P450-dependent Mixed-function Oxidase Research Articles

Catechol estrogen-forming enzyme of brain: demonstration of a cytochrome p450 monooxygenase.

A catechol estrogen-forming enzyme has been demonstrated in rat brain microsomes using a sensitive radioenzymatic assay. This method is based on conversion of the relatively labile catechol estrogens to their stable O-methylated derivatives. By employing a methyl donor of high specific activity (S-adenosyl-L-[methyl-3H]methionine), a partially purified preparation of catechol- O-methyltransferase (COMT), and selective solvent extraction this method has proven to be extremely sensitive. The specificity of the assay was established by subjecting the O-methylated product to thin layer chromatography in several solvent systems and further confirmed by mass spectral analysis of the chromatographed product. The enzymatic activity of rat brain is optimal using reduced NADP and is inhibited by well known inhibitors of P450-dependent mixed function oxidases, CO and SKF- 525A. The subcellular distribution, cofactor requirements, and the effects of various inhibitors strongly suggest that the enzymatic activity of b...

Evidence for the Clara cell as a site of cytochrome P450-dependent mixed-function oxidase activity in lung.

THE cellular localisation of pulmonary mixed-function oxidase (MFO) activity has aroused considerable interest, especially because of the increasing incidence of lung disease of environmental origin—including cancers—and the realisation that many chemical toxins and carcinogens require MFO-catalysed activation. The precise identification of pulmonary cellular populations possessing MFO activity has previously met with little success, however, probably because of the complexity and heterogeneity of the lung, which has been estimated to contain over 40 different specific cell types1. In this communication I describe studies with the pulmonary toxin, 4-ipomeanol (1-[3-furyl]-4-hydroxypentanone)2, which indicate that a highly reactive metabolite of this naturally occurring furan derivative is preferentially formed and covalently bound in pulmonary non-ciliated bronchiolar (Clara)3 cells, leading to their necrosis. Since previous studies4 have established that the toxic metabolite formed in vivo during cytochrome P450-dependent oxidative metabolism of 4-ipomeanol is formed in situ in the lung, and does not reach the lung by way of the circulation, the present results indicate that the Clara cell is a primary locus of P450-dependent MFO enzymes in lung.

Dehydrogenation: A previously unreported pathway of lindane metabolism in mammals

This study presents evidence for the dehydrogenation of lindane by a hepatic microsomal mixed-function oxidase system. Preliminary investigation established that the incubation of lindane with rat liver homogenates produces a chlorinated, nonpolar compound identified as hexachlorocyclohexene. Differential centrifugation resulted in the sedimentation of most of the dehydrogenase activity in the microsomal fraction. Optimum in vitro assay conditions were established and it was found that the dehydrogenase system required molecular oxygen and reduced pyridine nucleotide coenzyme for maximum activity. Inhibition by SKF 525-A and CO suggested that the enzyme was cytochrome P-450 dependent. Lack of inhibition by cyanide indicated that the cytochrome b 5 desaturase system was probably not involved. Pretreatment of rats with DDT, which stimulates lindane metabolism, also induced significantly higher dehydrogenase activity. Both the in vivo and in vitro metabolism of hexachlorocyclohexene produced previously identified lindane metabolites. The existence of a cytochrome P-450 dependent mixed-function oxidase which catalyzes the dehydrogenation of lindane has not previously been reported and may be of importance in the metabolism of other xenobiotics.

Mutagenicity of primary and secondary carcinogens altered by normal and induced hepatic microsomes.

To establish the role of the activity of the microsomal biotransformation system in chemical carcinogenesis, the mutagenic effect of primary (ultimate) and secondary (potential) carcinogens after exposure to isolated hepatic microsomes was studied. The principal enzyme system, suggested to be active in the metabolism of carcinogenic compounds, is the nonspecific cytochrome P-450 dependent mixed-function oxidase (1). This microsomal enzyme system is inducible by many substrates, including some environmental contaminants (2). The involvement of the microsomal enzymes in carcinogenesis is suggested by the observed increased carcinogen biotransformation in animals (3) and isolated cells (4) following exposure to inducing substrates. Most carcinogenic compounds are mutagenic for microorganisms (5). The primary are themselves mutagenic, the secondary are inactive until metabolized to a mutagenic form (6). Metabolism of primary carcinogens may reduce mutagenic and carcinogenic properties. Both types of metabolic...

On the binding of aflatoxin B 1 and its metabolites to hepatic microsomes

The metabolism of aflatoxin B 1 was studied using the cytochrome P450-dependent mixed function oxidase system of rat liver microsomes. An aflatoxin metabolite produced in the presence of microsomes and NADPH and not produced in the presence of SKF-525A seems to become covalently bound to microsomes. The bound metabolite is observed as a spectral peak at 412 nm by means of difference spectroscopy. This metabolite appears to be related to either aflatoxin B 2a or its precursor.

The effects of thiols on cholesterol synthesis by rat liver [formula omitted

Certain thiols such as β-mercaptoethylamine, β-mercaptoethanol, ethanethiol and dithiothreitol inhibit cholesterol synthesis from mevalonic acid (MVA) in rat liver homogenates and cause accumulation of a mixture of lanosta-8,24-dien-3β-ol (lanosterol) and lanost-8-en-3β-01 ( Δ 8-lanosterol). Cysteine and glutathione inhibit cholesterol synthesis without accumulation of lanosterol and Δ 8-lanosterol. Since it has been proposed that the demethylation of lanosterol involves hydroxylation of the methyl group this process must differ from certain P450-dependent mixed function oxidases which are stimulated by similar concentrations of β-mercaptoethylamine.