Benzo pyrene epoxide hydrolases

P. Bentley, F. Oesch, H. Glatt, Dual Role of Epoxide Hydrolase in Both Activation and Inactivation of Benzo[a]pyrene , Arch. Toxicol. 1977, 39, 65 - 75. 

An example of the second type of chiral effect in metabolism is afforded by benzofa]-pyrene, also discussed in more detail in chapter 7. This carcinogenic polycyclic hydrocarbon is metabolized stereos elec lively by a particular cytochrome P-450 isozyme, CYP1A1, to the (+)-7R,8S oxide (chap. 7, Fig. 5.2), which in turn is metabolized by epoxide hydrolase to the (—)-7R,8S dihydrodiol. This metabolite is further metabolized to (- -)-benzo[aIpyrene, 7R,8S dihydrodiol, 9S,10R epoxide in which the hydroxyl group and epoxide are trans and which is more mutagenic than other enantiomers. The (—)-7R,8S dihydrodiol of benzo[aIpyrene is 10 times more tumorigenic than the (+)-7R,8S enantiomer. It was reported that in this case the configuration was more important for tumorigenicity than the chemical reactivity. 

Although a number of the epoxides and diol epoxides are mutagenic, the 7,8-dihydrodiol 9,10-oxide, shown in Figure 7.2, is believed to be the ultimate carcinogen. It should be noted that there are several possible diastereoisomers of this metabolite, but as the action of epoxide hydrolase yields a trans dihydrodiol and the epoxide ring produced by the cytochrome P-450 may be cis or trans, there are two diastereoisomers produced metabolically. Thus, benzo(a)pyrene... 

Application of the exciton chirality rule has allowed the assignment of configuration (+ )-trans-(4R,5R) to dihydroxy-4,5-dihydrobenzo[a]pyrene (357), a mammalian metabolite of benzo[a]pyrene.217 Thus the prominent enantiomer of the 4,5-dihydrodiol formed from benzo [a] pyrene by rat-liver microsomes has the configuration (4R,5R) in contrast to the earlier report that the configuration was the opposite one.218 Consequently, ( + )-(358) has a (4S,5R) configuration. This epoxide is a better substrate for epoxide hydrolase than its enantiomer and has 1.5-5.5-fold less mutagenic activity as compared to the (—) enantiomer. 

The stereospecific metabolism of racemic benzo [a] pyrene 4,5-, 7,8-, and 9,10-oxides (28, 366, and 367, respectively) by rat liver microsomes or highly purified epoxide hydrolase has been examined by Thakker et al.22 The optical purity of the metabolically formed benzo [a] pyrene 7,8- and 9,10-dihydrodiols is relatively low (8 and 22%, respectively). The metabolically formed benzo [a] pyrene-4,5-dihydrodiol is highly enriched in the (—) enantiomer (78% optical purity). The low optical purity of benzo [a] pyrene-7,8-dihydrodiol is attributed to the ability of epoxide hydrolase to act upon position 8 of both optical isomers of 366 with almost equal ease. 

The oxidation of naphthalene was one of the earliest examples of an epoxide as an intermediate in aromatic hydroxylation. As shown in Figure 7.3, the epoxide can rearrange nonenzymatically to yield predominantly 1-naphthol, or interact with the enzyme epoxide hydrolase to yield the dihydrodiol, or interact with glutathione S-transferase to yield the glutathione conjugate, which is ultimately metabolized to a mercapturic acid. These reactions are also of importance in the metabolism of other xenobiotics that contain an aromatic nucleus, such as the insecticide carbaryl and the carcinogen benzo(a)pyrene. 

Dehydrogenation A-Demethylation Hydroxylation Epoxidation Sulfoxidation Oxidations Acetaminophen, benzidine, DES, epinephrine Dimethylaniline, benzphetamine, aminocarb Benzo[a]pyrene, 2-aminofluorene, phenylbutazone 7,8-Dihydrobenzo[a]pyrene Methylphenylsulfide FANFT, ANFT, bilirubin Esterases and Amidases Paraoxon, dimethoate, phenyl acetate Epoxide Hydrolase Benzo(a)pyrene epoxide, styrene oxide DDT-Dehydrochlorinase p,p- DDT Glutathione Reductase Disulfiram... 

The proximate carcinogens arising from the metabolic activation of benzo(a)pyrene are isomers of benzo(a)pyrene 7,8-diol-9,10-epoxide (Figure 10.1C). These metabolites arise by prior formation of the 7,8-epoxide, which gives rise to the 7,8-dihydrodiol through the action of epoxide hydrolase. The diol is further metabolized by the microsomal monooxygenase system to the 7,8-diol-9,10-epoxides, which are both potent mutagens and unsuitable substrates for the further action of epoxide hydrolase. 

