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British Journal of Industrial Medicine 1991;48:492-498

Thioether excretion in urine of applicators exposedto 1,3-dichloropropene: a comparison with urinarymercapturic acid excretion

R T H van Welie, CM van Marrewijk, F A de Wolff, N P E Vermeulen

AbstractThe excretion of thioethers in urine ofapplicators occupationally exposed to the soilfumigant 1,3-dichloropropene (DCP) wasdetermined by the thioether assay. The mer-capturic acid metabolite of E-1,3-dichloropropene, N-acetyl-S-(E-3-chloropro-penyl-2-)-L-cysteine (E-DCP-MA), was thereference compound in the thioether assay.The mean recovery of E-DCP-MA was 58X5%(coefficient of variation (CV) 9%, n = 4). Innon-exposed men mean background ofurinarythioethers was 6 05 mmol SHImol creatinine(n = 56). In applicators exposed to soilfumigants containing DCP, urinary excretionof thioethers followed first order eliminationkinetics. Urinary half lives of elimination ofthioethers were 8-0 (SD 2-5) hours based onexcretion rates and 9 5 (SD 3 1) hours based oncreatinine excretion. The urinary half life ofelimination of thioethers was almost twofoldhigher compared with halflives of eliminationof the mercapturic acids of Z- and E-1,3-dichloropropene. The post- minus pre-shiftthioether concentrations in urine and thecumulative urinary thioether excretionscorrelated well with exposure to DCP. In urinesamples the mean thioether concentration was1-38 higher than mean DCP mercapturic acidconcentration. This suggests the presence ofunidentified thioether metabolite(s) due toexposure to soil fumigants containing DCP.According to the present data, an eight hourtime weighted average exposure to the Dutchoccupational exposure limit of 5 mg/M3 DCP

Department of Pharmacochemistry, Division ofMolecular Toxicology, Free University, DeBoelelaan 1083, 1081 HV Amsterdam, The Nether-landsR T H van Welie, N P E VermeulenDepartment of Human Toxicology, UniversityHospital of Leiden, Leiden, The NetherlandsCM van Marrewijk, F A de Wolff

results in a post- minus pre-shift thioetherconcentration of 9-6 mmol SH/mol creatinine(95% confidence interval (95% CI) 7-411-8 mmol SHImol creatinine) and in acumulative thioether excretion of 139 pmol SH(95% CI 120-157 pmol SH). It is concluded thatthe thioether assay can be used to assess com-paratively high levels of exposure to DCP.

Exposure of man to electrophilic compounds mayultimately lead to the excretion of thioethers such ascysteine conjugates and mercapturic acids (S-sub-stituted N-acetyl-L-cysteine conjugates) in urine.Mercapturic acids are end products from metabolicdetoxication of electrophilic chemicals via conjuga-tion to glutathione. Glutathione conjugation eitherproceeds spontaneously or is catalysed by gluta-thione S-transferases present in liver, blood, andseveral other organs and tissues. Once glutathioneconjugates have been formed in vivo, metaboliccleavage of the glutamyl and glycine residue gen-erally takes place. N-acetylation of the remaining S-alkylated cysteine conjugates finally leads to theformation of the mercapturic acid.

Mercapturic acids excreted in urine can be deter-mined as thioethers using the thioether assay. Thedetermination of urinary thioethers can be used as anon-selective test to assess exposure to electrophiliccompounds.' The thioether assay has been usedfrequently to compare exposed and non-exposedpopulations, for example, factories producingrubber,2 pharmaceuticals and explosives, or uponexposure to bitumen fumes.34 Mostly, however, noinformation is available about the chemical structureof the various thioethers determined. Besides alimited selectivity, the sensitivity of the thioetherassay is also limited by background excretion ofthioethers. This is strongly influenced by lifestylefactors (for instance, smoking and medication). Bycontrast, for the determination of mercapturic acidsin urine, selective and sensitive analytical techniquessuch as high performance liquid chromatographyand gas chromatography with different detectiontechniques are available.56

