Enzymes ethanol of 1,1,1 - Occupational and Environmental...

Occupational and Environmental Medicine 1994;51:113-119

Enzymes induced by ethanol differently affect thepharmacokinetics of trichloroethylene and1, 1, 1 -trichloroethane

T Kaneko, P-Y Wang, A Sato

AbstractThis study was undertaken to clarifythe effect of enzymes induced byethanol consumption on the pharma-cokinetics of trichloroethylene (TRI, ahighly metabolised substance) and 1,1,1-trichloroethane (1,1,1-TRI, a poorlymetabolized substance). Rats maintainedon a control liquid diet or a liquid dietcontaining ethanol (2 glday/rat) for notless than three weeks were exposed toeither TRI (50, 100, 500, and 1000 ppm)or 1,1,1-TRI (50, 100, and 500 ppm) byinhalation for six hours and the concen-tration of each compound in the bloodand the urinary excretion of metabolites(trichloroethanol and trichloroaceticacid) were measured over several hours.Ethanol, which increased the in vitrometabolism of both compounds aboutfivefold, enhanced the in vivo metab-olism of TRI only at high levels of expo-sure (marginally at 500 and considerablyat 1000 ppm), whereas the metabolism of1,1,1-TRI was enhanced at all concentra-tions tested. Moreover, there was a defi-nite difference in the effect of inductionofenzymes between the two solvents: theenhanced metabolism of TRI in vivo wasshown by a decrease in the blood concen-tration ofTRI as well as by an increase inthe urinary excretion of its metabolites,whereas that of 1,1,1-TRI was shown byan increase in the urinary excretion of itsmetabolites alone. These results suggestthat the induction of enzymes differen-tially affects the pharmacokinetics ofTRIand 1,1,1-TRI in human occupationalexposure: TRI metabolism may beincreased only at concentrations muchhigher than the current occupationalexposure limit (mostly 50 ppm), whereas1,1,1-TRI metabolism may be increasedat an exposure similar to occupationalexposure.

(Occup Environ Med 1994:51:113-119)

Hepatic metabolism of organic solvents playsa decisive part in determining the disappear-ance rate of most organic solvents from thebody.' Enzymes that regulate the biotrans-formation are mono-oxygenases related tocytochrome P-450 and are most concentratedin the liver.2 The metabolic pathway wherebythe cytochrome P-450 system is -involved isconsidered to be the rate limiting step in the

overall metabolic processes, because itrequires energy to cleave chemical bonds.This enzyme system is susceptible to theinfluence of various environmental factorsincluding dietary and drinking habits, medi-cation, and exposure to environmentalchemicals.3

Ethanol consumption is probably one ofthe most important environmental factorsthat affect the pharmacokinetic behaviour oforganic solvents in humans as ethanol is theonly biologically active substance that largenumbers of industrial workers often consumein multigram quantities. Some workers mayhave consumed large amounts of alcoholicbeverages in the evening and are thenexposed to organic solvent vapours in theworkplace the next day.

Ethanol consumption induces a form ofcytochrome P-450 (P-450IIE1),4 which is oneof the constitutive microsomal enzymes inboth humans5 and other animals.6 It ischaracterized by its high affinity (low-Kmenzyme) for low molecular weight substances,including organic solvents.7

Experimental evidence has shown thatethanol given to rats accelerates the hepaticmetabolism in vitro of a variety of volatileorganic solvents including trichloroethylene(TRI) and 1,1,1-trichloroethane (1,1,1-TRI).8 The metabolism enhancing effect ofethanol has also been shown in in vivo metab-olism studies.910 For example, TRI disap-peared faster in ethanol treated, than incontrol rats when they were exposed to aircontaining TRI at 500 ppm or higher, andthe ethanol treated rats excreted much moretrichloroacetic acid (TCA) and trichloro-ethanol (TCE), the major metabolites ofTRI, than did the control rats.'0 These resultsshould be interpreted with caution, however,as occupational exposure to concentrations ashigh as 500 ppm is unusual.A simulation study with a physiologically

based pharmacokinetic model has shown thatthe effect of induction of enzymes on themetabolism of inhaled chemicals depends onthe chemical as well as on the level of expo-sure."1 12

The rate of 1,1,1-TRI metabolism is about1/40 that of TRI metabolism.8 TRI is cate-gorised as a highly metabolised substratewhereas 1,1,1-TRI is poorly metabolised. 12 Inour study, we further investigated the work-ing hypothesis that enzyme induction byethanol consumption may affect the pharma-cokinetics of 1,1,1-TRI differently from thatof TRI.

