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									                    Toxicologic Pathology

                Metabolic Detoxification: Implications for Thresholds
Franz Oesch, Maria Elena Herrero, Jan Georg Hengstler, Matthias Lohmann and Michael Arand
                               Toxicol Pathol 2000; 28; 382
                           DOI: 10.1177/019262330002800305

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                                                 Metabolic Detoxification:
                                                Implications for Thresholds
                                FRANZ      OESCH, MARÍA ELENA HERRERO, JAN GEORG HENGSTLER,
                                              MATTHIAS LOHMANN, AND MICHAEL ARAND
                Institute   of Toxicology, University of Mainz,                Obere Zahlbacher Strasse 67, D-55131 Mainz,                          Germany


          The fact that chemical carcinogenesis involves single, isolated, essentially irreversible molecular events as discrete steps, several of
       which 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 enzymatic
       hydrolysis 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 epoxide
       hydrolases 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 is
       favorable 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 a
       sudden 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 that
        observed in human liver are completely protected from any measurable genotoxic effect of the model compound styrene oxide (STO)
        up to a dose of 100 μM in the cell culture medium (toxicokinetic threshold). In V79 cells that do not express mEH, STO leads to
        the formation of DNA strand breaks in a dose-dependent manner with no toxicokinetic threshold observable. Above 100 μM, the
        genotoxic effect of STO in the mEH-expressing cell line parallels the one in the parental cell line. Thus, the saturable protection from
        STO-induced strand breaks by mEH represents a typical example of a practical threshold. However, it must be pointed out that even
        in the presence of protective amounts of mEH, a minute but definite level of STO is present that does not contribute sufficiently to
        the 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; α/β hydrolase fold;    ester   intermediate; epoxy compounds metabolism; V79 Chinese hamster lung
        fibroblasts; DNA damage; genotoxicity

                               INTRODUCTION                                                    for a pharmacologic effect decreases steeply beyond the
   It is often stated that genotoxic carcinogens do not                                        threshold dose and rapidly approaches 0. From this point
show a threshold for their effect (11, 12, 15). This is                                        of view, there is indeed no indication for a threshold in
based on the view of genotoxic effects as stochastic, ir-                                      chemical carcinogenesis (the case of nongenotoxic car-
reversible events, in contrast to, eg, pharmacologic and                                       cinogens for which threshold effects have been docu-
                                                                                               mented is not the subject of this paper). However, this
toxicological effects of a drug that are reversible, in most
cases. Figure 1 illustrates the principal difference be-                                       simple view neglects the kinetic properties of genotoxic
tween these 2 phenomena under the simplifying gener-                                           agents, the concentrations of which are often tightly con-
alization that many pharmacologic effects are evoked by                                        trolled by drug-metabolizing enzymes.
concentration-dependent receptor interactions, and the                                                 XENOBIOTIC-METABOLIZING ENZYMES CONTROL                      THE
genotoxic effect is based on a covalent, essentially non-                                                   GENOTOXICITY OF FOREIGN COMPOUNDS
reversible modification of the DNA. Receptor-mediated
effects most often require a minimum percentage of the                                               Although              there   are a   fair number ofdirect-acting mu-
respective receptor molecules to be occupied, the classi-                                        tagens/carcinogens,                most    genotoxic agents require meta-
 cal setting for a threshold, whereas the DNA alteration                                         bolic activation in order                  to become sufficiently reactive
 eventually giving rise to a discrete step in cancer devel-                                      to chemically interact with the DNA. In order to allow
 opment is the consequence of a single hit. Thus, the                                            elimination of initially lipophilic compounds via the
 chance for tumor induction decreases linearly with the                                          aqueous excretion systems of the mammalian body, a
 dose of the genotoxic carcinogen, whereas the likelihood                                        huge network of xenobiotic-metabolizing enzymes that
                                                                                                 take care of these compounds has evolved. During bio-
                                                                                                 transformation by these enzymes, some compounds are
  Address  correspondence to: Prof.        Franz Oesch, Institute of Toxicol-
ogy, University of Mainz, Obere            Zahlbacher Strasse 67, D-55131                         &dquo;accidentally&dquo; activated to genotoxic metabolites. Figure
Mainz, Germany.                                                                                  2 presents the prototypic course of the metabolism of a
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  FIGURE 1.-Threshold    or no   threshold? A) A typical pharmacologic drug (ligand) occupies a receptor reversibly in an equilibrium-driven process.
Below a certain concentration of the drug, the percentage of occupied receptors, such as, eg, ligand-dependent ion channels in the cell membrane,
does not evoke the drug-specific effect, which only becomes apparent after rising above the threshold concentration. B) A genotoxic carcinogen
(modifying agent) binds with a concentration-dependent probability, yet in a stochastic event, covalently to the DNA (target) of a cell. This process
is, by its nature, essentially irreversible in the sense that it is not equilibrium driven. Since a single hit can be sufficient to start the cascade of
events finally leading to carcinogenesis, there is no apparent threshold for this effect.