Recently, occupational exposure to Z- and E-1,3-

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Thioether excretion in urine of applicators exposed to 1,3-dichloropropene

dichloropropene (Z- and E-DCP) of applicators inthe Dutch flower bulb culture was determined andshown to result in urinary excretion of two mer-capturic acids-namely, N-acetyl-S-(Z- and E-3-chloropropenyl-2-)-L-cysteine (Z- and E-DCP-MA). A linear relation between exposure to Z- and E-DCP and excretion of Z- and E-DCP-MA in urinewas found. Urinary half lives of elimination were 5-0(SD 1-2) hours for Z-DCP-MA and 4-7 (SD 1 3)hours for E-DCP-MA.7The primary aim ofour study was to determine the

relation between exposure to soil fumigants con-taining DCP and urinary excretion of thioethers inoccupationally exposed applicators. The post- minuspre-shift thioether concentrations and cumulativethioether excretion were correlated with exposure toDCP. Half lives of elimination of thioethers in urinewere also determined. Finally, excretion of thioetherwas compared with urinary mercapturic acidexcretion.7

Materials and methodsCHEMICALSN - acetyl - S - (Z - and E - 3 - chloropropenyl - 2-)-L -cysteine were synthesised.8 Potassium dihydrogenphosphate and citric acid (trisodium salt dihydrate)were purchased from Merck (Darmstadt, Germany).5,-5'-Dithiobis-2-nitrobenzoic acid and creatininestock solutions were obtained from Sigma (St Louis,MO, USA). N-acetyl-L-cysteine was purchasedfrom Janssen Chimica (Beerse, Belgium).

POPULATION AND EXPOSUREThe population under investigation consisted of 12male Dutch applicators of Z- and E-DCP in theflower bulb culture and four male non-exposedvolunteers. The respiratory exposure to Z- and E-DCP of these applicators has been the subject of acombined biological, environmental, and biologicaleffect monitoring study.7910 Population characteris-tics, application techniques, and sampling strategiesof urine and air samples have been describedextensively in these papers. Briefly, the eight hourtime weighted average (TWA) exposures to Z- andE-DCP were determined by means of personal airsampling during full period consecutive sampling.The exposure to (Z + E)-DCP ranged from 0-3 to18 9 mg/m3 in shifts of one to 11 hours. The urinaryexcretion of the corresponding mercapturic acids,Z- and E-DCP-MA, was determined in samplescollected before, during, and after the exposuremeasurements. Mean urinary half lives of elimina-tion were 5-0 (SD 1 2) hours for Z-DCP-MIA and4.7 (SD 1-3) hours for E-DCP-MA. Maximumcumulative urinary excretion of (Z + E)-DCP-MAamounted to 296 pmol. Strong correlations (r . 0-93)between exposure to Z- and E-DCP and the excre-

tion of respective mercapturic acids in urine werefound.7

ANALYSIS OF THIOETHERSThe urinary excretion of thioethers was determinedas described previously with slight modifications."Samples of human urine were centrifuged for fiveminutes at 4000 g and 2 ml portions were transferredinto glass stoppered tubes. The pH was adjusted to1-5-2 0 with 0-25 ml 4 M hydrochloric acid. Two x4ml of ethyl acetate were used for extraction of theacidified urine samples by vortex mixing for 30seconds. The organic layers were separated bycentrifugation for five minutes at 4000 g andevaporated to dryness at 37°C under a mild nitrogenflow. The residues obtained were resuspended in 1 mldistilled water. Subsequently alkaline hydrolysis wasperformed on 0 5 ml portions in screw capped glasstubes by adding 0-25 ml 4 M sodium hydroxide,saturating with nitrogen, and heating at 100°C in aboiling water bath in the dark. After hydrolysing for50 minutes, samples were cooled on ice for 10minutes and 0 25 ml of 4 M hydrochloric acid wasadded. To the remaining, non-hydrolysed portionsof 0 5 ml of resuspended residue, 0 5 ml 4 M sodiumchloride was added.