Department ofEnvironmentalHealth, MedicalUniversity ofYamanashi, Tamaho,Yamanashi, 409-38JapanT KanekoP-Y WangA SatoRequests for reprints to:Akio Sato, Department ofEnvironmental Health,Medical University ofYamanashi, Tamaho,Yamanashi, 409-38 Japan.Accepted 5 April 1993

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Materials and methodsANIMAL AND DIETSEight week old male Wistar rats (ShizuokaLaboratory Animal Centre, Shizuoka) werekept individually in stainless steel wire bot-tomed metabolism cages in an air condi-tioned room (22(2)0C) with artificial lightingfrom 0600 to 1800. They were maintained onunlimited pellet food (Clea CE-2, NipponClea, Tokyo) and water. At 10 weeks of age,they were switched to two different liquiddiets; one group of rats to a well balancedliquid diet (control diet) and the other groupto a diet containing ethanol (ethanol diet).The control diet was prepared by modifyingthat described by Lieber and DeCarli."3Dextrin and maltose were replaced withsucrose and ethyl linoleate was substituted forsafflower oil. The control diet (1 kcallml, 80ml), which constituted food and water forone day, contained 3-31 g casein, 3d17 g oil(corn oil, olive oil, and ethyl linoleate), 9-22 gsucrose, 40 mg L-cystine, 24 mg DL-methio-nine, 42 mg choline bitartrate, 0-8 g fibre(KC-flock, Sanyo Kokusaku Pulp, Tokyo),240 mg xanthan gum, 7-2 mg vitamin mix-ture, and 570 mg mineral mixture. Theethanol diet (1 kcal/ml) was identical to thecontrol diet except that it contained 2-0 gethanol with a reduction of sucrose content to3 07 g and an increase of oil content to 4-34g/80 ml. Thus the ethanol diet was an ethanolcontaining, low carbohydrate, high fat diet, aprescription that is effective in inducing theethanol inducible cytochrome P-450."4

After three weeks on the liquid diets, ratswere either killed for a metabolism study invitro or exposed to TRI or 1,1,1-TRI byinhalation for kinetic studies in vivo. On theday of the kinetic studies, all the rats exposedto TRI or 1,1,1-TRI were maintained on thecontrol diet. The experiments were per-formed in accordance with the guidelines foranimal experiments, Medical University ofYamanashi.

METABOUSM OF TRI AND 1,1,I-TRI IN VITRORats were killed by decapitation at 1000 andthe liver was removed. A 25% (w/v) liverhomogenate in 1-15% KC1 -0-01 M phos-phate buffer (pH 7.4) was centrifuged at10 000 g for 10 minutes. The supernatant wasfurther centrifuged at 105 000 g for 60 min-utes to obtain a microsomal pellet that wassuspended in the buffer and centrifuged againat 105 000 g for 60 minutes. The washedmicrosomal pellet was resuspended in thesame buffer and the protein content was mea-sured by the method of Lowry et al.'5 Theprotein content was adjusted to a concentra-tion of 10 mg/ml with the buffer, aerated withN2, and stored frozen at - 85°C until use.

Microsomal cytochrome P-450 contentwas determined spectrophotometrically bythe method of Omura and Sato.'6 TRI and1,1,1-TRI metabolism in vitro was assessedby measuring the rate of substrate (TRI or1,1,1-TRI) disappearance by the method ofSato and Nakajima"7 with the slight modifica-tion that the final volume of reaction mixture

was reduced to 0 5 ml consisting of 0 1 ml ofmicrosomal solution (enzyme), 0 3 ml ofcofactor solution, and 0-1 ml of substratesolution. The reaction mixture (0 5 ml) con-tained a final concentration of 1-0 mMNADP, 50 mM MgCl,, 20 mM glucose 6-phosphate (G-6-P), 50 mM K/K phosphatebuffer, and 0-11 mM TRI or 010 mM 1,1,1-TRI in addition to two units of G-6-P dehy-drogenase and microsomes corresponding to1-0 mg protein.

