Metabolic Detoxification - Implications for Thresholds

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DOI: 10.1177/019262330002800305

2000 28: 382Toxicol PatholFranz Oesch, Maria Elena Herrero, Jan Georg Hengstler, Matthias Lohmann and Michael Arand

Metabolic Detoxification: Implications for Thresholds

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Metabolic Detoxification:

Implications for Thresholds

FRANZ OESCH, MARA ELENA HERRERO, JAN GEORG HENGSTLER,MATTHIAS LOHMANN,AND MICHAELARAND

Institute of Toxicology, University of Mainz, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany

Address correspondence to: Prof. Franz Oesch, Institute of Toxicol-

ogy, University of Mainz, Obere Zahlbacher Strasse 67, D-55131

Mainz, Germany.

ABSTRACT

The fact that chemical carcinogenesis involves single, isolated, essentially irreversible molecular events as discrete steps, several ofwhich must occur in a row to finally culminate in the development of a malignancy, rather suggests that an absolute threshold for

chemical carcinogens may not exist. However, practical thresholds may exist due to saturable pathways involved in the metabolic

processing, especially in the metabolic inactivation, of such compounds.An important example for such a pathway is the enzymatichydrolysis of epoxides via epoxide hydrolases, a group of enzymes for which the catalytic mechanism has recently been established.These enzymes convert their substrates via the intermediate formation of a covalent enzyme-substrate complex. Interestingly, the

formation of the intermediate proceeds faster by orders of magnitude than the subsequent hydrolysis, ie, the formation of the terminal

product. Under normal circumstances, this does not pose a problem, since the microsomal epoxide hydrolase (mEH), the epoxidehydrolases with the best documented importance in the metabolism of carcinogens, is highly abundant in the liver, the organ with the

highest capacity to metabolically generate epoxides. Computer simulation provides evidence that the high amount of mEH enzyme isfavorable for the control of the steady-state level of a substrate epoxide and can keep it extremely low. However, once the mEH is

titrated out under conditions of extraordinarily high epoxide concentration, the epoxide steady-state level steeply rises, leading to asudden burst of the genotoxic effect of the noxious agent. This prediction of the computer simulation is nicely supported by experi-mental work. V79 Chinese hamster cells that we have genetically engineered to express human mEH at about the same level as thatobserved in human liver are completely protected from any measurable genotoxic effect of the model compound styrene oxide (STO)

up to a dose of 100 M in the cell culture medium (toxicokinetic threshold). In V79 cells that do not express mEH, STO leads to

the formation of DNAstrand breaks in a dose-dependent manner with no toxicokinetic threshold observable.Above 100 M, the

genotoxic effect of STO in the mEH-expressing cell line parallels the one in the parental cell line. Thus, the saturable protection fromSTO-induced strand breaks by mEH represents a typical example of a practical threshold. However, it must be pointed out that evenin the presence of protective amounts of mEH, a minute but definite level of STO is present that does not contribute sufficiently tothe strand break formation to overcome the background noise of the detection procedure.As pointed out above, absolute thresholds

probably do not exist in chemical carcinogenesis.

Keywords. Epoxide hydrolase; / hydrolase fold; ester intermediate; epoxy compounds metabolism; V79 Chinese hamster lungfibroblasts; DNAdamage; genotoxicity

INTRODUCTION

It is often stated that genotoxic carcinogens do notshow a threshold for their effect (11, 12, 15). This isbased on the view of genotoxic effects as stochastic, ir-reversible events, in contrast to, eg, pharmacologic and

toxicological effects of a drug that are reversible, in mostcases. Figure 1 illustrates the principal difference be-tween these 2 phenomena under the simplifying gener-alization that many pharmacologic effects are evoked byconcentration-dependent receptor interactions, and the

genotoxic effect is based on a covalent, essentially non-

reversible modification of the DNA. Receptor-mediatedeffects most often require a minimum percentage of therespective receptor molecules to be occupied, the classi-cal setting for a threshold, whereas the DNAalteration