After exactly five minutes, the sulphydryl (SH)concentrations in the hydrolysed and non-hydrolysed samples were determined by addingEllman reagent and measuring the absorbance at412nm. Freshly prepared Ellman reagent consistedof 10 ml 10 M phosphate buffer (pH 71) and0-3 ml 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB)solution (0-4 mgDTNB per ml 1% sodium citrate).'2

Standard calibration curves of SH groups wereprepared by dissolving N-acetyl-L-cysteine inconcentrations ranging from 0 10 to 0 80 mmol/l in2 M sodium chloride and directly analysing withEllman reagent. Calibration curves of thioetherswere prepared by dissolving N-acetyl-S-(E-3-chloropropenyl-2-)-L-cysteine (E-DCP-MA) inconcentrations ranging from 0 10 to 0 80 mmol/l inhuman urine, and treating as described above. Bothtypes of calibration curve were prepared fresh daily.

Urinary thioether concentrations were calculatedusing differences in absorbance at412nmbetween thehydrolysed and the non-hydrolysed parts of thesamples in the regression equation of the calibrationcurve of E-DCP-MA. The recovery ofE-DCP-MAwas calculated from the ratios of the absorbance at412 nm ofE-DCP-MA v the absorbance at 412 nm ofthe equimolar SH concentrations of N-acetyl-L-cysteine.

ANALYSIS OF CREATININEUrinary creatinine was determined by the Jaffereaction."3

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van Welie, van Marrewijk, de Wolff, Vermeulen

Table I Concentration dependent within day and day to day variation in recovery ofE-DCP-MA in thioether assay

Within day Day to day

E-DCP-MA (mmol/l) Recovery (%) CV (%) No Recovery (%) CV (%) No

0-10 527 25 3 588 285 25020 585 66 3 609 178 25040 655 23 3 65-1 11*7 25053 -* - - 702 93 140-80 57-1 3-9 3 505 64 11Mean 58-5 90 61.1 12-0

*Not determined.

CALCULATIONSUrinary elimination rate constants (kei) of thethioethers were calculated by linear regressionanalysis from the linear parts of the semilogarithmicthioether excretion rate v time curves or from thethioether excretion expressed per mol creatinine vtime curves. Elimination rate constants were used tocalculate half lives of elimination (t112 = ln 2/ke,).Correlation coefficients (r) were calculated as ameasure for the strength of linear relations. Spear-man correlations (p) were calculated to test for thesignificance of the correlation coefficients. Levels ofsignificance between urinary half lives of eliminationof mercapturic acids of DCP and thioethers wereassessed by the two tailed Wilcoxon signed rank test.

.~

5040

v0.C

o 0 20

Time (h)

ResultsANALYSIS OF THIOETHERSBoth types of standard calibration curves, N-acetyl-1-cysteine in 2 M sodium chloride and E-DCP-MAin human urine, showed linear relations (r . 0-998; n= 25). Table 1 presents within day and day to dayrecoveries of E-DCP-MLA determined with thethioether assay. The mean within day recovery ofE-DCP-MA in concentrations ranging from 0 10 to0-80 mmol/l was 58 5% (coefficient of variation (CV)= 9%, n = 4). The CV was <6-6% for allE-DCP-AMA concentrations tested. Mean day to dayrecovery was 61-1% (CV = 12 0%, n = 5). Meanwithin day and mean day to day recoveries were notsignificantly different. The day to day CV increased

20

30

Figure 1 Urinary excretion of thioethers (per mol creatinine) of applicators exposed to 3-8 (0), 9-8 (0), and 189 (A)mg/m3 eight hour TWA (Z + E)-DCP in respiratory air. Dark shaded areas indicate exposure periods.

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Table 2 Urinary half lives of elimination of thioethers andmercapturic acids of (Z + E)-DCP based on excretion rates(mg/h) and on creatinine excretion (mmollmol creatinine) inapplicators occupationally exposed to soilfumigantscontaining DCP

t,,2 of elimination (h (SD))

Mercapturicacidst Thioethers

Excretion rate (n = 5) 4-6 (1-0) 8-0 (2.5)*Creatinine excretion (n = 9) 4 9 (0-8) 9-5 (3 1)**

*p < 0 05; **p < 0-01.tTaken from reference7.

with decreasing concentrations of E-DCP-MA,reaching a value of 28-5% at 0 10 mmol/l. The highvalues of the day to day CVs were probably due tovariation in the efficiency of the alkaline hydrolysisstep in the thioether assay. From day to day theefficiency of the alkaline hydrolysis varied from 60 to90%. The efficiency of the ethyl acetate extraction ofE-DCP-MA was 94 7 (SD 6 6)%; Z-DCP-MAappeared to have the same recovery in the thioetherassay as E-DCP-MA. Therefore, only E-DCP-MAwas selected as a reference compound for this study.