Figure 24.11. Metabolic activation of benzo[a]pyrene to the ultimate carcinogenic species. Benzo[a]pyrene is metabolized by cytochromes P-450 and epoxide hydrolase to form the ultimate carcinogen, (+)benzo[a]pyrene-7,8-diol-9,10 epoxide-2. (Adapted from Conney, A. H. Cancer Res. 42, 4875,1982.)...

The assay method described by Eaton and Stapleton (1989), measures the activities of both cytosolic glutathione 5-transferase and microsomal epoxide hydrolase toward benzo[a]pyrene-4,5-oxide as a substrate. These enzymes are important in the biotransformation of many epoxide xenobiotics, including potentially carcinogenic arene oxides. 

A number of K-region arene oxides have been detected as intermediates in the metabolism of the corresponding PAHs in liver systems for example, phenanthrene, benz[a]anthracene, pyrene, benzo [a]pyrene, and dibenz(a,h)anthracene. These K-region arene-oxide metabolites were generally only detected by trapping the radiolabeled intermediate. The arene-oxide metabolite 102 obtained from a-naphthoflavone was found to be sufficiently stable with respect to isomerization and resistant to attack by epoxide hydrolase so that it could be isolated and identified spectroscopically. ... 

The combination of a large number of chemical resolution and assignment studies (Table 3) of benzo-ring /rans-dihydrodiols along with studies designed to define the stereochemical course of metabolism of benzo[a]pyrene, ° benz[a]-anthracene, chrysene, phenanthrene, benzo[c]phenanthrene, anthracene, and naphthalene has led to the conclusion that the combined action of cytochrome P450c and epoxide hydrolase has a marked preference for the formation of (—)-(R,R)-dihydrodiols. This result pertains even when the initially... 

Figure 18. Stereochemical course of the microsomal epoxide-hydrolase catalyzed hydration of (-) )- and (—)-benzo[a]pyrene 4,5-oxide. About 99% of the attack by water occurs at the (5S)-carbon of the (—)-(4R,5S)-oxide and about 85% at the (4S)-carbon of the (-l-)-(4S,5R)-oxide. ...

Figure 2 Benzo[a]pyrene is metabolized highly stereoseiectively to the ultimate carcinogen (+)-benzo[a]pyrene- ntf-7R,8S-dihydriol-9S,10R-epoxide by cytochrome P450-IA1 and epoxide hydrolase. (From Jerina et al., 1985.)...

Varying degrees of substrate enantioselectivity have been described for microsomal epoxide hydrolases. For example, for benzo(a]pyrene-4,5-oxide a 40-fold difference between the rates of hydration for the +) and (-) enantiomers was observed in vitro (Armstrong et al., 1980). Mono-substituted epoxides (such as 1,2-epoxyhexane and its geometrical isomer. 

R-configured carbon atom vide supra), microsomal epoxide hydrolase attacks at R- as well as S-configuxed carbon atoms. With some arene oxides, both enantiomers are attacked at the same carbon, irrespective of configuration (e.g., benzo[c]phenanthrene). Other arene oxides (e.g., benzo[a]-pyrene 4,5-oxide) undergo the primary reaction, predominantly at S-con-figured carbon (Yang, 1988). 

Figure 7 Conversion of benzo(a)pyrene 7,8-oxide to benzo(a)pyrene trans-7,8-dihydrodiol by microsomal epoxide hydrolase.

Epoxidation is thought to be the major pathway for benzo[a]pyrene metabolism pertinent to macromolecular interaction. The metabolic attack consists of the cytochrome P-450/P-448-dependent MFO system converting the benzo[a]pyrene molecule into an epoxide the epoxide is acted upon by epoxide hydrolase to form a dihydrodiol, and a second cytochrome MFO reaction gives rise to the ultimate mutagenic/carcinogenicform, benzo[a]pyrene 7, 8-diol-9,... 

Epoxides are highly reactive. They have been shown to bind irreversibly to DNA, RNA, and proteins, and have been implicated in carcinogenesis. Many epoxides are hydrolyzed to diol products (molecules containing two adjacent OH groups) by epoxide hydrolase, a microsomal enzyme (Figure 10E). In most cases the diols that are formed are less reactive and less toxic than the parent epoxide. However, with some polycyclic hydrocarbons (e.g., benzo[a]pyrene) the diols that are formed are precursors for carcinogenic metabolites. 

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