eventually giving rise to a discrete step in cancer devel-

opment is the consequence of a single hit. Thus, thechance for tumor induction decreases linearly with thedose of the genotoxic carcinogen, whereas the likelihood

for a pharmacologic effect decreases steeply beyond tthreshold dose and rapidly approaches 0. From this poof view, there is indeed no indication for a threshold

chemical carcinogenesis (the case of nongenotoxic c

cinogens for which threshold effects have been docmented is not the subject of this paper). However, t

simple view neglects the kinetic properties of genotoxagents, the concentrations of which are often tightly cotrolled by drug-metabolizing enzymes.

XENOBIOTIC-METABOLIZING ENZYMES CONTROL THE

GENOTOXICITY OF FOREIGN COMPOUNDS

Although there are a fair number of direct-acting m

tagens/carcinogens, most genotoxic agents require mebolic activation in order to become sufficiently reactto chemically interact with the DNA. In order to alelimination of initially lipophilic compounds via

aqueous excretion systems of the mammalian bodyhuge network of xenobiotic-metabolizing enzymestake care of these compounds has evolved. During btransformation by these enzymes, some compounds&dquo;accidentally&dquo; activated to genotoxic metabolites. Fig2 presents the prototypic course of the metabolism o

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FIGURE 1.-Threshold or no threshold?A)Atypical pharmacologic drug (ligand) occupies a receptor reversibly in an equilibrium-driven procBelow a certain concentration of the drug, the percentage of occupied receptors, such as, eg, ligand-dependent ion channels in the cell membrdoes not evoke the drug-specific effect, which only becomes apparent after rising above the threshold concentration. B)Agenotoxic carcin

(modifying agent) binds with a concentration-dependent probability, yet in a stochastic event, covalently to the DNA(target) of a cell. This prois, by its nature, essentially irreversible in the sense that it is not equilibrium driven. Since a single hit can be sufficient to start the cascadevents finally leading to carcinogenesis, there is no apparent threshold for this effect.

lipophilic and-in the present case-genotoxic com-

pound. In the so-called phase 1 of drug metabolism, themolecule is functionalized either by introduction or lib-eration of a functional group that can be used as a kind

of handle in phase 2 of drug metabolism to conjugate themolecule, usually with a hydrophilic, negatively charged,endogenous chemical building block. The resulting ter-minal metabolite is generally nonreactive, nontoxic, and

FIGURE 2.-Carcinogen metabolism. This scheme is an adaptation ofthe well-known phase model of drug metabolism for the carcinogenmetabolism.As a general rule, carcinogens are chemically activated

during phase I metabolism (therefore the designation procarcinogen)and inactivated during phase II metabolism. However, for both rules

there are a number of exceptions. The table above gives a list of the

enzyme families with implication in the metabolism of carcinogeniccompounds.Although only few isoenzymes are known in some of these

families, others may contain more than 100 individual enzymes.

highly water soluble, and thus easily excreted. The futionalized intermediate arising from phase 1 metabolihowever, is chemically more or less reactive. If the futional group that has been introduced is electrophilinature, as is, for instance, the case with epoxidesFigure 3), it has a tendency to react with electron-moieties in the DNAand give rise to the formationDNAadducts, DNAstrand breaks, or both. One examfor a compound that is activated to a genotoxic inmediate in the human body is styrene (Figure 4), a c

pound produced in thousands of tons per year to satthe demand of the plastics industries. Luckily, the getoxic intermediate, an epoxide, is itself rapidly inactivby the microsomal epoxide hydrolase (mEH; EC 3.3.2an important enzyme that protects the body from the hardous effects of many exogenous or endogenous epides (13). The metabolic capacity of mEH determinpractical threshold for the genotoxicity of compouthat are inactivated by this enzyme.