BACKGROUND THIOETHER CONCENTRATIONSControl urine samples were obtained from fourvolunteers (n = 27) and 12 applicators (n = 29).Background thioether concentrations were lognormally distributed over a range from 1 26 to 16-33mmol SH/mol creatinine. The mean background was6-05 mmol SH/mol creatinine with a 95% confidenceinterval (95% CI) lower limit of 5-48 mmol SH/molcreatinine and an upper limit of 6-68 mmol SH/molcreatinine.

URINARY THIOETHER EXCRETIONFigure 1 shows the urinary thioether excretionprofiles expressed per mol creatinine, of threeapplicators, exposed to 3 8, 9 8, and 18 9 mg/m3 eighthours time weighted average (TWA) (Z + E)-DCPrespectively. Pre-shift thioether concentrationscorresponded with background concentrations ofthioethers seen in control urine samples. Thioetherexcretion increased rapidly after exposure to DCPstarted and thioether concentrations appeared to bedependent on duration and level of exposure.Maximum thioether concentrations measured at theexposures presented in fig 1 were 16-6, 27-3, and 48-0mmol SH/mol creatinine respectively.

Post-shift urinary excretion of thioethers followedfirst order elimination kinetics. Urinary eliminationrate constants were calculated if the correlationcoefficients ofthe semilog linear parts ofthe excretionv time curves were r 2 0 917 and if at least four post-shift data points with SH concentrations higher thanthe background excretion levels, observed in con-

0 246810- 12 14 16 18 20(Z+E) -DCCP concentration (mg/rn3)

Figure 2 Correlation between eight hour TWA (Z + E)-DCP concentration in respiratory air and urinary post-minuspre-shift thioether concentrations in applicators. Regressionliney = 437 + 1O05x (r = 0O79;n = 19;p = 0001)and 95% CIs are drawn.

trols, were available. Urinary elimination rate con-stants were used to calculate t112 of elimination. Only afew excretion profiles met all these requirements,primarily due to background excretion of thioethers.The t112 of elimination was calculated both fromexcretion rates and creatinine excretion. Based onexcretion rates, half lives of elimination were 9 8,8 1,and 9 7 hours, respectively (fig 1). Mean t,v2 ofelimination based on excretion rates was 8-0 (SD 2 5)hours (n = 5) and based on creatinine excretion 9-5(SD 3 1) hours (n = 9). A roughly twofold largerstatistically significant difference (p < 0 05) in t112 ofelimination of thioethers was seen when comparedwith the urinary t1l2 of elimination of mercapturicacids determined previously (table 2).

THIOETHER EXCRETION IN RELATION TO DCP EXPOSUREFigure 2 shows the relation between the eight hourTWA exposure to (Z + E)-DCP and the thioetherconcentration, expressed per mol creatinine, of thefirst urine sample collected post-shift minus thethioether concentration of the sample collected pre-shift. Subtraction of the pre-shift thioether concen-tration served as an individual correction of the

0 15

,, 100.

00s-'0E_1

liny= 4!3 O1-0 x r=0-7 n=A19 1 0-001

4 6 8 10c t 14 16( Z + E) - DCP concentration ( mg/m3)

Figure 3 Correlation between eight hour TWA (Z + E) -DCP concentration in respiratory air and cumulativeurinary thioether excretion in applicators. Regression liney = 56 5 + 165 x (r = 0-93; n = 17; p < 0 001) and95% CIs are drawn.

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c 0-706

c 05c 0-4-0

' 0--,c O0 1

OI I I

0-1 0-2 0 3 0 4(Z+ E) - DCP-MA concentration (,umol/1)

Figure 4 Correlation between (Z + E) DCP-MAconcentration and thioether concentration in urine samples ofapplicators. Regression liney = 0-09 + 1 38 x (r = 0 90;n = 221; p < 0 001) is drawn.

background thioether excretion and improved thecorrelation with exposure to DCP. The correlationcoefficient between DCP exposure and post- minuspre-shift thioether concentration was r = 0 79.

