UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)

UvA-DARE (Digital Academic Repository)

Individual variation in biotransformation: relation to styrene kinetics and solvent-induced neur

Wenker, M.A.M.

Link to publication

Citation for published version (APA):Wenker, M. A. M. (2001). Individual variation in biotransformation: relation to styrene kinetics and solvent-induced neur.

General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.

Download date: 02 Jan 2020

Chapterr 10

Summarisingg discussion

Interindividuall variability in the disposition and toxicity of xenobiotics has gained more and moree interest in recent years. It has been identified as one of the most important factors determiningg the efficacy of drugs, adverse effects of xenobiotics, and disease susceptibility [1,2].. Thus, interindividual variability is likely to play a major role in the risk assessment and biologicall monitoring of exposure to chemicals. Polymorphisms in biotransformation enzymess seem to be an important factor in determining the interindividual variability in kineticss and toxicity. While research in the past two decades has focused mainly on the efficacyy of drugs and on susceptibility to environmentally-induced cancer, littl e is known aboutt the influence of differences in metabolic capacity on the kinetics and neurotoxicity of organicc solvents.

Thiss thesis focused on the relation between interindividual differences in metabolic capacityy and the toxicokinetics of styrene, as well as the relation between genetic polymorphismm in biotransformation enzymes and susceptibility to solvent-induced neurotoxicity.. The main study objectives were: (1) interindividual variability in styrene metabolismm in vitro and the relationship with genetic polymorphism of the two involved enzymes,, (2) the relation between individual metabolic capacity, assessed by phenotyping and genotyping,, and interindividual differences in the toxicokinetics of styrene in vivo, and (3) the relationn between the genetic polymorphism of several biotransformation enzymes and the risk off chronic toxic encephalopathy.

Inn addition, in the in vitro and in vivo studies, stereochemical aspects of styrene metabolismm were investigated.

Gass chromatographic methods for SO and MA enantiomers (chapters 2 and 5) Inn order to measure SO and MA enantiomers, enantiospecific and sensitive gas-chromatographicc (GC) methods needed to be developed. In chapter 2, a GC method for the analysiss of styrene oxide (SO) enantiomers is described. The method was based on base-catalysedd hydrolysis with sodium methoxide. In this way, opening of the epoxide ring was achievedd without racemisation, in contrast to other methods which employed acid-catalysed hydrolysis.. The resulting methoxy alcohols were derivatised to enable sensitive determination byy electron capture detection (ECD). Derivatization proceeded also without racemisation and aa good baseline separation was achieved by serial coupling of two columns, a chiral CP

157 7

ChapterChapter 10

Chirasil-Dex-CBB and a non-chiral AT 1705. With this method, the rate and enantioselectivity off the CYP450-dependent oxidation of styrene to SO enantiomers in human liver microsomes couldd be determined.

Inn chapter 5, a GC method for the analysis of mandelic acid (MA) enantiomers in urinee is described. In contrast to generally used methods [3-5], esterification of MA with isopropanoll was performed directly in urine, without previous solvent extraction. No racemisationn of the enantiomers occurred during esterification, and a good baseline separation off the isopropyl esters of the MA enantiomers was obtained using a Chirasil-Dex column. To achievee more sensitive ECD determination, subsequent acylation of the isopropyl esters of MAA was performed. With this GC method, a 24-h follow-up of the excretion of MA enantiomerss in urine of volunteers exposed to 104 and 360 mg/m3 was possible.

Interindividuall variability in styrene biotransformation in vitro (chapters 3 and 4) Inn chapters 3 and 4 the in vitro biotransformation of styrene in the livers of twenty Caucasian maless is described. The observed interindividual variability in the enzyme kinetic parameters wass correlated to the genetic polymorphism of the enzymes involved in styrene oxidation and styrenee oxide hydrolysis.

Thee oxidation of styrene was shown to be mediated by CYP2E1 (chapter 3), although att higher styrene concentrations other CYP450s also contributed, however, with much lower catalyticc efficiency. The Vmax and Km of CYP2E1-mediated styrene oxidation varied 8- and 6-fold,, respectively, between the livers. The variability in Vmax correlated well with the variationn in CYP2E1 activity, as measured by p-nitrophenol hydroxylation. However, the CYP2E11 polymorphisms did not seem to be responsible for the observed variability. This couldd indicate that the polymorphisms do not change the catalytic activity of the CYP2E1 enzyme,, although the number of livers studied and the low frequency of the mutations in Caucasianss preclude any definite conclusions.