lipophilic  and-in the present case-genotoxic com-                                       highly  water soluble, and thus easily excreted. The func-
pound. In  the so-called phase 1 of drug metabolism, the                                  tionalized intermediate arising from phase 1 metabolism,
molecule is functionalized either by introduction or lib-                                 however, is chemically more or less reactive. If the func-
eration of a functional group that can be used as a kind                                  tional group that has been introduced is electrophilic in
of handle in phase 2 of drug metabolism to conjugate the                                  nature, as is, for instance, the case with epoxides (see
molecule, usually with a hydrophilic, negatively charged,                                 Figure 3), it has a tendency to react with electron-rich
endogenous chemical building block. The resulting ter-                                    moieties in the DNA and give rise to the formation of
minal metabolite is generally nonreactive, nontoxic, and                                  DNA adducts, DNA strand breaks, or both. One example
                                                                                          for a compound that is activated to a genotoxic inter-
                                                                                          mediate in the human body is styrene (Figure 4), a com-
                                                                                          pound produced in thousands of tons per year to satisfy
                                                                                          the demand of the plastics industries. Luckily, the geno-
                                                                                          toxic intermediate, an epoxide, is itself rapidly inactivated
                                                                                          by the microsomal epoxide hydrolase (mEH; EC,
                                                                                          an important enzyme that protects the body from the haz-
                                                                                          ardous effects of many exogenous or endogenous epox-
                                                                                          ides (13). The metabolic capacity of mEH determines a
                                                                                          practical threshold for the genotoxicity of compounds
                                                                                          that are inactivated by this enzyme.

                                                                                                   THE MICROSOMAL EPOXIDE HYDROLASE-FAST
                                                                                                 DETOXIFICATION DESPITE LOW TURNOVER NUMBER
                                                                                          Mechanistic             Background
                                                                                              The very broad substrate specificity of mEH perfectly
                                                                                          suits its central role in the detoxification of genotoxic
                                                                                          epoxides. On the other hand, the enzyme displays a com-
   FIGURE 2.-Carcinogen metabolism. This scheme is an adaptation of
the well-known phase model of drug metabolism for the carcinogen
                                                                                          paratively low turnover number with most of its sub-
                                                                                          strates, usually smaller than 1 s-1 (16). This seems to be
metabolism. As a general rule, carcinogens are chemically activated
during phase I metabolism (therefore the designation procarcinogen)                       partly compensated by the fact that the mEH concentra-
and inactivated during phase II metabolism. However, for both rules
                                                                                          tion in the human liver, the prominent organ for inter-
there are a number of exceptions. The table above gives a list of the                     mediate epoxide formation in the body, is very high ( 10-
enzyme families with implication in the metabolism of carcinogenic                        50 ~LM, simplified, taking the whole organ as the &dquo;sol-
compounds. Although only few isoenzymes are known in some of these                        vent&dquo;) but still compromises its role as a rapid detoxifier.
families, others may contain more than 100 individual enzymes.                                Recent analyses have led to a detailed understanding