Figure 3 shows the relation between the eight hourTWA exposure to (Z + E)-DCP and the cumulativethioether excretion in urine. Cumulative excretion ofthioethers was calculated as the area under theurinary thioether excretion rate v time curve (AUC)from the start of the shift until eight hours post-shift.Data for two applicators were omitted; one applicatorwore a respirator and the excretion curve of the otherwas too irregular to interpret. A correlation co-

efficient of r = 0 93 was found between exposure toDCP and cumulative excretion ofthioethers in urine.

COMPARISON OF THIOETHER AND MERCAPTURIC ACIDEXCRETIONIn a previous study,7 concentrations of Z- andE-DCP-MA were determined in the same urinesamples in which the presently reported thioetherconcentrations were measured. Figure 4 shows therelation between (Z + E)-DCP-MA concentrationsand the thioether concentrations. Urine samples inwhich no mercapturic acids were detected and sam-

ples in which the thioether concentration was in the

350.2 300-

u 250-

E200-:PR150

.2 100 trHn

0 50 100 150 200 250 300 350

(Z+ E) DCP-MA excretion (,umol)

Figure 5 Correlation between cumulative urinary(Z + E)-DCP-MA excretion and cumulative urinarythioether excretion. Regression line y = 56-48 + 1l11 x(r = 095;n = 19;p < 0001) is drawn.

range of the background level (< 6-68 mmol SH/molcreatinine), were omitted from the regressionanalysis. The regression line was y = 0 09 + 1-38 x(r = 0 90).

Figure 5 compares the cumulative urinary excre-tion of (Z + E)-DCP-MA and of thioethers, bothcalculated as the area under the excretion rate v timecurves, for 19 shifts. Cumulative excretion of thethioethers was calculated from the start of the shiftuntil eight hours post-shift. Cumulative excretion ofmercapturic acid was also calculated from the start ofshift. Terminal excretion, however, was calculatedby extrapolating time to infinity using the eliminationrate constant.7 The regression line was y = 56-48 +1-1 1 x (r = 0 95).

DiscussionIn this study, the excretion of thioethers in urine ofapplicators ofDCP was investigated in relation to theexposure to this soil fumigant. Urinary half lives ofelimination of thioethers were calculated, andthioether and mercapturic acid excretion werecompared. Also, the thioether assay was evaluatedquantitatively.At present, no data concerning extraction

recoveries and detection limits of thioethers in thethioether assay are available. N-acetyl-S-(E-3-chloropropenyl-2-)-L-cysteine (E-DCP-MA), amercapturic acid metabolite of E-DCP, wastherefore used as a synthetic standard to evaluatethe analytical performance of the thioether assay.Standard calibration curves of SH groups andcalibration curves of thioethers in human urine allshowed good linearity. The recovery ofE-DCP-MAfrom human urine was only 58-5% (CV = 9%, n =4) but did not differ significantly day to day. Thewithin day recovery CV remained below 6-6% for E-DCP-MA concentrations ranging from 0 10 to 0-80mmol/l urine. Day to day recovery CV of E-DCP-MA, however, amounted to 28-5% at 0-10 mmol/lE-DCP-MA, stressing the need for preparingcalibration curves daily. The detection limit of E-DCP-MA in the thioether assay was 010 mmol/l.Lower concentrations could be detected as thioetherbut the CV exceeded 10%. Previously, recoveries ofZ- and E-DCP-MA from urine were found to be69% (CV = 7%, n = 4) and 70% (CV = 8%, n = 4)when measured by gas chromatography. With thistechnique, detection limits of Z- and E-DCP-MAwere 0 45 Mmol/l (CV = 7-2%, n = 8).5

In urine samples obtained from non-exposedvolunteers and applicators, a mean thioether back-ground of 6-05 mmol SH/mol creatinine was found.Comparable background concentrations rangingfrom 3-8 to 5-1 mmol SH/mol creatinine were foundpreviously in other reference populations.4"'Smoking, medication, and other confounding factors

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of lifestyle were not taken into account in our presentstudy. None of the subjects was taking medicines,however, and only four of 12 applicators weremoderate smokers. Moderate smoking did not resultin significantly increased urinary excretion ofthioether in the group of non-exposed volunteers.The shapes of the urinary thioether excretion

profiles appeared to be similar at different exposures inthe applicators. As already found with Z- andE-DCP-MA excretion, thioether excretion rapidlyincreased after exposure toDCP started and followedfirst order excretion kinetics. The height of thioetherexcretion depended on the level and duration ofexposure. Pre-exposure and, in general, more thaneight hours post-exposure, normal backgroundexcretion concentrations of thioethers were seen inapplicators.