Thee hydrolysis of styrene oxide to styrene glycol, a detoxification step, proceeded at a highh rate in vitro (chapter 4). The measured intrinsic clearance (CLi, Vmax/Km) was much higherr than the CLi for the CYP450 step, implying that the CYP450 step is the rate-limiting stepp in the metabolism of styrene. Mendrala et al. [6] showed that humans had the highest capacityy to hydrolyse SO and the lowest capacity to form SO, compared to laboratory animals,, indicating that humans are less susceptible to styrene oxide toxicity. The variability observedd in mEH-mediated SO hydrolysis in the present study was 5-fold for Vmax and 3- to 4-foldd for Km. No relation with the polymorphisms of the mEH gene was found, possibly due too the low number of livers studied.

Fromm these studies, the conclusion was drawn that a potential for large interindividual variationn in styrene metabolism in humans exists. As the rate-limiting step in the biotransformationn of styrene seems to be its oxidation, variation in CYP4502E1 could be of cruciall importance in susceptibility to styrene.

158 8

SummarisingSummarising discussion

Interindividuall variability in styrene biotransformation in vivo (chapter 7 and 8) Inn chapter 7, the interindividual variability in the toxicokinetics of styrene was studied in vivovivo in relation to individual metabolic capacity. Twenty healthy male volunteers were exposedd to 104 and 360 mg/m3 styrene for 1 h and 50 W physical exercise. Individual metabolicc capacity was assessed with the probe substrates chlorzoxazone (CYP2E1), caffeine (CYP1A2),, dextromethorphan (CYP2D6) and antipyrine (CYP450), and by genotyping for CYP2E11 polymorphism. Although the variation in the kinetics of styrene was quite large for a relativelyy homogeneous group, no correlation with either one of the model substrates, nor withh the genotyping was observed. This is thought to be due to perfusion-limited kinetics of styrene.. From the fact that apparent clearance of styrene in vivo (84 1/h) almost equals the bloodd flow (96 1/h) and that the intrinsic metabolic clearance (580 1/h), calculated from in vitrovitro data, is much larger than the liver blood flow, the conclusion is drawn that styrene is completelyy metabolised while passing through the liver and that the blood flow limits its extentt of metabolism. This implies that induction of CYP2E1 by chronic ingestion of ethanol, previouss exposure to other solvents, medication, dietary habits or genetic polymorphism has littl ee or no effect on the clearance of styrene and formation of styrene oxide, as long as the exposuree concentration is low. When exposure to styrene increases, reaching internal concentrationss that saturate the biotransformation enzymes, the capacity of the enzymes becomess the rate-limiting factor instead of hepatic blood flow. Then, interindividual variationss in Vmax will become apparent. In this light it is interesting to note that peak exposuress have been implicated as an etiological factor in the development of chronic toxic encephalopathyy [7]. This means that an increased activity of CYP2E1 could have an impact onn the risk of neurotoxicity in the case of peak exposures. However, the increased enzyme activityy will be detrimental if styrene oxide is responsible for the neurotoxic effects, but beneficiall when styrene itself is the toxic factor.

Att low exposure concentrations, inhibition of CYP2E1 will have a more pronounced effectt on the clearance of styrene than enzyme induction, because it will decrease the intrinsic clearance,, thereby changing metabolism from flow-limited to capacity-limited. Co-exposure too ethanol, other solvents or drugs may inhibit styrene metabolism, although the dose of both styrenee and the co-substance determines the extent of interaction. In addition, genetic polymorphismss which significantly reduce or even delete enzyme activity will affect styrene metabolism,, if involved in styrene biotransformation. This has been shown for methyl chloride,, where subjects with the GSTT1 null genotype have a lower uptake, lower metabolic clearance,, higher concentrations in the blood and a slower post-exposure decay compared to subjectss with the normal genotype [8].

Otherr factors can also contribute to the absence of a relation between the model substratess and the kinetic parameters of styrene. Our study was hampered by the fact that SO inn blood could not be measured. Therefore, indirect parameters were taken, which were consideredd to reflect SO formation. Also factors like intraindividual variation in the metabolismm of the model substrates and the kinetics of styrene (see below) and the uncertainty

159 9

ChapterChapter 10

off the enzyme specificity of the model substrates could have contributed to the negative result. .