EXPOSURE TO TRI AND 1,1,1-TRI BYINHALATIONBoth control and ethanol treated rats wereexposed to air containing TRI at a concentra-tion of 50, 100, 500, or 1000 ppm, or 1,1,1-TRI at a concentration of 50, 100, or 500ppm for six hours from 1000 to 1600. Theexposure was in a dynamic flow exposurechamber as described previously.'8 At pre-selected intervals after the end of exposure, asmall amount of blood (20 pl) was taken froma cut in the tail, and TRI or 1,1,1-TRI con-centration in the blood was measured bymeans of a gas chromatographic syringe equi-libration method.'9 The operating conditionsof the gas chromatograph were: 2 m x 3 mmglass column packed with PEG-400 onUniport B (Gasukuro Kogyo, Tokyo) at80°C; injection port temperature, 1 00°C;carrier gas, N2 at 70 ml/min; H2 at 20 ml/min.

Samples of urine collected at predeter-mined intervals from the start of exposure for48 hours were each diluted with distilledwater to 100 ml, then centrifuged at 3000rpm for five minutes. The major urinarymetabolites of both TRI and 1,1,1-TRI(TCA and TCE) were analysed in the urineessentially according to the head space gaschromatographic method originally describedby Breimer et al20 as modified by Ohara etal.2' In brief, 0 1 ml of diluted urine wasplaced in a head space vial (Perkin-Elmer;22 ml in volume) containing 0.05 mlmethanol and 0-25 ml concentrated H2SO4.The vial, capped with a Teflon lined stopper,was kept at 85°C to make the methyl ester ofTCA and to hydrolyse TCE-glucuronide.The head space gas was transferred to a gaschromatograph (Hitachi 263-30) equippedwith an electron capture detector (63N), andanalysed for TCA and TCE. This series ofprocedures was performed with an auto-sampler (Perkin-Elmer Headspace SamplerHS 40). The resultant peak areas were mea-sured with an integrator (Hitachi Chromato-Integrator D-2500). Operating conditions ofthe autosampler were: thermostat tempera-ture 85°C; thermostat time 65 minutes;transfer temperature 1 20°C; pressurisationtime 05 minutes; injection time 4-8 seconds.The analytical conditions of the gas chro-matograph were: column 0-32 mm x 30 mTC-WAX capillary (GL Sciences, Tokyo);column temperature 130°C; carrier gas, N2 ata flow rate of 2 ml/min; split ratio, 40:1.

STATISTICSThe results are represented by means (SD).

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Enzymes induced by ethanol differently affect the pharmacokinetics of tnichloroethykne and 1,1,1-trichloroethane

Effects ofethanol on the metabolism in vitro of trichloroethylene (TRI) and1,1,1-trichlorethane (1,1, I-TRI)

Rate of metabolismMicrosomal Cytochrome (nmollmg protein/min)protein P-450

Group (mg/g liver) (nmollmg protein) TRI 1,1,1-TRI

Control 17-2(1-6) 0 69(0 07) 0-82(0-21) 0.04(0.02)Ethanol 20-6(1-9)* 0 88(0 07)** 4-48(0-15)** 0-19(0-04)**

Values represent the mean (SD) for five rats.*p<005; **p < 001 v Control.

Means were tested by the Student-Newman-Keuls' test.22 The 0 05 level of probabilitywas chosen as the criterion of significance.

ResultsTRI AND 1,1,1-TRI METABOLISM IN VITROEthanol consumed in combination with a lowcarbohydrate and high fat diet increased liver

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microsomal protein and cytochrome P-450contents (table). The ethanol treatmentenhanced both TRI and 1,1,1-TRI metabo-lism in vitro about fivefold. The rate ofmetabolism of 1,1,1-TRI was about 1/20 thatof TRI when measured at almost the samesubstrate concentration of 0 1 mM.

TRI AND 1,1,1-TRI KINETICS AFTERINHALATION EXPOSUREWhen rats were exposed to TRI at a concen-tration of 50 or 100 ppm the three weekethanol consumption, which increased thehepatic metabolism of TRI in vitro about fivefold, had no influence on the kinetics of TRI:no significant difference was found betweencontrol and ethanol treated rats either in theTRI concentration in the blood or in theamounts of TCA and TCE excreted into theurine (figs 1 and 2). At an exposure level of

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Figure 1 Trichloroethylene (TRI) concentration in blood(A); cumulative amounts of trichloroacetic acid (TCA)(B); and trichloroethanol (TCE) (C) excreted into urineafter exposure to 50ppm of TRI. Vertical lines depict SD(n = 5).

2-0

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Figure 2 Trichloroethylene (TRI) concentration in blood(A); cumulative amounts of trichloroacetic acid (TCA)(B); and trchloroethanol (TCE) (C) excreted into urineafter exposure to 100 ppm of TRL Vertical lines depict SD(n = 5).