Urinary tl,2 values for elimination of thioetherswere 8 0 (SD 2 5) hours and 9 5 (SD 3 1) hours basedon excretion rates or on creatinine excretions,respectively. Compared with the t,,2 of elimination ofthe mercapturic acids of DCP, t1/2 of elimination ofthioethers was almost twice as high. This significantdifference can be explained by the presence of a(DCP) thioether metabolite(s) with a longer t1,2 ofelimination than those of (Z + E)-DCP-MA.Exposure to impurities in commercial DCP formula-tions could be the reason for differences betweenurinary thioether excretion and urinary mercapturicacid excretion.Two urinary thioether excretion parameters-

namely, post- minus pre-shift thioether concentra-tion and cumulative thioether excretion-proved tobe successful in assessing exposure to DCP. Bothparameters showed good linear relations withexposure to DCP. Cumulative Z- and E-DCP-MA excretion also correlated well with exposure toDCP.7 In line with both findings, thioether andmercapturic acid concentrations in urine samples ofapplicators showed a strong linear relation. Theintercept of the regression equation corresponded tothioether concentrations found in blank urine sam-ples. The slope of 1-38 supported the postulatedpresence of an unidentified thioether metabolite(s).Cumulative thioether excretion also exceededcumulative mercapturic acid excretion. In calculat-ing the first, however, no corrections were made forthe background excretion and in calculating thesecond, excretion to infinity was taken into considera-tion.The question remains at whichDCP exposures the

mercapturic acid and thioether assay can be appliedsuccessfully. The determination of the mercapturicacids of DCP by gas chromatography with differentdetection techniques proved to be much moresensitive and selective than the determination ofthioethers with the thioether assay. Moreover, themercapturic acids of DCP were not detectable in

urine of subjects not exposed to this soil fumigant.Exposure to < 0 09 mg/m3 (eight hour TWA) for 4-9hours, however, resulted in detectable Z-DCP-MAconcentrations in urine of volunteers. By contrast,thioethers are excreted with background concentra-tions depending on lifestyle. The upper limits of the95% CI of the intercepts of the post- minus pre-shiftconcentrations (7-5 mmol SH/mol creatinine) andthe cumulative excretion (88 ,umol SH) indicate theincrements in thioether excretion that are necessaryto distinguish between samples from exposed andnon-exposed subjects. According to the respectiveregression equations 7-5 mmol SH/mol creatininecorresponded to an eight hour TWA exposure of 3-0mg/m3 DCP and 88 imol SH to an eight hour TWAexposure of 1 9 mg/m3 DCP. The thioether assay canbe used to assess comparatively high DCP exposurelevels.An eight hour TWA exposure to the Dutch

occupational exposure limit of 5 mg/m3 DCP wouldresult in a post- minus pre-shift thioether concentra-tion of 9-6 mmol SH/mol creatinine with a 95% CI of7-4 to 11-8 mmol SH/mol creatinine. The sameexposure level would result in an excretion of 139imol SH with a 95% CI of 120 to 157 ,umol SH.These values could regarded as warning limits to beused by occupational hygienists.

This investigation was financially supported by theDirectorate General of Labour of the Ministry ofSocial Affairs and Employment and the Ministry ofHousing, Physical Planning, and the Environment.

Requests for reprints to: N P E Vermeulen, Depart-ment of Pharmacochemistry, Division of MolecularToxicology, Free University, De Boelelaan 1083,1081 HV Amsterdam, The Netherlands.

1 Doom van R, Leijdekkers Ch-M, Bos RP, Brouns RME,Henderson PTh. Detection of human exposure to electro-philic compounds by assay of thioether detoxication productsin urine. Ann Occup Hyg 1981;24:77-92.