THE MICROSOMAL EPOXIDE HYDROLASE-FAST

DETOXIFICATION DESPITE LOW TURNOVER NUMBER

Mechanistic Background

The very broad substrate specificity of mEH perfesuits its central role in the detoxification of genotoepoxides. On the other hand, the enzyme displays a c

paratively low turnover number with most of itsstrates, usually smaller than 1 s-1 (16). This seems t

partly compensated by the fact that the mEH concention in the human liver, the prominent organ for imediate epoxide formation in the body, is very high50 ~LM, simplified, taking the whole organ as the &dvent&dquo;) but still compromises its role as a rapid detoxif

Recent analyses have led to a detailed understan



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FIGURE 3.-Genotoxic effect of epoxides. Epoxides, ie, 3-membered

heterocycles with an oxygen as the hetero atom, are reactive compoundsdue to the strain arising from the unfavorable bond angles and the dif-ference in electronegativity of the binding partners in the ring. The ringcarbon atoms represent electrophilic centers that can chemically reactwith electron-rich partners, such as the exocyclic amino groups of DNAbases or the N7 position of purine bases. The latter modification desta-bilizes the bond between base and sugar in the DNAand thus leads to

apurinic sites that can be transformed into single strand breaks underalkaline conditions. The substitution pattern (R,-R4) of the epoxidemodulates its reactivity in that asymmetric substitution increases the

reactivity at one of the electrophilic centers, whereas symmetric substi-

tution usually has a stabilizing effect. Epoxide hydrolases are enzymesspecialized in the hydrolysis of the epoxide ring. This reaction elimi-nates the electrophilic reactivity and thus the genotoxicity of the ep-oxide. The resulting metabolites can usually be excreted either directlyor after conjugation to glucuronic acid or sulfate. It should be men-

tioned, however, that some specific diols formed during the metabolismof polycyclic aromatic hydrocarbons can be further metabolized to the

corresponding diol epoxides, a class of potent ultimate carcinogens.

of the enzymatic mechanism, by which mEH and the re-

lated soluble epoxide hydrolase hydrolyze their substrates(1-3, 5, 7-10, 17, 18). These enzymes belong to the largestructural family of /~ hydrolase fold enzymes (14).These hydrolytic enzymes harbor a so-called catalytic tri-ad. In the first step of the enzymatic reaction (Figure 5),the catalytic nucleophile, which is an aspartic acid residuein the case of the epoxide hydrolases, attacks the sub-strate to form an enzyme-substrate ester intermediate.This is subsequently hydrolyzed by an activated watermolecule. Water activation is achieved by proton abstrac-tion through a charge-relay system composed of a histi-dine residue that is hydrogen bonded to an acidic residue,either glutamic or aspartic acid. Experimental evidence

clearly shows that in the case of enzymatic epoxide hy-

drolysis, the first step of the reaction proceeds signifi-cantly faster than the second step, which therefore be-comes rate limiting.Armstrong and colleagues (18) havecalculated the rate constant of step 1 to be 3 orders of

magnitude higher than the rate constant for step 2 with

glycidyl-4-nitrobenzoate as the substrate. According toour own work, similar reaction kinetics exist for the turn-over of styrene oxide and 9,10-epoxystearic acid (2).What conclusions can be drawn from this new insight

in the enzymatic mechanism ofmEH? Certainly, the mostimportant point is to realize that the rate of product for-mation does not adequately mirror the detoxification ef-

FIGURE 4.-Metabolism of styrene in the human body. The majroute of styrene metabolism in man is represented. The predominaexcretion products are mandelic acid and phenyl glyoxylic acid. Tfirst step in the metabolism is epoxidation in the 7,8-position, ie,

vinyl group, to the 7,8-epoxide. This reaction is catalyzed by a numbof different cytochrome P450-dependent monooxygenases. The epoxcan be readily hydrolyzed by epoxide hydrolases. Subsequently,resulting glycol is further oxidized by the sequential action of an alco

dehydrogenase (ADH) and an aldehyde dehydrogenase to mande

acid, which is either directly excreted or metabolized again by anAto phenyl glyoxylic acid.

