Cytochrome P-450, isozymes

Nakajima T, Wang RS, Elovaara E, et al. 1992a. A comparative study on the contribution of cytochrome P-450 isozymes to metabolism of benzene, toluene and trichloroethylene in rat liver. Biochem Pharmacol 43 251-257. 

Parkinson, A., Thomas, P.E., Ryan, D.E. etal. (1983) Differential time course of induction of rat liver microsomal cytochrome P 450 isozymes and epoxide hydrolase by Aroclor 1254. Archives of Biochemistry and Biophysics, 225, 203-215. 

Figure 1. The major pathways in the metabolism of BaP to BaP epoxides, dihydrodiol, and 7,8-dihydrodiol-9,10-epoxides. The absolute configurations are as shown. The position of trans-addition of water is shown by an arrow. The optical purity of the 4,5-epoxide formed in BaP metabolism is dependent on the cytochrome P-450 isozymes present in the microsomal enzyme system. EH epoxide hydrolase.

BA trans-3.4-dihvdrodiol cannot be separated from BA trans-8.9-dihydrodiol in several HPLC conditions (27-29). Quantification of BA trana-3,4-dihydrodiol by HPLC can only be accomplished after converting the 3,4-dihydrodiol to its diacetate (25.26). The BA trans-3.4-dihydrodiol formed in BA metabolism by liver microsomes from pheno-barbital-treated rats was determined to have a 3R,4R/3S,4S enantiomer ratio of 69 31 (30). Recently we have determined the optical purity of the BA trans-3.4-dihvdrodiol formed in the metabolism of BA by three liver microsomes prepared from untreated rats and rats that had been pretreated with an enzyme inducer. As shown in Table II, cytochrome P-450 isozymes contained in liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats had similar stereoselectivity toward the 3,4-double bond of BA. BA trans-3.4-dihydrodiol is formed via the 3,4-epoxide intermediate (31). 

The 8,9- and 10,11-dihydrodiols formed in the metabolism of BA and DMBA respectively are all highly enriched (>90%) in R,R enantiomers (Table III). Labeling experiments using molecular oxygen-18 in the in vitro metabolism of the respective parent compounds and subsequent mass spectral analyses of dihydrodiol metabolites and their acid-catalyzed dehydration products indicated that microsomal epoxide hydrolase-catalyzed hydration reactions occurred exclusively at the nonbenzylic carbons of the metabolically formed epoxide intermediates (unpublished results). These findings indicate that the 8,9- and 10,11-epoxide intermediates, formed in the metabolism of BA and DMBA respectively, contain predominantly the 8R,9S and 10S,11R enantiomer, respectively. These stereoselective epoxidation reactions are relatively insensitive to the cytochrome P-450 isozyme contents of different rat liver microsomal preparations (Table III). 

Environmental agents that influence microsomal reactions will influence hexachloroethane toxicity. The production of tetrachloroethene as a metabolite is increased by agents like phenobarbital that induce certain cytochrome P-450 isozymes (Nastainczyk et al. 1982a Thompson et al. 1984). Exposure to food material or other xenobiotics that influence the availability of mixed function oxidase enzymes and/or cofactors will change the reaction rate and end products of hexachloroethane metabolism and thus influence its toxicity. 

The metabolism of 77-hexane takes place in the liver. The initial reaction is oxidation by cytochrome P-450 isozymes to hexanols, predominantly 2-hexanol. Further reactions convert 2-hexanol to 2-hexanone, 2,5-hexanediol, 5-hydroxy-2-hexanone, 4,5-dihydroxy-2-hexanone and the neurotoxicant 2,5-hexanedione. Hydroxylation at the 1- and 3- positions can be considered detoxification pathways hydroxylation at the 2- position is a bioactivation pathway. A diagram of the proposed pathway for mammalian metabolism of -hexane is presented in Figure 2-3. 

Because many other chemicals can affect the enzymes responsible for n-hexane metabolism (see Section 2.3.3, Metabolism), the possibility of interactions is a significant concern. The initial step in n-hexane metabolism is oxidation to a hexanol by a cytochrome P-450 isozyme other chemicals can induce these enzymes, possibly increasing the rate of metabolism to the neurotoxic 2,5-hexanedione, or competing with M-hexanc and its metabolites at enzyme active sites, reducing the rate of metabolism. Interactive effects can be concentration and/or duration dependent. 

Oral administration of acetone has been reported to potentiate the neurotoxicity caused by oral exposure to the /2-hexane metabolite 2,5-hexanedione in rats (Ladefoged et al. 1989, 1994). Oral exposure to acetone alone in rats at 650 mg/kg/day resulted in a statistically significant decrease in motor nerve conduction velocity after 6 weeks co-exposure to acetone and 2,5-hexanedione resulted in greater effects than those seen with 2,5-hexanedione alone (Ladefoged et al. 1989). It is possible that acetone may potentiate /2-hexane neurotoxicity by decreasing body clearance of 2,5-hexanedione (Ladefoged and Perbellini 1986). Acetone also influences the action of many chemicals by its induction of the cytochrome P-450 isozyme CYP2E1 (Patten et al. 1986). /2-Hexane is metabolized by P-450 isozymes... 

Toftgard R, Haaparanta T, Eng L, etal. 1986. Rat lung and liver microsomal cytochrome P-450 isozymes involved in the hydroxylation of M-hexane. Biochem Pharmacol 35(21) 3733-3738. 

Toftgard R, Halpert J, Gustafsson JA. 1983. Xylene induces a cytochrome P-450 isozyme in rat liver similar to the major isozyme induced by phenobarbital. Mol Pharmacol 23(1) 265-271. 