Noo correlation was observed between mEH genotype and styrene kinetics (styrene glycoll in blood or urinary excretion of metabolites), this not being mentioned in chapter 7. However,, the influence of mEH on styrene kinetics is difficult to assess in vivo. Styrene glycoll concentrations in blood are dependent on the hydrolysis of styrene oxide to styrene glycol,, but also on the further metabolism of styrene glycol to mandelic acid and conjugated glycol.. Since we were unable to measure SO concentrations and since we did not analyse MA concentrationss in blood, it is impossible to assess which step determines the SG concentrationss in blood. In addition, mEH-mediated hydrolysis is probably not a rate-limiting stepp in the biotransformation of styrene, therefore, only the absence or a very low enzyme activityy of mEH would have an impact on styrene kinetics.

Inn light of the mechanism of styrene toxicity, i.e. styrene oxide is responsible for the genotoxicityy and both styrene and styrene oxide are implicated in the neurotoxicity, we have chosenn styrene concentration in blood and the 3-h and 24-h cumulative excretion of MA and PGAA as surrogate target doses, in chapter 7. SO concentrations in blood could not be measured.. However, Mutti et al. [9,10] implicate PGA as the metabolite responsible for the neurotoxicityy of styrene, although much controversy exists regarding this research. They observedd a marked depletion of striatal and tubero-infundibular dopamine after dosing rabbits withh styrene and mandelic and phenylglyoxylie acid, and, in addition, found that PGA condensess non-enzymatically with dopamine in vitro. If PGA is a neurotoxic metabolite, then interindividuall variation in the biotransformation of MA to PGA, which might be capacity-limited,, is very important with respect to susceptibility to styrene-induced neurotoxicity. In thiss light it is interesting to mention that (although not reported in chapter 7) metabolism of antipyrine,, caffeine and dextromethorphan at 104 mg/m3, and metabolism of antipyrine and chlorzoxazonee at 360 mg/m3 were significantly correlated to the formation of PGA. It is evidentt that much more needs to be known about the mechanism by which styrene induces toxicity,, in order to identify biomarkers of susceptibility.

Ass stated in the introduction, several studies did find a relationship between genetic polymorphismm and solvent metabolism. This can be explained in several ways; (1) capacity-limitedd metabolism of the solvents studied (1,1,1-trichloroethane and perchloroethylene in the studyy of Berode et al [11]), (2) ethnic variation, as in the study of Kawamoto et al. [12], wheree an association was found between ALDH2 activity and toluene metabolism in Japanese;; about 50% of the Orientals lack ALDH2 activity, but this polymorphism does not existt in Caucasians, and (3) type of gene polymorphism studied; with null-genotypes or gene mutationss that lead to absence of enzyme activity, the effect on metabolism will be much moree pronounced [8,13].

Despitee the fact that the variability in styrene toxicokinetics in vivo in volunteers was nott related to oxidative biotransformation capacity in our study, still a relatively large interindividuall variation was observed. We currently have no conclusive explanation for the observedd variability, but the kinetics of solvents in general depends on many physiological

160 0

SummarisingSummarising discussion

factors,, like body build (body size and body fat), physical activity, tissue blood flow and renal clearancee [14,15]. The variability in styrene concentrations in blood observed in our study couldd perhaps be attributed to physiological variability in liver blood flow. Explaining the variabilityy in urinary excretion presents a bigger challenge, as the //ifraindividual variability in excretionn was also large (calculated by a mixed effects model). Interindividual and day-to-day variationn in renal clearance, or changes in behaviour of the volunteers could perhaps explain thee intra- and interindividual variability, associated with urinary metabolite excretion. This inter-- and intra-variability renders MA and PGA excretion less suitable as biomarkers for individualindividual styrene exposure.

Inn order to integrate the in vitro and in vivo data on styrene kinetics and variability, pharmacokineticc modelling was applied (chapter 8). For styrene, an existing physiologically-basedd pharmacokinetic (PBPK) model by Ramsey and Andersen was used [19]. In order to describee metabolite kinetics that model was extended with one-compartment models. The in vitrovitro determined enzyme kinetic parameters of CYP450 and mEH were scaled to the in vivo situationn and incorporated in the model. For CYP450, these parameters were in good agreementt with the parameters used by Ramsey and Andersen and described the styrene kineticss well, but the Vmax of mEH had to be divided by four in order to obtain a good fit of thee experimental data. The variability in Vmax of styrene oxidation observed in vitro was incorporatedd into the model, and styrene concentrations in blood and excretion of MA and PGAA in urine were predicted. As expected, the interindividual variation observed in vitro did nott result in large changes in styrene or MA and PGA concentrations in vivo, thereby confirmingg the theory of flow-limited metabolism.