FIGURE 5.-Catalytic mechanism of enzymatic epoxide hydrolysimEH. The course of the reaction is described in detail in the main

The indices of the individual amino acid residues designate their

spective positions in the mEH amino acid sequence.



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FIGURE 6.-Computer simulation of the reaction kinetics of enzy-matic epoxide hydrolysis.A) Time course of the reaction after a singleapplication of epoxide (from t = 0 to 1 second). Parameters for thesimulation were the following: enzyme concentration = 50 p,M; sub-strate concentration at the beginning = 5 pLM: kp = 500 pLM; k, (rateconstant for the formation of the ester intermediate) = 1000 s-; k, (rate

ficiency of the enzyme. Because step 1 is so much fasthan step 2 of the enzymatic reaction, the ester intermdiate will accumulate at the cost of the substrate, ie, tfree epoxide. Thus, the enzyme works like a moleculsponge in that it first consumes much more epoxide th

diol, the terminal reaction product is formed.As longthe enzyme is in excess over its substrate, which cantaken as a realistic setting under physiological condition

the epoxide is eliminated much faster than the diolformed. This theoretical consideration can be tested i

first step by computer simulation (Figure 6).As antipated, the in silico analysis reveals a much faster decrein epoxide concentration compared with the increasediol formation (Figure 6A). Thus, the effective dosethe genotoxic agent, as represented by the area undertime-concentration curve, is actually much smaller tthe calculation from the formed diol predicts (Figure 6Also very important is the finding that there is a dirproportional relationship between epoxide steady-stconcentration and epoxide hydrolase concentration, aunder conditions well below substrate saturation (Fig6C).

Experimental ProofAconvenient way to test the importance of a gidetoxifying enzyme in the control of a genotoxic agis its recombinant expression in a suitable indicatorline and the subsequent analysis of the susceptibilitythe recombinant cell line, as well as the parental cell ltoward the genotoxic effect of the compound in questi

(-

constant for the hydrolysis of the ester intermediate) = 1 s-. The m

important observation from this simulation is that the free subst

disappears rapidly,while the ester intermediate accumulates at the s

time. From this reservoir, the product is formed at a much slower

because of the large difference between k, and k2. B) Comparisontween the authentic concentration of the substrate over time and

substrate concentration calculated from the appearance of the term

product (the usual analytical approach) from the data shown in (Ais apparent that the authentic substrate calculation is orders of matude lower than that obtained by calculation from the formation oterminal product. Since the area under the curve is proportional t

biological effect of the compound in the given system, the conventi

approach to determine the substrate concentration from productmation heavily overestimates the substrate concentration and undetimates the protective effect of the enzymatic detoxification. C) Slation of the influence of mEH concentration on the substrate epounder steady state conditions. Conditions were as described underwith the exception that the substrate was continuously supplied at a

of I plum s and the half-life of the substrate due to spontaneoudrolysis was set to 1 s (this is about the shortest half-life reporteepoxides to date; the shorter the half-life, the lower the contributi

enzymatic hydrolysis). Despite these stringent conditions, the epohydrolase still drastically reduces the steady state concentration o

epoxide by at least I order of magnitude under conditions resemthe physiological situation. E = enzyme concentration: S = subs

concentration; ES = concentration of the Michaelis-Menten (non

lent) complex between substrate and enzyme; E-S = concentratithe substrate-enzyme ester intermediate: P = concentration of the

uct. Calculations were performed as described elsewhere (M.Ara

al, in preparation).