Lewandowski M, Levi P, Hodgson E. 1989. Induction of cytochrome P-450 isozymes by mirex and chlordecone. J Biochem Toxicol 4(3) 195-199. 

P. (1986) The molecular mechanisms of two common polymorphisms of drug oxidation—evidence for functional changes in cytochrome P-450 isozymes catalysing bufuralol and mephenytoin oxidation. Xenobiotica 16, 449-464. 

Polybrominated Biphenyls. A recent study has used caffeine as a potential tool to characterize exposure and/or effect of PBBs (Lambert et al. 1990). In this test, caffeine is used as a metabolic probe of cytochrome P-450 isozymes activity from the CYPIA family, which in animals is significantly induced by PBBs (Safe 1984). Tire caffeine breath test (CBT) is primarily useful for detecting induction of CYP1A2 activity in human liver, and for that reason, it also has been used as a marker for exposure to PCBs, CDDs, and CDFs (Lambert et al. 1992). A volunteer population of 50 Michigan subjects with previously high serum PBB levels and 50 with undetectable or low serum levels was compared to a control population not exposed to PBBs (Lambert et al. 1992). Two tests were conducted, the CYP1A2-dependent caffeine... 

Parkinson A, safe SH, Robertson LW, et al. 1983. Immunochemical quantitation of cytochrome P-450 isozymes and epoxide hydrolase in liver microsomes from polychlorinated or polybrominated biphenyl-treated rats. J Biol Chem 258(9) 5967-5976. 

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. 

Thus in some cases, enzyme expression is directly influenced in a particular organ by testosterone or estrogens. For example, in the mouse kidney, testosterone directly regulates the expression of cytochrome P-450 isozymes, and this leads to the particular sensitivity of the female kidney to the nephrotoxicity of paracetamol. 

Hietanen, E., Kobljakov, V. Bartsch, H. (1986) Role of different cytochrome P-450 isozymes in the demethylation of varions snbstrates. Gen. Pharmacol., 17, 565-568... 

Goldstein JA, Linko P, Hahn ME, et al. 1986. Structure-activity relationships of chlorinated benzenes as inducers of hepatic cytochrome P-450 isozymes in the rat. IARC Sci Publ 77 519-526. 

Beaune P, Kremers PG, Kaminsky LS et al. (1986) Comparison of monooxygenase activities and cytochrome P-450 isozyme concentration in human liver micrsosomes. Drug Metab Disp 14 437M42... 

Stegeman, J.J. Klopper-Sams, P.J., Cytochrome P-450 isozymes and monooxygenase activity in aquatic animals Environ. Health Perspect. 1987, 71, 87-95. 

Swinney, D. C., Ryan, D, E., Thomas, E E., and Levin, W. (1987). Regioselective Progesterone Hydroxylation Catalyzed by Eleven Rat Hepatic Cytochrome P-450 Isozymes, BiodKmistiy, 26 7073-7083. 

Yang, S. K. (1988). Stereoselectivity of Cytochrome P-450 Isozymes and Epoxide Hydrolase in the Metabolism of Polycyclic Aromatic Hydrocarbons, Biochetn. Pharmacol, 37 61-70. 

Yang, S. K. and Bao, Z.-P. (1987). Stereoselective Formations of K-Region and Non-K-Region Epoxides in the Metabolism of Chrysene by Rat Liver Microsomal Cytochrome P-450 Isozymes, Mol. Pharmacol, 32 73-80. 

Acute hepatotoxicity has been reported with isoflurane (14). The authors hypothesized that induction of cytochrome P-450 by phenytoin had caused enhanced transformation of isoflurane to trifluoroacetic acid. They suggested that caution should be taken in the use of halogenated anesthetics in patients taking drugs that induce cytochrome P-450 isozymes. 

Some cytochrome P-450 isozymes, such as CYP lAl are inhibited by enoxacin, resulting in potentially important interactions with other drugs. For example, enoxacin has been reported to decrease theophylline clearance, causing increas plasma levels and increa.sed toxicity. Enoxacin forms insoluble chelates with divalent metal ions present in antacids and hcmalinic.s. which reduce its oral bioavailabilily. 

The mechanism of chloroform nephrotoxicity involves the oxidation of chloroform to trichloro-methanol by renal cytochrome P-450 isozymes (Figure 6). The trichloromethanol readily eliminates HCl to form the highly reactive toxicant phosgene (COCI2). The phosgene can (1) be detoxified by conjugation with two molecules of glutathione, (2) react with water to form two molecules of HCl and one molecule of CO2, or (3) covalently bind to renal macromolecules to disrupt cellular function and induce nephrotoxicity. 

Huang M-T, Johnson EF, Muller-Eberhard U, Koop DR, Coon MJ, Conney AH. 1981. Specificity in the activation and inhibition by flavonoids of benzo-[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes. J. Biol. Chem. 256 10897-901... 

Getchell ML, ChenY, DingX, SparksDL, Getchell TV. 1993. Immunohistochemi-cal localization of a cytochrome P-450 isozyme in human nasal mucosa age-related trends. Ann. Otol. Rhinol. Laryn-gol. 102 368-74... 

Aliphatic EC>8-EC16 Fraction. Hydrocarbons in this fraction are oxidatively metabolized to fatty acids and alcohols, apparently mediated by cytochrome P-450 isozymes (see Miller et al. 1996 for review). Studies regarding the metabolism of hydrocarbons in this fraction in humans or animals provide suggestive evidence that metabolism may be slow. In a study of humans exposed to 100 ppm white spirit 6 hours/day for 5 days (white spirit is a mixture comprised predominately of C10-C12 linear and branched alkanes), only minor differences were observed in the GC-MS spectrum of hydrocarbons in biopsied fatty tissue, than in the spectrum of hydrocarbons in the test material (Pedersen et al. 

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