TRI) under controlled repeated exposure in volunteers, and under occupational conditions. METHODS: Volunteers were exposed to TRI, using repeated 15 min exposures at 50 and 100 ppm. This exposure schedule resulted in internal doses of 1.30 and 2.40 mmol of TRI respectively. Urine samples were collected for a minimum of 45 h. Urine samples were also collected from occupationally exposed workers. The samples were analysed for the known human metabolites of TRI, trichloroethanol (TCE), trichloroacetic acid (TCA) and both regio-isomeric forms of the mercapturic acid N-acetyl-S-(dichlorovinyl)-L-cysteine (DCV-NAC), and for (dichlorovinyl)-L-cysteine (DCVC). In order to further elucidate the metabolism of TRI in humans, we analysed samples for dichloroacetic acid and for the proposed break-down products of 1,2 and 2,2-dichlorovinyl-L-cysteine after deamination: the S-conjugates of 3-mercaptolactic acid, 3-mercaptopyruvic acid and 2-mercaptoacetic acid. RESULTS: None of the glutathione metabolites was found in urine of volunteers. In workers occupationally exposed to TRI at levels between 0.4 and 21 ppm [8-h time-weighted average (TWA)], levels of DCV-NAC in urine samples collected at the end of the 4th working day and also next morning were below detection limit (0.04 mumol/l). This confirms the findings of Bernauer et al. (1996) that these metabolites are excreted at very low levels in humans. Urinary levels of DCVC and six postulated metabolites of dichlorovinyl-S-cysteine conjugates via deamination were also below 0.04 mumol/l, indicating that at most 0.05% of the dose is excreted in the form of these metabolites. These data further strengthen the argument for a very low activity of glutathione-mediated metabolism for chronically exposed workers. CONCLUSIONS: This study gives additional data which indicate that glutathione-mediated metabolism is of minor importance in humans exposed to TRI. In spite of indications to the contrary, significant metabolism of the cysteine conjugate via beta-lyase, which could result in a toxic metabolite, cannot be ruled out completely.

Dermal absorption of neat liquid solvents on brief exposures in volunteers.

The dermal absorption of liquid 1,1,1-trichloroethane (111TRI), trichloroethene (TRI), tetrachloroethene (TETRA), toluene (TOL), and m-xylene (XYL) was studied in volunteers. The solvents were applied for 3 min on the volar forearm over an area of 27 cm2. An inhalation exposure with a known input rate served as a reference exposure. Using the linear system dynamics method, permeation rates were calculated from exhaled air concentration courses measured after both inhalation and dermal exposure. The permeation time courses of the solvents showed two different patterns. TRI, TOL, and 111TRI in three subjects showed fast increase in permeation, reaching maximal permeation rates a few minutes after initiation of exposure. Slower permeation was seen in the other three subjects exposed to 111TRI and in all subjects exposed to TETRA and XYL with the time of maximal permeation rates of 15-25 min. These differences in the permeation may partly be explained by the irritation of the skin observed in all subjects showing fast permeation kinetics. The flux into the skin averaged over the exposure period amounted to 56, 430, 69, 223, and 46 nmol/cm2/min for 111TRI, TRI, TETRA, TOL, and XYL, respectively. Comparing the dermal uptake with the respiratory uptake at the TLV, all solvents showed substantial skin absorption, although at present only TOL has a skin indication in the American Conference of Governmental Industrial Hygienists threshold limit value list.

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.

Skin absorption of some vaporous solvents in volunteers.