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FIGURE 7.-Influence of mEH expression in V79 Chinese hamstercells on the rate of styrene 7,8-oxide-induced DNAstrand breaks. Pa-

rental V79 cells (open circles) and derivatives thereof stably expressingmEH at a rate comparable to that in human liver (closed circles) were

exposed to styrene 7,8-oxide at different concentrations for 1 hour, and

the rate of DNAstrand break formation was determined by the alkaline

elution technique (6). Each circle represents the mean of 4 separatedeterminations, and the error bars represent the standard deviation. The

data clearly document a protective effect by mEH expression up to atleast a styrene oxide concentration of 100 ~,M. The graph is the rep-resentative example of 3 independent experiments.

We have used V79 Chinese hamster fibroblast cells (4)as the indicator cell line and tested the influence of re-

combinant mEH expression on the styrene oxide-inducedDNAstrand breaks, as measured by the alkaline elution

technique (6; Figure 7). The results of these experimentsclearly show that the expression of mEH protects V79cells from styrene oxide-induced DNAstrand break for-

mation up to a concentration of at least 100 RM.Above

this, a steep rise in the genotoxic effect of the agent isnoted. In contrast, the parental cell line that is devoid of

any mEH expression shows a monophasic, dose-depen-

dent increase inDNA

strand breaks without the initiallag phase observed with the mEH-expressing cells. Thus,mEH expression introduces a threshold for the suscepti-bility of V79 cells to styrene oxide genotoxicity.

The above-described enzymatic mechanism is 1 im-

portant factor contributing to the high clearance of sty-rene oxide by mEH; yet it may not be sufficient as astand-alone explanation for the observed threshold. In theexperiment shown in Figure 7, the threshold concentra-tion of 100 )JbM equals a total amount of 500 nmol styreneoxide in the entire culture dish (cells plus medium). Thetotal amount of mEH in one dish, however, is only about

FIGURE 8.-Immunofluorescence analysis of the mEH-expressV79 fibroblasts. The recombinant cells were permeabilized, and the

calization of the mEH in the ER was visualized by immunodetect

with an mEH-specific antibody in combination with a second fluor

cein isothiocyanate-conjugated antibody. The ER is represented adense network tightly surrounding the nucleus of the cell.

500 pmol, although the mEH concentration within a s

gle cell is similar to that of the epoxide, namely ab50 j..LM. Since in this setting there is a large substrexcess in terms of numbers of substrate molecules in

entire culture dish, but in the cell there are approximatequimolar concentrations of substrate and enzyme at

practical threshold level, this threshold may be domina

by the cellular concentrations of the partners. This

plies that the replenishment of substrate at the siteenzyme may not be substantially faster than the regeration of the free enzyme.As the immunofluoresce

analysis of the recombinant cells shows (Figure 8),mEH is localized in the endoplasmic reticulum (ER)surrounds the cell nucleus, the target structure of getoxic agents. This potentially leads to a filter effect

many lipophilic epoxides, such as the styrene oxide,fore they can enter the cell nucleus. To reach the nuclethey have to travel along the ER, where they meet mETherefore, the mEH may turn the ER into a barrier

is hard for epoxides to overcome, which eventually leto a large difference in epoxide concentration betwthe 2 sites separated by this barrier. Once the capacitthis barrier is exhausted, however, the genotoxic effecthe epoxide becomes apparent.The detoxification of

styreneoxide

bymEH

represan illustrative example on how practical thresholdschemical carcinogenesis are determined by the metabofate of genotoxic carcinogens.Anumber of factors, sas the enzymatic mechanism and the subcellular c

partmentalization of the protective enzyme or enzymsynergize in the protection of the cells against these c

lenges. Likewise, the kinetics of processes in the acttion of carcinogens may lead to nonlinearity in the d

response curve of such compounds. These aspects shbe considered in the discussion about the existenc

practical thresholds in carcinogenesis.



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ACKNOWLEDGMENTS

We thank the Deutsche Forschungsgemeinschaft (SFB519/ project Bl) and the European Community (4thframework &dquo;Biotechnology,&dquo; contract PL950005).

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Metabolic Detoxification - Implications for Thresholds