2 Kilpikari I. Correlation of urinary thioethers with chemicalexposure in a rubber plant. Br J Ind Med 1981;38:98-100.

3 Ahlborg G, Bergstrom B, Hogstedt C, Einisto P, Sorsa M.Urinary screening for potentially genotoxic exposures in achemical industry. Br J Ind Med 1985;42:691-9.

4 Burgaz S, Bayhan A, Karakaya AE. Thioether excretion ofworkers exposed to bitumen fumes. Int Arch Occup EnvironHealth 1988;60:347-9.

5 Welie van RTH, Duyn van P, Vermeulen NPE. Determinationoftwo mercapturic acid metabolites of 1 ,3-dichloropropene inhuman urine with gas chromatography and sulphur-selectivedetection. J Chrom Biomed Appl 1989;496:463-71.

6 Vermeulen NPE. Analysis of mercapturic acids as a tool inbiotransformation, biomonitoring and toxicological studies.Tips 1989;10:177-81.

7 Welie van RTH, Duyn van P, Brouwer DH, Hemmen van JJ,Brouwer EJ, Vermeulen NPE. Inhalation exposure to 1,3-dichloropropene in the Dutch flower-bulb culture. Part II.Biological monitoring by measurement of urinary excretion oftwo mercapturic acid metabolites. Arch Environ ContamToxicol 1991;20:6-12.

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8 Onkenhout W, Mulder PPJ, Boogaard PJ, Buijs W, VermeulenNPE. Identificationand quantitative determination ofmercap-turic acids formed from Z- and E-1,3-dichloropropene by therat, using gas chromatography with three different detectiontechniques. Arch Toxicol 1986;59:235-41.

9 Brouwer DH, Brouwer EJ, Vreede de JAF, Welie van RTH,Vermeulen NPE, Hemmen van JJ. Inhalation exposure to 1,3-dichloropropene in the Dutch flower-bulb culture. Part I.Environmental monitoring. Arch Environ Contam Toxicol1991;20: 1-5.

10 Brouwer EJ, Evelo CIA, VerplankeAJW, Welie van RTH, Wolffde FA. Biological effect monitoring ofoccupational exposure to1 ,3-dichloropropene: effects on liver and renal function and on

glutathione conjugation. Br J Ind Med 1991;48:167-72.11 Doom van R, Leijdekkers Ch-M, Bos RP, Brouns RME,

Henderson PTh. Enhanced excretion of thioethers in urine ofoperators of chemical waste incinerators. Br J Ind Med1981;38:187-90.

12 Ellman GI. Tissue sulfhydryl groups. Arch Biochem Biophys1959;82:70-7.

13 Tietz NW. Textbook of clinical chemistry. Philadelphia: WBSaunders, 1986:1279.

Accepted 26 November 1990

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international biomedical journals, has agreed toaccept articles prepared in accordance with theVancouver style. The style (described in full in BrMed J, 24 February 1979, p 532) is intended tostandardise requirements for authors.

References should be numbered consecutivelyin the order in which they are first mentioned inthe text by Arabic numerals above the line on eachoccasion the reference is cited (Manson' confirmedother reports". . .). In future references to paperssubmitted to the Br J IndMed should include: the

names of all authors if there are six or less or, ifthere are more, the first three followed by et al; thetitle of journal articles or book chapters; the titlesof journals abbreviated according to the style ofIndexMedicus; and thefirstandfinal pagenumbersof the article or chapter.Examples ofcommon forms of references are:

1 International Steering Committee of Medical Editors.Uniform requirements for manuscripts submitted tobiomedical journals. Br Med J 1979;1:532-5.

2 Soter NA, Wasserman SI, Austen KF. Cold urticaria:release into the circulation of histamine and eosino-philchemotactic factor of anaphylaxis during cold challenge.N Engi JMed 1976;294:687-90.

3 Weinstein L, Swartz MN. Pathogenic properties ofinvadingmicro-organisms. In: Sodeman WA Jr, Sodeman WA,eds. Pathologic physiology: mechanisms ofdisease. Philadel-phia: W B Saunders, 1974:457-72.

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