Inn conclusion, (genetic) polymorphism in styrene oxidation or styrene oxide hydrolysiss does not seem to influence the kinetics of styrene and, therefore, is unlikely to modifyy susceptibility to styrene-induced toxicity, or compromise biological monitoring. Physiologicall factors and genetic and environmental factors that interfere further down in the metabolicc pathway apparently determine the interindividual variability in styrene kinetics. However,, interindividual variability in styrene biotransformation keeps attracting considerablee attention, as witnessed from ongoing studies in this field [16,17].

Thee oxidative metabolism of many commonly used solvents, e.g. toluene, xylene, trichloroethylene,, methylene chloride, is perfusion-limited [18], thus interindividual variation inn biotransformation will probably play a minor role in the overall variability observed in the dispositionn of, and susceptibility to, these chemicals.

Stereochemistryy of styrene metabolism in vitro and in vivo (chapters 3,4,6 and 8) Inn the in vitro studies (chapters 3 and 4) the enantioselectivity of the styrene biotransformationn reactions was described. Enantioselectivity is an important aspect, because enantiomerss can differ in their interaction with enzymes, receptors, DNA and proteins, and thereforee kinetics and toxicity can differ appreciably between enantiomers. This is the case withh styrene oxide, with the (R)-enantiomer being four times as mutagenic to Salmonella

161 1

ChapterChapter 10

typhimuriumtyphimurium TA 100 as the (S)-enantiomer. In chapter 3, we have shown that (S)-SO is preferentiallyy formed, with a S/R ratio of 1.3, but that this enantioselectivity can change towardss a preference for the (R)-enantiomer at high concentrations of styrene, probably becausee of involvement of different CYP450 enzymes. In chapter 4, the hydrolysis of SO wass shown to be enantiospecific as well as enantioselective, with Vmax and Km for (S)-SO beingg five times higher than for (R)-SO. This means that the intrinsic clearance (V^/Km) of (R)-- and (S)-SO hydrolysis is similar, when incubated separately at non-saturating concentrations.. However, (R)-SO inhibited the hydrolysis of (S)-SO when incubated together inn a racemic mixture. The stereochemical metabolism of styrene was also measured in vivo in thee volunteer study (chapter 6). Concentrations of SO could not be measured. The maximal concentrationn and area under the curve (AUC) of unbound (R)-SG in blood proved to be higherr than the concentration of (S)-SG, indicating either the inhibitory effect of (R)-SO on (S)-SOO hydrolysis, or enantioselective metabolism of SG to MA. The shorter half-life found forr (S)-SG compared to (R)-SG points towards the latter explanation. For MA, an excess of thee (S)-enantiomer was observed in cumulative 24-h urine (S/R ratio of 1.6). In theory, the cumulativee excretion of MA represents the ratio of SO enantiomers formed in the liver. However,, the S/R ratio of MA enantiomers in the volunteers was 1.6, approaching the 1.3 foundd in vitro. This could mean that either the ratio of SO enantiomers formed in vivo is 1.6 insteadd of 1.3, or that enantioselective metabolism of MA to PGA occurs. Either way, the data showw that the less toxic (S)-SO is the preferred enantiomer formed in vivo. However, as long ass the enantioselectivity of several steps in styrene metabolism remains unknown, measurementt of the enantiomeric ratio of metabolites like MA cannot be used to predict the enantiomericc ratio of styrene oxide.

Thee model in chapter 8 was also used to describe stereochemical metabolism. The S/RR ratio of 1.3 found in vitro for the SO enantiomers and the enantioselective parameters of thee SO hydrolysis (without inhibition because, amongst others, the inhibition constant was unknown)unknown) were incorporated. With these assumptions, the model indeed predicted enantioselectivee metabolism of SG to MA, with (S)-SG being preferentially metabolised. Withoutt assuming enantioselective oxidation of MA to PGA, the S/R ratio of MA enantiomerss observed with the model was indeed the 1.3 of the SO enantiomers.

