Carbon monoxide P450 inhibition

Figure 11-6. Cytochrome P450 hydroxylase cycle in microsomes. The system shown is typical of steroid hydroxylases of the adrenal cortex. Liver microsomal cytochrome P450 hydroxylase does not require the iron-sulfur protein FejSj. Carbon monoxide (CO) inhibits the indicated step.

Formaldehyde generated from nitromethane was found only in trace amounts after incubation with microsomes from from Fischer 344 rat liver, but none was found after incubation with rat nasal microsomes (Dahl Hadley, 1983). Nitromethane inhibited rabbit liver cytochrome P450 activity, apparently competing for the same ferrohaemo-chrome-binding sites as carbon monoxide (Wade et al, 1977). 

In the fall army worm, Spodopterafrugiperda, sulfoxidation of phorate requires NADPH. It is inhibited by carbon monoxide and piperonyl butoxide, and induced by cytochrome P450 inducers (e.g., indole 3-carbinol and indole 3-acetonitrile) (Yu, 1985). 

Carbon monoxide inhibited the 6/3-. la-, and 16a-hydroxylation of testosterone by rat liver microsomes to different extents. A C0/02 ratio of 0.5 inhibited the la-, 6/i-, and 16a-hydroxylation reactions by 14%, 25%, and 36%, respectively, and the ratio of C0/02 needed for 50% inhibition of testosterone hydroxylation in the 16a-, 6/3-, and 7a-positions was 0.93, 1.54, and 2.36, respectively (36,48). Studies on the photochemical action spectrum revealed that CO inhibition of the three hydroxylation reactions was maximally reversed by monochromatic light at 450 nm, but there were differences in the shape of the photochemical reactivation spectra for the 6/3-, la-, and 16a-hydroxylation reactions (36,48). The data from our laboratory summarized above and at the First International Symposium on Microsomes and Drug Oxidation in 1968 pointed to multiple cytochromes P450 with different catalytic activities that were under separate regulatory control (36,45,46), and we indicated that the actual number of cytochromes that participate in the multiple hydroxylation reactions must await the solubilization and purification of the microsomal system (36). The use of different inducers of liver microsomal monooxygenases caused selective increases in the concentration of specific cytochromes P450 in fiver microsomes that greatly facilitated the isolation and purification of these hemoproteins. 

In vitro studies showed that rat liver microsomes activated with NADPH and molecular oxygen metabolized MMT (Hanzlik et al. 1980b). Preliminary studies with pooled liver microsomes from 5-6 normal or pheno-barbital-induced rats showed that reaction rates of metabolism were linear for the first 20 minutes. MMT and aminopyrine, a positive control compound that is metabolized exclusively by cytochrome P450, showed parallel responses to changes in incubation conditions (i.e. NADPH dependence, inhibition by carbon monoxide, induction by phenobarbital). Liver microsomes metabolized MMT with an estimated of 78 pM and a Vni of 3.12 nmol/mg protein/min. When the studies were done with liver microsomes from phenobarbital-treated rats, the remained the same, but the doubled (Hanzlik et al. 1980b). Lung microsomes were equally capable of metabolizing MMT, but phenobarbital induction did not enhance the response. 

Leeman, T, P. Bonnabry, and P. Dayer (1994). Selective inhibition of major drug metabolizing cytochrome P450 isozymes in human liver microsomes by carbon monoxide. Life Sci. 54, 951 956. 

The major metabolic pathway of terminal acetylenic derivatives is via oxidation to the corresponding acetic acid derivatives. Thus, Sullivan and coworkers and Wade and coworkers showed in 1979 that the major metabolites of ethynylbiphenyls in the rat were biphenyl-4-yl acetic acids. These metabolites are further oxidized to 4 -hydroxybiphenyl-4-yl acetic acids before being excreted in the urine (Scheme 4). Because the in vitro metabolism of biphenylacetylenes by rat liver microsomes requires nicotine-adenine-dinucleotide phosphate (NADPH) and molecular oxygen and is inhibited by carbon monoxide, it was concluded that the oxidative metabolism of the acetylenes to the corresponding acetic acid derivatives is mediated by cytochrome P450 . Acetic acid... 

The possible role of cytochrome P450 as an enzyme that can hydroxylate tropane alkaloids has been examined in vitro by use of a rat liver microsomal preparation. 3a-Tigloyloxytropane (40) was converted into meteloidine (41). The reaction was dependent on NADPH and was inhibited by carbon monoxide, thus suggesting that cytochrome P450 was the enzyme responsible. 

The enzyme reaction is inhibited by the typical cytochrome P450 inhibitors. Light reverses the inhibition by carbon monoxide. The obtained data strongly indicate that the DH is a novel cytochrome P450-dependent monooxygenase (pH optimum 8.0). The analysis of the presence of the hydroxylase in nine ceU cultures of seven different plant famUies indicates a very limited taxonomic distribution of this enzyme. 

Estabrook, demonstrated the involvement of cytochrome P450 enzymes in steroid biochemistry when they showed that carbon monoxide inhibited the 21-hydroxylase activity in adrenal microsomes [3], Radiolabeled steroids became commercially available in the 1960s, which fa-cihtated experiments to confirm precursor-product relationships. Despite these initial advances, further progress was slow, due to low abundance of enzymes, the need to obtain animal adrenals as the source, the tedious nature of the assays, species-specific variations in the pathways, and the inability to purify the enzymes, which limited the interpretation of messy experiments. 

Cinnarizine, l-(diphenylmethyl)-4-(3-phenyl-2-propenyO-piperazine is a selective calcium entry blocker, and is extensively used in the treatment of cerebral and peripheral insufficiency (Godfraind et al. 1982, Singh 1986). Its oxidative metabolism to l-(diphenylmethyl)piperazine (M-1), l-(diphenyl-methyl)-4-[3-(4 -hydroxyphenyl)-2-propenyl]-piper-azine (M-2b benzophenone (M-3) and l-[4 -hydro-xyphenyl)-phenylmethyl] - 4 -(3-phenyl-2-propenyl) piperazine (M-4) in rat liver microsomes required NADPH, and was inhibited by carbon monoxide and SKF 525-A, typical inhibitors of P450 (Kariya et al. 1992). Only M-2 formation was suppressed by sparteine or metoprolol, and was significantly lower in female Dark Agouti rats than in Wistar rats of both sexes. 

In the first step of bile acid formation, cholesterol is hydroxylated in the 7 position by a microsomal hydroxylase that requires NADPH, thus yielding the 5-cholestene-3j5, 7a-diol. Cytochrome c reductase and cytochrome P450 have been suspected to be involved because the reaction is inhibited by carbon monoxide. It is not certain whether the 7a hydroxylation preferentially attacks cholesterol or cholesterol esters. 

Inducible nitric oxide synthase (iNOS) suppression by CO. iNOS is a cytochrome p450 type protein, and is responsible for the synthesis of nitric oxide (NO), which is known to cause an inflammatoiy response. Carbon monoxide has been shown to suppress the activity of this en me. It is clear that the CO binds to the haem in this protein and inhibits the two step reaction that occurs at the protein haem centre. CO can be considered as an anti inflammatoiy molecule as it can suppress the production of a molecule associated with pro-inflammatoiy action. This is useful and important when potentially using CO as a therapeutic molecule. 

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