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Kaneko, Wang, Sato

Control

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Figure 3 Trichloroethylene (TRI) concentration in blood.(A); cumulative amounts of trichloroacetic acid (TCA)(B); and trichloroethanol (TCE) (C) excreted into urineafter exposure to 500 ppm of TRI. Vertical lines depict S)(n = 5).

500 ppm the ethanol induced enhancementof TRI metabolism in vitro made its appear-ance in the TRI metabolism in vivo but theeffect was only marginal with an increase ofurinary excretion of TCA alone (fig 3).Ethanol consumption had no effect on eitherthe TRI concentration in the blood or theamount of TCE excreted in the urine at thislevel of exposure. When the exposure was

raised to 1000 ppm, however, ethanol con-sumption definitely enhanced the TRI metab-olism in vivo as shown by a significantdecrease in the blood TRI concentration aswell as by a significant increase of both TCAand TCE excretion in the urine of the ethanoltreated rats (fig 4). These findings suggestthat enzyme induction significantly affects thepharmacokinetics of TRI only when the expo-sure is 500 ppm or higher.

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Figure 4 Trichloroethylene (TRI) concentration in blood(A); cumulative amounts of trichloroacetic acid (TCA)(B); and trichlorethanol (TCE) (C) excreted into urineafter exposure to 1000 ppm of TRI. Vertical lines depictSD (n = 5).

By contrast, in the case of 1,1,1-TRI,ethanol treated rats excreted more TCA andTCE in the urine than did the control rats atany exposure level studied (figs 5-7). Theinduction of enzymes however was notreflected in the concentration of 1,1,1-TRI inthe blood at any level of exposure. Thesefindings indicate that the effect of inductionof enzymes on the pharmacokinetics of 1,1,1 -TRI sharply contrasts with that of TRI.

DiscussionA three week ethanol consumption enhancedthe hepatic metabolism of TRI and 1,1,1-TRI in vitro about five fold (table), almost tothe same extent as that previously reported.8The metabolism study in vitro also indicatesthat 1,1,1-TRI is poorly metabolised by the

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Enzymes induced by ethanol differently affect the pharmacokinetics of trichIoroethylene and 1,1,1-trichloroethane

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Figure 5 1,1,1-Trichloroethane (1,1,1-TRI)concentration in blood (A); cumulative amounts oftrichloroacetic acid (TCA) (B); and trichloroethanol(TCE) (C) excreted into urine after exposure to 50ppm of1,1,1-TRI. Vertical lines depict SD (n = 5).

hepatic microsomal enzymes, by contrastwith TR, which is a highly metabolised sub-strate.12Enzymes induced by ethanol consumption

had an impact on the metabolism of TRI invivo only at high exposure (500 ppm orhigher; (figs 1-4), a finding that is in goodagreement with previous experimental evi-dence from m-xylene, another highlymetabolised substrate.2' These findingstogether support the working hypothesis thatthe effect of induction of enzymes on themetabolism of some inhaled chemicalsdepends on the level of exposure and that theinduction of enzymes or the enhancement ofmetabolism found in vitro is reflected in vivoonly at a high level of exposure.By contrast, enzymes induced by ethanol

considerably affected the metabolism of1,1, 1-TRI in vivo at any exposure level from

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Figure 6 1,1,1-Trichloroethane (1,1,1-TRI)concentration in blood (A); cumulative amounts oftrichloroacetic acid (TCA) (B); and trichloroethanol(TCE) (C) excreted into urine after exposure to 100 ppmof 1,1,I-TRI. Vertical lines depict SD (n = 5).

50 to 500 ppm, although it had no influenceon the blood concentration of the parentcompound (figs 5-7). The reason why theenhancement of metabolism was not reflectedin the blood 1,1,1-TRI concentration is thatthe clearance of 1,1,1-TRI by metabolismnormally accounts for only a few per cent ofthe total elimination from the body, contrast-ing with that of TRI, which accounts for75%.'

Clearance of foreign chemicals by hepaticmetabolism depends on the relation betweenthe intrinsic metabolic clearance (VmaxfKm)and the hepatic blood flow (QH), and themetabolic clearance of foreign chemicals isoften rate limited by QH independently ofenzyme capacity (Vmax) when the exposureconcentration is low (perfusion limitedmetabolism).24 Compounds with Vmax/Kmlarger than QH are highly metabolised

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Figure 7 1,1,1-Trchloroethane (1,1,1-TRI) concentration inblood (A); cumulativeamounts of trichloroaceticacid (TCA) (B); andtrichloroethanol (TCE)(C) excreted into urineafter exposure to SOOpppmof 1,l,1 -TRI. Verticallines depict SD (n = 5).