Thee enantioselective variation in vivo is somewhat smaller than the variation in total metabolitee excretion, described in chapter 7. Therefore, we conclude that the 'additional' susceptibilityy to styrene-induced toxicity associated with its stereochemical metabolism is relativelyy small.

Geneticc polymorphism and risk of chronic toxic encephalopathy (chapter 9) AA possible relationship between differences in metabolic capacity and solvent-induced neurotoxicityy was investigated by genotyping CTE patients and an exposed control group for severall polymorphic biotransformation enzymes (chapter 9). A higher risk of CTE was demonstratedd in persons with the CYP2E1 mutated alleles. The mechanism behind this increasedd risk is unclear. CYP2E1 is involved in the metabolism of many solvents and the

162 2

SummarisingSummarising discussion

CYP2E1*5BCYP2E1*5B mutated allele has been shown to increase transcriptional activity and mRNA expressionn and increase the elimination rate of ethanol and paracetamol [20-22]. Thus, the polymorphismm could increase the biotransformation rate of solvents to reactive intermediates. However,, our conclusions from chapter 7 and 8 are contradictory to this theory; with perfusion-limitedd kinetics an increase in the activity of biotransformation enzymes has no effectt on metabolism. One possible explanation for the observed association is that the exposuree of the patients might have consisted mainly of capacity-limited solvents. The majorityy of the patients and the controls were painters; exposure in painters consists mainly of mixturess and the assessment of individual solvents is difficult. Therefore, we can neither confirmm nor deny this explanation. Another possible explanation is that the CYP2E1 polymorphismss might be linked to other mutations that affect CTE risk. Yet another possibilityy is that the increased risk might be due to an increased amount of reactive metabolitess formed directly at the target site, the brain [23]. The presence of CYP450 enzymess has been demonstrated in the brain, although in much smaller quantities than in liver (makingg brain metabolism probably capacity-limited) [24-26]. Metabolism of parathion [27], n-hexanee [28], imipramine [29] and m-dinitrobenzene [30] has been demonstrated in the brain inin vitro, using brain slices or brain microsomes. In vitro and in vivo studies have shown metabolismm of toluene and, consequently, generation of reactive oxygen species in the brain [31,32].. Indirect evidence has been obtained for metabolism of styrene in the brain; after i.p. injectionn of radioactive styrene in rats, the liposoluble fraction of radioactivity in the brain decreasedd at a faster rate than the water soluble fraction [33]. This may indicate metabolism off styrene in the brain, because with passive redistribution both fractions would decrease at thee same rate.

Beforee the CYP2E1 alleles can be used as biomarkers of susceptibility to CTE, the resultss should be confirmed in other studies, and the rational behind the association should be elucidated.. However, because of ethical, social and legal issues, care should be taken in implementingg genetic screening as a tool in occupational health practice [34,35].

Furtherr research Organicc solvents are still extensively used in the industrialised world, and much concern has beenn raised about the interindividual variation in the disposition of solvents and their metabolitess and the possibility of individual susceptibility to solvent-induced toxicity.

Variabilityy in styrene toxicokinetics was observed in this study, although this variation seemedd not to be related to individual biotransformation capacity. The focus in studying interindividuall variability in styrene, or perhaps perfusion-limited solvents in general, should shiftt from metabolism to physiological factors affecting absorption, distribution and excretion.. More knowledge should be gathered about the interindividual variation in these physiologicall factors. In order to improve biological monitoring of styrene, sources of the largee intra- and interindividual variation should be determined. The metabolism of SG to MA andd PGA should be studied in more detail. This is of particular importance if PGA is a metabolitee responsible for the neurotoxic effects of styrene. Studies with genetic

163 3

ChapterChapter 10

polymorphismss of biotransformation enzymes can best be performed by studying either capacity-limitedd solvents (although metabolic clearance normally accounts for only a few percentt of total elimination with these solvents), or studying solvents metabolised by enzymes showingg 'null' polymorphisms. In vitro studies and PBPK modelling can be of assistance in determiningg whether a solvent exhibits capacity-limited or perfusion-limited kinetics.