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   FIGURE 3.-Genotoxic effect of epoxides. Epoxides, ie, 3-membered
heterocycles  with an oxygen as the hetero atom, are reactive compounds
due to the strain arising from the unfavorable bond angles and the dif-
ference in electronegativity of the binding partners in the ring. The ring
carbon atoms represent electrophilic centers that can chemically react
with electron-rich partners, such as the exocyclic amino groups of DNA
bases or the N7 position of purine bases. The latter modification desta-
bilizes the bond between base and sugar in the DNA and thus leads to
apurinic sites that can be transformed into single strand breaks under
alkaline conditions. The substitution pattern (R,-R4) of the epoxide
modulates 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 enzymes
specialized 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 directly
 or after conjugation to glucuronic acid or sulfate. It should be men-                       FIGURE 4.-Metabolism of styrene in the human body. The major
 tioned, however, that some specific diols formed during the metabolism                   route of   styrene metabolism in man is represented. The predominant
 of polycyclic aromatic hydrocarbons can be further metabolized to the                    excretion products are mandelic acid and phenyl glyoxylic acid. The
 corresponding diol epoxides, a class of potent ultimate carcinogens.                     first step in the metabolism is epoxidation in the 7,8-position, ie, the
                                                                                          vinyl group, to the 7,8-epoxide. This reaction is catalyzed by a number
                                                                                          of different cytochrome P450-dependent monooxygenases. The epoxide
 of the enzymatic mechanism, by which mEH and the re-
                                                                                          can be readily hydrolyzed by epoxide hydrolases. Subsequently, the
 lated soluble epoxide hydrolase hydrolyze their substrates
                                                                                          resulting glycol is further oxidized by the sequential action of an alcohol
 (1-3, 5, 7-10, 17, 18). These enzymes belong to the large                                dehydrogenase (ADH) and an aldehyde dehydrogenase to mandelic
 structural family of Œ/~ hydrolase fold enzymes (14).                              acid, which is either directly excreted or metabolized again by an ADH
 These hydrolytic enzymes harbor a so-called catalytic tri-                               to phenyl glyoxylic acid.
 ad. In the first step of the enzymatic reaction (Figure 5),
 the catalytic nucleophile, which is an aspartic acid residue
 in the case of the epoxide hydrolases, attacks the sub-
 strate to form an enzyme-substrate ester intermediate.
 This is subsequently hydrolyzed by an activated water
 molecule. 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) have
  calculated 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 to
  our 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                                      FIGURE 5.-Catalytic mechanism of enzymatic epoxide hydrolysis by
  in the enzymatic mechanism of mEH? Certainly, the most                                    mEH. The course of the reaction is described in detail in the main text.
  important point is to realize that the rate of product for-                               The indices of the individual amino acid residues designate their re-
  mation does not adequately mirror the detoxification ef-                                  spective positions in the mEH amino acid sequence.

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                                                                                       ficiency  of the enzyme. Because step 1 is so much faster
                                                                                       than step 2 of the enzymatic reaction, the ester interme-
                                                                                       diate will accumulate at the cost of the substrate, ie, the
                                                                                       free epoxide. Thus, the enzyme works like a molecular
                                                                                       sponge in that it first consumes much more epoxide than
                                                                                       diol, the terminal reaction product is formed. As long as
                                                                                       the enzyme is in excess over its substrate, which can be
                                                                                       taken as a realistic setting under physiological conditions,
                                                                                       the epoxide is eliminated much faster than the diol is
                                                                                       formed. This theoretical consideration can be tested in a
                                                                                       first step by computer simulation (Figure 6). As antici-
                                                                                       pated, the in silico analysis reveals a much faster decrease
                                                                                       in epoxide concentration compared with the increase in
                                                                                       diol formation (Figure 6A). Thus, the effective dose of
                                                                                       the genotoxic agent, as represented by the area under the
                                                                                       time-concentration curve, is actually much smaller than
                                                                                       the calculation from the formed diol predicts (Figure 6B).
                                                                                       Also very important is the finding that there is a direct
                                                                                       proportional relationship between epoxide steady-state
                                                                                        concentration and epoxide hydrolase concentration, also
                                                                                        under conditions well below substrate saturation (Figure
                                                                                         Experimental Proof
                                                                                            A convenient way to test the importance of a given
                                                                                         detoxifying  enzyme in the control of a genotoxic agent
                                                                                         is its recombinant expression in a suitable indicator cell
                                                                                         line and the subsequent analysis of the susceptibility of
                                                                                         the recombinant cell line, as well as the parental cell line,
                                                                                         toward the genotoxic effect of the compound in question.