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substrates.'2 These compounds are almostcompletely metabolised while passing throughthe liver as long as the concentration in thehepatic tissue (CH) is low. Thus the bloodflow (QH) limits the extent of their metabo-lism and enzyme induction (Vmax isincreased) has little or no effect on themetabolism, provided that the exposure con-centration is low.

When the concentration in the inhaled airis raised so that the metabolic clearanceexpressed in terms of Vmax/(Km+ CH)becomes smaller than QH, the Vmax becomesa factor limiting the metabolism. In otherwords, the enzyme induction found in an invitro metabolism study would be fullyreflected in the in vivo metabolism only athigh exposure concentrations where themetabolism is solely dependent on the capac-ity of enzymes.On the other hand, for those solvents

whose Vmax/Km is smaller than QH (poorlymetabolised substrates) the enzyme activitylimits the metabolism independently of the

level of exposure (capacity limited metabo-lism), and induction of enzymes can increasetheir metabolism in vivo even at a low level ofexposure.'2

Although the Vmax/Km values are notavailable for TRI and 1,1, 1-TRI metabolism,our study indicates that the Vmax/Km of TRIis larger than QH and thus the perfusion lim-ited metabolism prevails when the exposureremains below 100 ppm, whereas theVmax/Km of 1,1,1-TRI is smaller than QHand thus the capacity limited metabolism is anormal occurrence independently of level ofexposure.

Experimental evidence has been publishedindicating that induction of enzymes does notalways produce a significant effect on themetabolism of inhaled chemicals in a livingbody. Phenobarbitone is another potentinducer of hepatic cytochrome P-450.Treatment of rats with phenobarbitone accel-erated in vivo metabolism of a variety oforganic solvents such as m-xylene,2' ben-zene,26 27 toluene,26 and n-hexane,28 but onlyat high doses of these solvents.

Phenobarbitone inducible cytochrome P-450 isozymes (primarily, P-45011IB1) havehigh Km values for organic solvents, by con-trast with ethanol inducible P-450IIE1, whichis a low Km enzyme for these compounds.629Our present findings together with the otherreports mentioned earlier indicate thatenzymes induced by ethanol (a typicalinducer of low Km isozymes) or phenobarbi-tone (a typical inducer of high Km isozymes)will significantly affect the pharmacokineticsof highly metabolised organic solvents onlywhen the exposure concentration is high.

In conclusion, enzyme induction byethanol consumption may differently affectthe pharmacokinetics of TRI and 1,1,1-TRIin human occupational exposure: TRI metab-olism may be increased only at an exposuremuch higher than the current occupationalexposure limit (mostly 50 ppm), whereas1,1,1-TRI metabolism may be increased at anexposure similar to occupational exposure.

1 Sato A, Nakajima T. Pharmacokinetics of organic solventvapors in relation to their toxicity. ScandJ Work EnvironHealth 1987;13:81-93.

2 Lu AYH, West SB. Multiplicity of mammalian micro-somal cytochrome P-450. Pharmacol Rev 1980;31:277-95.

3 Sato A. The effect of environmental factors on the phar-macokinetic behaviour of organic solvent vapours. AnnOccup Hyg 1991;35:525-41.

4 Koop DR, Coon MJ. Ethanol oxidation and toxicity: roleof alcohol P-450 oxygenase. Alcohol Clin Exp Res 1986;10:44-9.

5 Wrighton SA, Thomas PE, Molowa DT, et al.Characterization of ethanol-inducible human liver N-nitrosodimethylamine demethylase. Biochemistry 1986;25:6731-5.

6 Nakajima T, Elovaara E, Park SS, Gelboin HV, HietanenE, Vainio H. Immunochemical characterization ofcytochrome P.450 isozymes responsible for benzeneoxidation in the rat liver. Carcinogenesis 1989;10:1713-7.

7 Sato A, Nakajima T. Enhanced metabolism of volatilehydrocarbons in rat liver following food deprivation,restricted carbohydrate intake and administration ofethanol, phenobarbital, polychlorinated biphenyl and 3-methylcholanthrene: a comparative study. Xenobiotca1985;15:67-75.

8 Sato A, Nakajima T, Koyama Y. Effects of chronicethanol consumption on hepatic metabolism of aromaticand chlorinated hydrocarbons in rats. Br j Ind Med1980;37:382-6.