Researchh on the mechanism by which solvents cause (neuro)toxicity has been limited soo far in toxicology. This is, however, of vital importance as it provides information on whetherr the parent compound or the metabolites (and which metabolite) are responsible for thee toxicity. This information can be used in studies investigating susceptibility, in refining PBPKK models and in the risk assessment of the solvent. One of the mechanistic points to be studied,, is to determine whether brain metabolism plays an important role in the toxicity of organicc solvents and whether interindividual variability in brain metabolism is an important aspectt in susceptibility.

Inn the study of susceptibility to the toxic effects of chemicals, interindividual variation inn toxicokinetics is usually highlighted. However, as the toxicity of any chemical is determinedd by both toxicokinetics and toxicodynamics, the importance of interindividual variationn in the toxicodynamics of organic solvents should be emphasised.

164 4

SummarisingSummarising discussion

References s (1)) Hattis D and Silver K. Human interindividual variability - A major source of

uncertaintyy in assessing risks for non-cancer health effects. Risk Anal 1994; 14:421-

431. .

(2)) Renwick AG. Variability in toxic response - Human and environmental. Environ

ToxicolToxicol Pharmacol 1996; 2:75-242.

(3)) Kivistö H, Pekari K, and Aiti o A. Analysis and stability of phenylglyoxylic and mandelicc acids in the urine of styrene-exposed people. Int Arch Occup Environ Health 1993;64:399-403. .

(4)) Dill s RL, Wu RL, Checkoway H, and Kalman DA. Capillary gas chromatographic methodd for mandelic and phenylglyoxylic acids in urine. Int Arch Occup Environ HealthHealth 1991;62:603-606.

(5)) Kom M, Wodarz R, Schoknecht W, Weichardt H, and Bayer E. Styrene metabolism in man:: Gas chromatographic separation of mandelic acid enantiomers in the urine of exposedd persons. Arch Toxicol 1984; 55:59-63.

(6)) Mendrala AL, Langvardt PW, Nitschke KD, Quast JF, and Nolan RJ. In vitro kinetics off styrene and styrene oxide metabolism in rat, mouse, and human. Arch Toxicol 1993; 67:18-27. .

(7)) Health Council of the Netherlands. Peak exposures to organic solvents. The Hague: Healthh Council of the Netherlands, 1999; publication no. 1999/12.

(8)) Löf A, Johanson G, Rannug A, and Warholm M. Glutathione transferase Tl phenotypee affects the toxicokinetics of inhaled methyl chloride in human volunteers. PharmacogeneticsPharmacogenetics 2000; 10:645-653.

(9)) Mutti A, Falzoi M, Romanelli A, Bocchi MC, Ferroni C, and Franchini I. Brain dopaminee as a target for solvent toxicity: Effects of some monocyclic aromatic hydrocarbons.. Toxicology 1988; 49:77-82.

(10)) Mutti A, Falzoi M, Romanelli A, and Franchini I. Regional alterations of brain catecholaminess by styrene exposure in rabbits. Arch Toxicol 1984; 55:173-177.

(11)) Berode M, Boillat MA, Guillemin P, Wu MM, and Savolainen H. Demethylation pathwayss in caffeine metabolism as indicators of variability in 1,1,1-trichloroethane oxidationn in man. Pharmacol Toxicol 1990; 67:41-46.

165 5

ChapterChapter 10

(12)) Kawamoto T, Koga M, Murata K, Matsuda S, and Kodama Y. Effects of ALDH2,

CYP1A1,, and CYP2E1 genetic polymorphisms and smoking and drinking habits on

toluenee metabolism in humans. Toxicol ApplPharmacol 1995; 133:295-304.

(13)) Rossi AM , Guarnier C, Rovesti S, Gobba F, Ghittori S, Vivol i G, and Barale R.

Geneticc polymorphisms influence variability in benzene metabolism in humans.

PharmacogeneticsPharmacogenetics 1999; 9:445-451.

(14)) Sato A, Endoh K, Kaneko T, and Johanson G. A simulation study of physiological

factorss affecting pharmacokinetic behaviour of organic solvent vapours. Br J Ind Med

1991;48:342-347. .

(15)) Droz PO. Quantification of biological variability. Ann Occup Hyg 1992; 36:295-306.

(16)) Hirvonen A, Norppa H, Tuimala J, and Voho A. Genetic polymorphisms and

biomonitoringg of styrene. In: Current Research Projects 2000, Finnish Institute of

Occupationall Health.