OBJECTIVES: To determine the dermal absorption rates of vaporous 1,1,1-trichloroethane (111TRI), trichloroethene (TRI), tetrachloroethene (TETRA), hexane (HEX), toluene (TOL) and m-xylene (XYL) in humans. The determined absorption data were used for the validation of two published models for prediction of non-steady-state skin absorption. METHODS: Five volunteers were dermally exposed on an area of about 1,000 cm2 (forearm and hand) for 20 or 30 min. An inhalation exposure with a known dose rate served as a reference. Using the solvent concentrations in exhaled air, measured after both inhalation and dermal exposure, we calculated the maximum absorption rate into the blood, and the average absorption rates into the skin throughout the exposure, using the linear system dynamics method. RESULTS: The absorption rates into the skin, normalised for exposure concentration, amounted to 0.021 cm/h (111TRI), 0.049 cm/h (TRI), 0.054 cm/h (TETRA), 0.013 cm/h (HEX), 0.14 cm/h (TOL), and 0.12 cm/h (XYL). The maximum absorption rates into the blood ranged from 0.005 nmol/h for 111TRI and HEX to 0.050 nmol/hr for TOL. The ratios between the predicted and experimental values of the absorption rates into the skin ranged, for the model of Cleek and Bunge [4], from 0.3 (HEX) to 1.1 (TRI and TETRA), and for the model of Wilschut and Ten Berge [22], from 1.1 (HEX) to 4.7 (XYL). CONCLUSION: The linear system dynamics method allowed us to calculate not only the total amount absorbed by the skin but also the maximum absorption rate into the blood. The steady-state absorption rate, usually described by a permeability constant, will be below the absorption rate into the skin and above the maximum absorption rate into the blood. The skin absorption rates predicted by the models showed a good agreement with the experimental values. A comparison of the estimated whole-body skin uptake with the inhalatory uptake from the same atmosphere, revealed that the dermal uptake contributed from 0.1% (HEX) to 1% (TOL and XYL) to the total uptake.

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.

New technologies in the use of exhaled breath analysis for biological monitoring.

Breath analysis is an attractive non-invasive procedure for screening workers exposed to solvents. It has been used in numerous laboratory based studies and for field research. Despite the obvious advantages in routine biological monitoring it has failed to become widely accepted as a tool in occupational hygiene. Recent advances in breath sampling and analysis are such that it is likely to become more widely used in the future. In this paper, the past 5 years have been reviewed to try to assess what developments might now contribute to the increased use of breath analysis in biological monitoring; in particular, the development of a selected ion flow tube mass spectrometer for real time direct analysis of trace gases in breath and the more immediately available and less expensive indirect methods involving collection devices with adsorbent tubes is important. The introduction of guidance values for biological monitoring with clear advice on sampling times and the recognition of the importance of quality assurance programmes will help improve confidence in the technique.

Dermal absorption of vaporous and liquid 2-methoxyethanol and 2-ethoxyethanol in volunteers.

OBJECTIVES: To estimate dermal absorption of vaporous and liquid 2-methoxyethanol (ME) and 2-ethoxyethanol (EE) in volunteers. METHODS: Five volunteers (two men and three women) were dermally exposed to vaporised and liquid ME and EE. Dermal exposure on an area of about 1000 cm2 (forearm and hand) to vapours of ME and EE (4000 mg/m3 ME and 3700 mg/m3 EE) lasted for 45 minutes. Duration of exposure to liquid ME and EE on an area of 27 cm2 (forearm) was 15 minutes. Dermal uptake was assessed by measurement of the main metabolites in urinary methoxyacetic acid (MAA) and ethoxyacetic acid (EAA). For each volunteer, excretion of metabolites was compared with a reference inhalatory exposure. RESULTS: Mean (SD) absorption rates of ME and EE vapour were 36 (11) and 19 (6) cm/h respectively. The mean (SD) absorption rates of the liquid ME and EE amounted to 2.9 (2.0) and 0.7 (0.3) mg/cm2.h. CONCLUSIONS: Vaporised and liquid ME and EE are readily absorbed through the skin. In the combined inhalatory and dermal exposure when whole body surface is exposed to vapour, the uptake through the skin is estimated to be 55% of the total uptake of ME and 42% of EE. Dermal uptake resulting from skin contact of both hands and forearms (about 2000 cm2) with liquid ME and EE for 60 minutes would exceed inhalatory uptake of the eight hour occupational exposure limit by 100 times at 16 mg/m3 of ME and 20 times at 19 mg/m3 of EE. The substantial skin uptake of ME and EE indicates that in assessing the health risks biological monitoring and use of biological exposure indices are preferable to environmental monitoring.

Dermal absorption of cis-1,3-dichloropropene vapour: human experimental exposure.

1. The relevance of skin absorption of cis-1,3-dichloropropene (cis-1,3-DCP) vapour as a route of entry compared to inhalatory uptake has been assessed in human volunteers under controlled exposure conditions. 2. Five adults (four males and one female) were dermally exposed on the forearm and hand during 45 min to 86 mg/m3 cis-1,3-DCP. 3. Dermal uptake was assessed by determination of the main cis-1,3-DCP metabolite in urine: the mercapturic acid conjugate of cis-1,3-DCP (cis-1,3-DCP-MA). 4. When whole-body dermal exposure to vapour is compared to inhalatory exposure, the uptake through the skin is estimated to be about 2-5% of the inhalatory absorption.