                                                                                           constant for the hydrolysis of the ester intermediate)
                                                                                                                                                  1 s-’. The most
                                                                                         important observation from this simulation is that the free substrate
                                                                                         disappears rapidly, while the ester intermediate accumulates at the same
                                                                                         time. From this reservoir, the product is formed at a much slower rate
                                                                                         because of the large difference between k, and k2. B) Comparison be-
                                                                                         tween    the authentic concentration of the substrate over time and the
                                                                                          substrate concentration calculated from the appearance of the terminal
                                                                                          product (the usual analytical approach) from the data shown in (A). It
                                                                                          is apparent that the authentic substrate calculation is orders of magni-
                                                                                          tude lower than that obtained by calculation from the formation of the
                                                                                          terminal product. Since the area under the curve is proportional to the
                                                                                          biological effect of the compound in the given system, the conventional
                                                                                          approach to determine the substrate concentration from product for-
                                                                                          mation heavily overestimates the substrate concentration and underes-
                                                                                          timates the protective effect of the enzymatic detoxification. C) Simu-
                                                                                          lation of the influence of mEH concentration on the substrate epoxide
                                                                                          under steady state conditions. Conditions were as described under (A),
                                                                                          with the exception that the substrate was continuously supplied at a rate
                                                                                          of I plum s and the half-life of the substrate due to spontaneous hy-
                                                                                          drolysis was set to 1 s (this is about the shortest half-life reported for
                                                                                          epoxides to date; the shorter the half-life, the lower the contribution of
                                                                                          enzymatic hydrolysis). Despite these stringent conditions, the epoxide
                                                                                          hydrolase still drastically reduces the steady state concentration of the
                                                                                           epoxide by at least I order of magnitude under conditions resembling
   FIGURE 6.-Computer simulation of the reaction kinetics of enzy-                         the physiological situation. E =
                                                                                                                             enzyme concentration: S    =
matic epoxide hydrolysis. A) Time course of the reaction after a single                    concentration; ES     concentration of the Michaelis-Menten (noncova-

application of epoxide (from t    =
                                     0 to 1 second). Parameters for the                    lent) complex between substrate and enzyme; E-S      =
                                                                                                                                                    concentration of
simulation were the following: enzyme concentration        50 p,M; sub-
                                                                                           the substrate-enzyme ester intermediate: P =
                                                                                                                                          concentration of the prod-
strate concentration at the beginning  =
                                           5 pLM: kp =
                                                       500 pLM; k, (rate                   uct. Calculations were performed as described elsewhere (M. Arand et
constant for the formation of the ester intermediate) =
                                                        1000 s-’; k, (rate                 al, in preparation).

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                                                                                           FIGURE 8.-Immunofluorescence analysis of the mEH-expressing
                                                                                        V79 fibroblasts. The recombinant cells were permeabilized, and the lo-
                                                                                        calization of the mEH in the ER was visualized by immunodetection
                                                                                        with an mEH-specific antibody in combination with a second fluores-
                                                                                        cein isothiocyanate-conjugated antibody. The ER is represented as a
                                                                                        dense network tightly surrounding the nucleus of the cell.