(17)) Lison D and Haufroid V. Individual variations in the expression of enzymes involved

inn the biotransformation of styrene and influence on the interpretation of biomarkers

off exposure. Singapore: 26th International Congress on Occupational Health, 2000:

171. .

(18)) Löf A and Johanson G. Toxicokinetics of organic solvents: a review of modifying

factors.. CritRev Toxicol 1998; 28:571-650.

(19)) Ramsey JC and Andersen ME. A physiologically based description of the inhalation

pharmacokineticss of styrene in rats and humans. Toxicol Appl Pharmacol 1984;

73:159-175. .

(20)) Watanabe J, Hayashi S, and Kawajiri K. Different regulation and expression of the

humann CYP2E1 gene due to the Rsa\ polymorphism in the 5-flanking region. J

BiochemBiochem 1994; 116:321-326.

(21)) Ueno Y, Adachi J, Imamichi H, Nishimura A, and Tatsuno Y. Effect of the

cytochromee P-450IIE1 genotype on ethanol elimination rate in alcoholics and control

subjects.. Alcoholism: Clin Exp Res 1996; 20:17A-21A.

(22)) Ueshima Y, Tsutsumi M, Takase S, Matsuda Y, and Kawahara H. Acetaminophen

metabolismm in patients with different cytochrome P-4502E1 genotypes. Alcohol Clin

ExpExp Res 1996; 20:25A-28A.

166 6

SummarisingSummarising discussion

(23)) Lowndes HE, Philbert MA, Beiswanger CM, Kauffman FC, and Reuhl KR. Xenobioticc metabolism in the brain as mechanistic bases for neurotoxicity. In: Chang LWW and Dyer RS editors. Handbook of Neurotoxicology. New York: Marcel Dekker,Inc,, 1995: 1-27.

(24)) Tindberg N and Ingelman-Sundberg M. Expression, catalytic activity, and inducibility off cytochrome P450 2E1 (CYP2E1) in the rat central nervous system. J Neurochem 1996;; 67:2066-2073.

(25)) Farin FM and Omiecinski CJ. Regiospecific expression of cytochrome P-450s and microsomall epoxide hydrolase in human brain tissue. J Toxicol Environ Health 1993; 40:317-335. .

(26)) Ghersi-Egea J, Perrin R, Leininger-Muller B, Grassiot M, Jeandel C, Floquet J, Cuny G,, Siest G, and Minn A. Subcellular localization of cytochrome P450, and activities of severall enzymes responsible for drug metabolism in the human brain. Biochem PharmacolPharmacol 1993; 45:647-658.

(27)) Soranno TM and Sultatos LG. Biotransformation of the insecticide parathion by mousee brain. Toxicol Lett 1992; 60:27-37.

(28)) Crosbie SJ, Blain PG, and Williams FM. Metabolism of n-hexane by rat liver and extrahepaticc tissues and the effect of cytochrome P-450 inducers. Hum Exp Toxicol 1997;; 16:131-137.

(29)) Sequeira D and Strobel HW. In vitro metabolism of imipramine by brain microsomes: effectss of inhibitors and exogenous cytochrome P450 reductase. Brain Res 1996; 738:24-31. 738:24-31.

(30)) Hu H-L, Bennett N, Lamb JH, Ghersi-Egea J, Schlosshauer B, and Ray DE. Capacity off rat brain to metabolize /w-dinitrobenzene: an in vitro study. Neurotoxicology 1997; 18:363-370. .

(31)) Mattia CJ, LeBel CP, and Bondy SC. Effects of toluene and its metabolites on cerebral reactivee oxygen species generation. Biochem Pharmacol 1991; 42:879-882.

(32)) Mattia CJ, Adams JD, and Bondy SC. Free radical production in the brain and liver by productss of toluene catabolism. Biochem Pharmacol 1993; 46:103-110.

(33)) Savolainen H and Vainio H. Organ distribution and nervous system binding of styrene andd styrene oxide. Toxicology 1977; 8:135-141.

(34)) Soskolne CL. Ethic, social, and legal issues surrounding studies of susceptible populationss and individuals. Environ Health Perspect 1997; 105:837-841.

167 7

ChapterChapter 10

(35)) Van Damme K, Casteleyn L, Heseltine E, Huici A, Sorsa M, Larebeke van N, and Vineiss P. Individual susceptibility and prevention of occupational diseases: Scientific andd ethical issues. JOEM 1995; 37:91-99.

168 8