Epoxide metabolite identification

Research in PAH carcinogenesis has made major advances in the past decade. Most notable has been identification of diol epoxide metabolites as the active forms of benzo[a]pyrene, 7,12-dimethylbenz[tf]anthracene, and other carcinogenic PAH. This finding has stimulated enormous research activity and opened the way to determination of the detailed molecular mechanism of action of this important class of carcinogenic molecules. 

Recent advances in PAH carcinogenesis research over the past decade have led to identification of diol epoxide metabolites as the principal active forms of the PAH investigated to date Q,2). Benzo-(a)pyrene (BP) has been most intensively investigated, and it has been demonstrated that a diol epoxide metabolite anti-BPDE is the active intermediate which binds covalently to DNA in human and other mammalian tissues 0,4). Anti-BPDE was also demonstrated to be a powerful mutagen in both bacterial and mammalian cells (15) These findings stimulated an outpouring of research directed towards elucidation of the molecular mechanism of PAH carcinogenesis. 

As far as the direct identification of these epoxide metabolites is concerned, it has to be pointed out that for polycyclic hydrocarbons there is some direct evidence for the formation of relatively stable epoxides in the K region of these molecules... 

Studies on the comparative abilities (13) of B[a]P metabolites to bind to DNA in microsomal systems showed that the 7,8-dihydrodiol was the most efficient. This led to the proposal (69) that dihydrodiol epoxides were the ultimate carcinogenic metabolites. Chemical synthesis of all possible isomers (70.71) has allowed complete structural identification of the adducts (72-74). 

Analytical methods exist for measuring heptachlor, heptachlor epoxide, and/or their metabolites in various tissues (including adipose tissue), blood, human milk, urine, and feces. The common method used is gas chromatography (GC) coupled with electron capture detection (ECD) followed by identification using GC/mass spectrometry (MS). Since evidence indicates that heptachlor is metabolized to heptachlor epoxide in mammals, exposure to heptachlor is usually measured by determining levels of heptachlor epoxide in biological media. A summary of the detection methods used for various biological media is presented in Table 6-1. 

In cattle feces, 64% of the total residues was identified as diethylstilbestrol, 23% as 3-(p-hydroxyphenyl)-2-hexene-4-one, and less than 1% as 4 -hydroxypro-piophenone (43). The identification of 4 -hydroxypropiophenone as a metabolite of diethylstilbestrol implies that dienestrol is formed through an epoxide-diol pathway and that these metabolites show electrophilic reactivity (45). These observations have to be seen in connection with the mutagenic and carcinogenic activity of diethylstilbestrol and possibly also the other stilbene estrogens. 

Identification of DNA-Reactive Metabolites Generated in a Target Tissue, Mouse Skin, In Vivo. Our initial studies focused on activation of DMBA in mouse embryo cells in culture because of the ease of isolation of sufficient DNA for adduct characterization. The cells were exposed to DMBA and the isolated DNA enzymatically hydrolyzed to deoxyribonucleosides. DMBA-deoxyribonucleoside adducts were characterized by fluorescence measurements (11,22), by photosensitivity studies (12) and by column chromatography (23,24). These studies provided evidence that the DNA-reactive metabolite generated in these cells is a bay region dihydrodiol epoxide. The enzymatic steps in this activation pathway (Figure 1) involve oxidation of DMBA by mixed function oxidases to a 3,4-epoxide which is converted by epoxide hydrase to a 3,4-dihydro-diol. This is, in turn, oxidized by mixed function oxidases to the dihydrodiol epoxide. 

Because the half-life of the epoxide intermediate is short, immediate rearrangement or reaction may lead to a single metabolite or a variety of substituted metabolites. The intermediacy of an epoxide intermediate can be inferred by the identification of para-and meta-hydroxylated and dihydrodiol metabolites, although their relative abundances will vary with substitution and steric considerations. Acetanilide, like phenobarbital discussed previously, exemplifies the aromatic compounds that rearrange rapidly following CYP-mediated arene epoxide formation leading to a single metabolite, as shown in Scheme 11.10. 

Continued studies from several laboratories of the binding, mutagenicity and tumorigenicity of benzo [a] pyrene and its derivatives led to the identification of (+)-(7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[aJpyrene (a diol epoxide-2 diastereomer) as the principal metabolite responsible for the carcinogenic activity of benzo[a]pyrene. Only one of the four metabolically possible isomers of the 7,8-diol-9,10-epoxide was found to have high tumorigenic activity... 

The metabolism of trichloroethylene has been the subject of several reviews (3, 38, 196, 217, 255, 471). Trichloroethylene was shown to be metabolically activated via epoxidation of its olefinic bond. The formation of trichloroethylene oxide in the presence of liver microsomes was determined by spectral studies of the cytochrome P-450 complex (465, 466). The identification of chloral as an in vitro metabolite (61, 403) and of trichloromethanol and trichloroacetic acid as in vivo metabolites in rats (94) and dogs (60) supports the metabolic activation pathway outlined in Fig. 20. 

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