C. Roland Wolf

Identification of Cytochrome P-450 Isozymes in Nonciliated Bronchiolar Epithelial (Clara) and Alveolar Type II Cells Isolated from Rabbit Lung

Two forms of cytochrome P-450 (P-450I and P-450II) have been shown by several techniques to be present in both nonciliated bronchiolar cells (Clara) and alveolar type II cells isolated from rabbit lung. In contrast, the alveolar macrophage contains little or none of these cytochromes. Cross-reactivity between antibodies to cytochrome P-450I or P-450II and detergent-digested microsomes prepared from 80% type II or 70% Clara cell fractions was shown by Ouchterlony double immunodiffusion. The presence of both cytochromes was also demonstrated by histochemical immunofluorescence in smears of type II cells stained by a modified Papanicolaou procedure and Clara cells stained with nitroblue tetrazolium. However, this same fluorescent antibody technique used for localization of rabbit pulmonary cytochromes P-450I and P-450II in tissue sections showed most of the immunofluorescence in the Clara cells of the bronchiolar epithelium. SDS-polyacrylamide gel electrophoresis of microsomes from either the type II or Clara cell fractions produced bands which corresponded to cytochrome P-450I (52,000 daltons) and cytochrome P-450II (58,000 daltons).

Cytochrome P-450 mediated genetic activity and cytotoxicity of seven halogenated aliphatic hydrocarbons in Saccharomyces cerevisiae

Cells of Saccharomyces cerevisiae, harvested from log-phase cultures, contain cytochrome P-450 and are capable of metabolizing promutagens to genetically active products. The activities of 7 halogenated aliphatic hydrocarbons in the yeast system have been investigated. All of the compounds tested (methylene chloride, halothane, chloroform, carbon tetrachloride, trichloroethylene, tetrachloroethylene and s-tetrachloroethane) induced mitotic gene convertants and recombinants and, to a lesser extent, gene revertants when incubated with log-phase cells of the yeast strain D7. An examination of the difference spectra observed upon the addition of carbon tetrachloride, halothane and trichloroethylene to whole-cell or microsomal suspensions of yeast suggested that cytochrome P-450 mediated the metabolism of the hydrocarbons tested to cytotoxic and genetically active compounds.

The rabbit pulmonary monooxygenase system: characteristics and activities of two forms of pulmonary cytochrome P-450.

Two forms of rabbit pulmonary cytochrome P-450 have been characterized spectrally and their activities in reconstituted monooxygenase systems investigated. The presence of both microsomal phospholipids and sodium cholate was required to obtain optimum activity. Only one of the cytochromes (I) was active in the N-demethylation of benzphetamine and the O-deethylation of 7-ethoxycoumarin. However, cytochrome II was 20% more active than cytochrome I in the metabolism of benzo[a]pyrene. The profile of the metabolites formed from benzo[a]pyrene indicated that metabolism at the 9 and 10 positions was insignificant in the case of cytochrome I but represented about 40% of the metabolites produced by cytochrome II. The two forms of the cytochrome are present in pulmonary microsomes in approximately equal amounts.

Purification and structural comparison of pulmonary and hepatic cytochrome P-450 from rabbits

A procedure is described for the purification of a major form of cytochrome P-450 from the livrs of rabbits treated with phenobarbital and a major form of the cytochrome from the lungs of untreated rabbits. Preparations in good yield (13--17%) and of high purity (up to 21 nmol of cytochrome per mg of protein) that were free of lipid and contained minimal amounts of non-ionic detergent were obtained from either tissue. The two cytochromes cannot be distinguished from each other on the basis of absorption spectra, extinction coefficients, apparent molecular weights (52 000), amino acid compositions, or peptide fragments produced by treatment of the proteins with cyanogen bromide. These data are consistent with a major indigenous form of rabbit pulmonary cytochrome P-450 being the same as the major form of hepatic cytochrome induced by phenobarbital.

Interaction of 4-methylbenzaldehyde with rabbit pulmonary cytochrome p-450 in the intact animal, microsomes, and purified systems: Destructive and protective reactions

Abstract About 50 per cent of rabbit pulmonary cytochrome P-450 is destroyed by treatment of the intact animal, microsomes, or systems reconstituted from purified pulmonary mono-oxygenase components with 4-methylbenzaldehyde. The loss of the cytochrome is accompanied by an equimolar loss of heme. The action of 4-methylbenzaldehyde requires the presence of NADPH and O2 and appears to result from cytochrome P-450-catalyzed metabolism. Selective destruction of one of the known forms of rabbit pulmonary cytochrome P-450 does not account for the lack of complete destruction of pulmonary P-450 by 4-methylbenzaldehyde; loss of about 50 per cent of each form of the cytochrome occurs in vivo and in reconstituted systems. However, form II is affected to a greater extent than form I when microsomes are incubated with 4-methylbenzaldehyde. The portion of the cytochrome not degraded by 4-methylbenzaldehyde appears to be protected by some factor produced from 4-methylbenzaldehyde during the incubation. This factor also protects against complete destruction of the cytochrome by cumene hydroperoxide

Cumene hydroperoxide and yeast cytochrome P-450: spectral interactions and effect on the genetic activity of promutagens.