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Enzymes induced by ethanol differently affect the pharmacokinetics of trichloroethylene and 1,1,1-trichloroethane

9 Sato A, Nakajima T, Koyama Y. Dose-related effects of asingle dose of ethanol on the metabolism in rat liver ofsome aromatic and chlorinated hydrocarbons. ToxicolAppl Pharmacol 1981;60:8-15.

10 Nakajima T, Okino T, Okuyama S, Kaneko T, YonekuraI, Sato A. Ethanol-induced enhancement oftrichloroethylene metabolism and hepatotoxicity: differ-ence from the effect of phenobarbital. Toxicol ApplPharmacol 1988;94:227-37.

11 Sato A, Endoh K, Kaneko T, Johanson G. Effects of con-sumption of ethanol on the biological monitoring ofexposure to organic solvent vapours: a simulation studywith trichloroethylene. BrJ Ind Med 1991;48:548-56.

12 Sato A. Confounding factors in biological monitoring ofexposure to organic solvents. Int Arch Occup EnvironHealth 1993;65:561-7.

13 Lieber CS, DeCarli LM. 1986 Update: the feeding ofethanol in liquid diets. Alcohol Clin Exp Res 1986;10:550-3.

14 Sato A, Nakajima T, Koyama Y. Interaction betweenethanol and carbohydrate on the metabolism in rat liverof aromatic and chlorinated hydrocarbons. Toxicol ApplPharmacol 1983;68:242-9.

15 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Proteinmeasurement with the Folin phenol reagent. BiolChem 1951;193:265-75.

16 Omura T, Sato R. The carbon monoxide-binding pigmentof liver microsomes. 1. Evidence for its hemoproteinnature. Biol Chem 1964;239:2370-8.

17 Sato A, Nakajima T. A vial-equilibration method to evalu-ate the drug-metabolizing enzyme activity for volatilehydrocarbons. Toxicol Appl Pharmacol 1979;47:41-6.

18 Sato A, Nakajima T, Fujiwara Y, Murayama N. Kineticstudies on sex difference in susceptibility to chronicbenzene intoxication-with special reference to body fatcontent. BrJ Ind Med 1975;32:321-8.

19 Sato A, Nakajima T, Fujiwara Y. Determination ofbenzene and toluene in blood by means of a syringe-

equilibration method using a small amount of blood.BrJ Ind Med 1975;32:210-4.

20 Breimer DD, Ketelaars HCJ, Van Rossum JM. Gas chro-matographic determination of choral hydrate,trichloroethanol and trichloroacetic acid in blood andurine employing head-space analysis. Chromatogr1974;88:55-63.

21 Ohara A, Michitsuji H, Jyoyama Y, Yamaguchi K, FujikiY. Determination of trichloroacetic acid andtrichloroethanol by head-space gas chromatography.Japanese Journal ofIndustrial Health 1991;33:94-103.

22 Steel RGD, Torrie JH. Principles and procedures forstatistics. New York: McGraw-Hill, 1960:110-1.

23 Kaneko T, Wang P-Y, Sato A. Enzyme induction byethanol consumption affects the pharmacokinetics ofinhaled m-xylene only at high levels of exposure. ArchToxicol 1993 (in press).

24 Hoyumpa AM Jr, Schenker S. Major drug interactions:effect of liver disease, alcohol and malnutrition. Ann RevMed 1982;33:113-49.

25 David A, Flek J, Frantik E, Gut I, Sedivec V. Influence ofphenobarbital on xylene metabolism in man and rats.Int Arch Occup Environ Health 1979;44:117-25.

26 Ikeda M, Ohtsuji H. Phenobarbital-induced protectionagainst toxicity of toluene and benzene in the rat.ToxicolApplPharmacol 1971;20:30-43.

27 Gut I, Hitle K, Zizkova L. Effect of phenobarbital pre-treatment on benzene biotransformation in the rat. II.9,000 g supernatant and isolated perfused liver versusliving rat. Arch Toxicol 1981;47:13-24.

28 Perbellini L, Leone R, Fracasso ME, Brugnone F,Venturini MS. Metabolic interaction between n-hexaneand toluene in vivo and in vitro. Int Arch Occup EnvironHealth 1982;50:351-8.

29 Nakajima T, Wang RS, Murayama N, Sato A. Threeforms of trichloroethylene-metabolizing enzymes in ratliver induced by ethanol, phenobarbital, and 3-methyl-cholanthrene. ToxicolAppl Pharmnacol 1990;102:546-52.

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