Evaluation of biological monitoring parameters for occupational exposure to toluene.

A survey was conducted in a rotogravure printing plant with inhalatory and percutaneous exposure to toluene. Workers (n = 9) were followed for 2 consecutive days and the frequency and duration of skin contact with toluene were monitored. In order to assess percutaneous absorption an airstream helmet was worn during one day. Urine and exhaled air samples were collected simultaneously 5 times each day for toluene (urine and breath) and hippuric acid (urine). The mean (personal air sampling) exposure concentration was between 30 mg/m3 and 600 mg/m3. The best biological monitoring parameter of external exposure (without a helmet) was the concentration toluene in exhaled air 8 h after work (r = 0.99). While wearing the airstream helmet the relationship between external exposure (measured in the helmet) and concentrations in exhaled air and urine deviated from the preceding relations. This was likely the result of the high body burden and not of skin contact with toluene. Skin contact with toluene (usually by cleaning of the hands) was limited to 0-30 minutes a day, with an average of about 5 minutes. During experimental exposure (n = 6) in which the hands were washed with toluene for 5 minutes the toluene in exhaled air (max after 1040 min) clearly demonstrated skin absorption of toluene. The next morning 0.1 mg/m3 toluene was still detectable; this was less than the concentration measured the next morning in exhaled air of workers: between 0.5 and 10 mg/m3.

Biological exposure and/or effect limits, facts, fallacies and uncertainties: general principles.

When looking for facts, fallacies and uncertainties of the use of biological exposure limits one has at first to discuss the general principles of biological monitoring (BM) and biological effect monitoring (BEM) because they determine the validity of the data that underpin the biological exposure limits. A difference between countries in preferred BM-methods can be observed. The terminology is still confusing: in addition to BM and BEM, biomonitoring and biological markers also are used. There are a number of problems in respect of the inter- and intra-individual variability in internal exposure and effect at similar exposure levels due to differences in for example, physical workload, body composition and genetics. Toxicokinetic models based on data from individual workers should be developed in order to get information on the variability and the cause of this. Both kinetics and dynamics may be sex-dependent. To date, BM methods have been tentatively suggested for only about 10 per cent of the regulated industrial chemicals. BM and BEM programmes yield important extra information on exposure and health risk, not to be gained by environmental monitoring alone.

Biological exposure and/or effect limits, facts, fallacies, and uncertainties: practical aspects.

In the preceding article general principles in setting biological occupational exposure limits (BOEL) and effect limits (BOEEL) were discussed. Here monitoring in every day occupational health practice is discussed. The specific objectives of biological monitoring (BM) and biological effect monitoring (BEM) determine to a large extent the choice of the parameters to be measured. According to the objective, the assessment may be either simple or sophisticated. The choice of an appropriate reference is essential for a valid evaluation of internal exposure, health risk and state of health. The measurement strategy depends on the working mechanism and the kinetics of the chemical. Protocols for BM and BEM-programmes should be regularly updated. Different compounds of the same metal may carry widely different health risks. In general it is necessary to correct the excretion of chemicals for dilution of the urine.

Determination of methyl ethyl ketone and its metabolites in urine using capillary gas chromatography.

A method is described for the determination of the concentration of methyl ethyl ketone and its metabolites: 2-butanol, 3-hydroxy-2-butanone and the meso- and d,l-isomers of 2,3-butanediol in urine. The analytes were isolated from urine by solid-phase extraction and analysed by capillary gas chromatography. The recovery rates were 50-70% for the 2,3-butanediol isomers and 88-96% for the other analytes. The precision of the method ranged from 5 to 12% (S.D.%). The detection limit was 1.0 and 1.4 mg/l for meso- and d,l-2,3-butanediol, respectively, and ranged from 0.1 to 0.15 mg/l for the other analytes.

Biological markers of solvent exposure.

The important limitation of many epidemiologic studies is the relative inaccuracy of the assessment of the magnitude of exposure. For some solvents, the concentration in biological media is an indication of the internal exposure and is an indirect indication of the health risk, at least for acute effects. For long-term effects, e.g., carcinogenicity, biological monitoring data can also be used as showed with the individual occupational data on the level of trichloroacetic acid (TCA) in urine. Occupational epidemiology can improve the methods for the assessment of the actual total exposure and health risk in environmental epidemiology by providing higher dose cohort data.