Abstract Cells of Saccharomyces cerevisiae , harvested from log phase cultures, contain cytochrome P-450 and are capable of activating promutagens to products that are genetically active in the same cell. The effect of cumene hydroperoxide, a compound known to support cytochrome P-450-mediated reactions, on the activation of a variety of the promutagens was investigated. In all cases the genetic activity of the promutagens was increased. With dimethyl-nitrosamine as the promutagen, the increased rate of gene conversion was linear for at least 1 hr. Yeast cytochrome P-450 was stable in intact cells in the presence of cumene hydroperoxide. However, in microsomal preparations the cytochrome was rapidly destroyed. When cumene hydroperoxide was added to a suspension of intact yeast cells, a spectrum with a Soret maximum at 455 nm — indicative of an interaction with cytochrome P-450 — was observed.

THE REDUCTIVE METABOLISM OF CARBON TETRACHLORIDE IN RECONSTITUTED MONOOXYGENASE SYSTEMS

The reduction of carbon tetrachloride (CCl4) to chloroform (CHCI3) by reconstituted monooxygenase systems4 demonstrates that cytochrome P-450 is the enzyme that catalyzes the reaction. The reaction is totally inhibited by air and significantly inhibited by free radical scavengers. CCl4 metabolism, determined by both CHCl3 formation and CCl4 disappearance, could not be detected under aerobic conditions although significant levels of CCl4 -induced lipid peroxidation could be. These findings appear to contradict the accepted theory that a trichloromethyl radical is responsible for the initiation of the lipid peroxidation reaction.

THE INTERACTION OF POLYHALOGENATED METHANES WITH FERROUS CYTOCHROME P450

ABSTRACT The interaction of a series of halogenomethanes, CCl 4 , CBr 4 , CCl 3 Br, CCl 3 F, CHCl 3 , CHBr 3 , CHI 3 , CCl 3 CN, CH 2 Cl 2 and CH 2 Br 2 with ferrous cytochrome P450 was investigated in anaerobic rat liver microsomal preparations in the presence of sodium dithionite or NADPH. Apart from CH 2 Cl 2 and CH 2 Br 2 these compounds exhibited complexes with ferrous cytochrome P450 having absorption maxima in the difference spectra between 450 and 470 nm. The proposal that these complexes result from a cytochrome P450 mediated two electron reduction resulting in the formation of a carbene intermediate was strengthened by the finding that carbon monoxide was a product of the reaction. Carbon monoxide is a known hydrolysis product of dihalocarbenes.

CHARACTERIZATION OF PURIFIED FORMS OF RABBIT PULMONARY CYTOCHROME P-450 AND COMPARISON WITH THE HEPATIC CYTOCHROME P-450 INDUCED BY PHENOBARBITAL

Purified rabbit pulmonary cytochrome P-450 I (P-450 I ) and the major form of rabbit hepatic phenobarbital-induced cytochrome P-450 (P-450 PB ) were shown to be indistinguishable on the basis of monomeric molecular weighty, amino acid composition, and peptide maps. Based upon these criteria, however, pulmonary P-450 II was shown to differ from P-450 I and P-450 PB .

PURIFICATION OF RABBIT PULMONARY AND HEPATIC CYTOCHROME P-450 BY HYDROPHOBIC COLUMN CHROMATOGRAPHY

Procedures have been developed for the purification of two forms of rabbit pulmonary cytochrome P-450 and the hepatic form induced by phenobarbital. The procedures take advantage of the hydrophobic nature of cytochrome P-450 through the utilization of octyl- and phenyl-Sepharose 4B. Pulmonary cytochrome P-450I and the hepatic cytochrome can be purified to 21 nmol/mg protein by the reported method and the final preparations of these hemoproteins contain up to 17% of the microsomal P-450 content. This suggests a yield of up to 33% for the form of the cytochrome purified. Pulmonary cytochrome P-450I and the hepatic form of P-450 induced by phenobarbital appear to be the same enzyme as determined by a number of criteria. Pulmonary cytochromes P-450I and P-450II are clearly different proteins.