                                                                                         500   pmol, although the mEH concentration within a sin-
                                                                                         gle  cell is similar to that of the epoxide, namely about
   FIGURE 7.-Influence of mEH expression in V79 Chinese hamster
                                                                                         50 j..LM. Since in this setting there is a large substrate
cells on the rate of styrene 7,8-oxide-induced DNA strand breaks. Pa-
                                                                                         excess in terms of numbers of substrate molecules in the
rental V79 cells (open circles) and derivatives thereof stably expressing
mEH at a rate comparable to that in human liver (closed circles) were
                                                                                         entire culture dish, but in the cell there are approximately
exposed to styrene 7,8-oxide at different concentrations for 1 hour, and                 equimolar concentrations of substrate and enzyme at the
the rate of DNA strand break formation was determined by the alkaline                    practical threshold level, this threshold may be dominated
elution technique (6). Each circle represents the mean of 4 separate                     by the cellular concentrations of the partners. This im-
determinations, and the error bars represent the standard deviation. The                 plies that the replenishment of substrate at the site of
data clearly document a protective effect by mEH expression up to at
                                                                                         enzyme may not be substantially faster than the regen-
least a styrene oxide concentration of 100 ~,M. The graph is the rep-
                                                                                         eration of the free enzyme. As the immunofluorescence
resentative example of 3 independent experiments.
                                                                                         analysis of the recombinant cells shows (Figure 8), the
                                                                                         mEH is localized in the endoplasmic reticulum (ER) that
                                                                                         surrounds the cell nucleus, the target structure of geno-
 We have used V79 Chinese hamster fibroblast cells (4)
                                                                                         toxic agents. This potentially leads to a filter effect for
 as the indicator cell line and tested the influence of re-
                                                                                         many lipophilic epoxides, such as the styrene oxide, be-
 combinant mEH expression on the styrene oxide-induced
                                                                                         fore they can enter the cell nucleus. To reach the nucleus,
 DNA strand breaks, as measured by the alkaline elution
 technique (6; Figure 7). The results of these experiments                                they have to travel along the ER, where they meet mEH.
                                                                                          Therefore, the mEH may turn the ER into a barrier that
 clearly show that the expression of mEH protects V79                                     is hard for epoxides to overcome, which eventually leads
 cells from styrene oxide-induced DNA strand break for-
                                                                                          to a large difference in epoxide concentration between
 mation up to a concentration of at least 100 RM. Above
                                                                                          the 2 sites separated by this barrier. Once the capacity of
 this, a steep rise in the genotoxic effect of the agent is
 noted. In contrast, the parental cell line that is devoid of                             this barrier is exhausted, however, the genotoxic effect of
                                                                                          the epoxide becomes apparent.
 any mEH expression shows a monophasic, dose-depen-
 dent increase in DNA strand breaks without the initial                                      The detoxification of styrene oxide by mEH represents
                                                                                          an illustrative example on how practical thresholds in
 lag phase observed with the mEH-expressing cells. Thus,
 mEH expression introduces a threshold for the suscepti-                                  chemical carcinogenesis are determined by the metabolic
 bility of V79 cells to styrene oxide genotoxicity.                                       fate of genotoxic carcinogens. A number of factors, such
     The above-described enzymatic mechanism is 1 im-                                      as the enzymatic mechanism and the subcellular com-

 portant factor contributing to the high clearance of sty-                                partmentalization of the protective enzyme or enzymes,
  rene oxide by mEH; yet it may not be sufficient as a                                     synergize in the protection of the cells against these chal-
  stand-alone explanation for the observed threshold. In the                               lenges. Likewise, the kinetics of processes in the activa-
  experiment shown in Figure 7, the threshold concentra-                                   tion of carcinogens may lead to nonlinearity in the dose-
  tion of 100 )JbM equals a total amount of 500 nmol styrene                               response curve of such compounds. These aspects should
  oxide in the entire culture dish (cells plus medium). The                                be considered in the discussion about the existence of
  total amount of mEH in one dish, however, is only about                                  practical thresholds in carcinogenesis.

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                         ACKNOWLEDGMENTS                                                          epoxide hydrolases are members of the same family of C-X bond
                                                                                                  hydrolase enzymes. Chem Res Toxicol 7: 121-124.
  We thank the Deutsche Forschungsgemeinschaft (SFB                                          9.   Laughlin LT, Tzeng H-F. Lin S, Armstrong RN (1998). Mechanism
519/ project Bl) and the European Community (4th                                                  of microsomal epoxide hydrolase. Semifunctional site-specific mu-
framework &dquo;Biotechnology,&dquo; contract PL950005).                                          tants affecting the alkylation half-reaction. Biochemistry 37: 2897-
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