Inhibition of microsomal cytochrome c reductase activity by a series of alpha, beta-unsaturated aldehydes.

alpha, beta-Unsaturated aldehydes are reactive and cytotoxic compounds which occur in the environment and are also formed in vivo. Many of these aldehydes have been reported to inhibit hepatic cytochrome P-450. Our laboratory has shown that trans,trans-muconaldehyde (a possible metabolite of benzene) as well as acrolein and crotonaldehyde, when added to hepatic microsomes, decreased cytochrome P-450 (measured spectrophotometrically). Additional studies showed that several alpha, beta-unsaturated aldehydes also inhibited hepatic microsomal NADPH-cytochrome c reductase. Acrolein, crotonaldehyde and trans,trans-muconaldehyde all decreased NADPH-cytochrome c reductase activity in vitro. Concentrations of 0.5, 1.0 and 1.5 mM acrolein decreased activity to 60, 26 and 11% of control respectively. Similar concentrations of trans,trans-muconaldehyde inhibited NADPH-cytochrome c reductase. Crotonaldehyde was a less effective inhibitor of this enzyme. Propionaldehyde, a saturated aldehyde, had no effect on NADPH-cytochrome c reductase activity. Time course experiments with acrolein over a period of 5-45 min suggest that the loss of NADPH-cytochrome c reductase activity is non-linear. The addition of reduced glutathione protected against the inhibition of reductase activity by acrolein. Formation of these aldehydes and their subsequent inhibition of these enzymes may have important consequences in xenobiotic metabolism.

Mutagenicity and toxicity studies of several alpha,beta-unsaturated aldehydes in the Salmonella typhimurium mutagenicity assay.

alpha,beta-Unsaturated aldehydes are reactive compounds which are ubiquitous in the environment. This class of compounds has been tested for mutagenicity in Salmonella typhimurium by a number of groups who have obtained differing results. The present studies were undertaken to test the mutagenicity and toxicity of two novel alpha, beta-unsaturated aldehydes, specifically trans, trans-muconaldehyde and trans-4-hydroxynonenal, and to re-examine the mutagenicity of crotonaldehyde. Trans, trans-muconaldehyde is a newly found microsomal metabolite of benzene, and trans-4-hydroxynonenal is a toxic aldehyde formed endogenously during lipid peroxidation. Compounds were tested in S. typhimurium strain TA 100 using a 30-min liquid preincubation procedure. The present mutagenicity studies indicate that these alpha, beta-unsaturated aldehydes at first appear to be mutagenic, although only at concentrations which decrease survival counts, and result in a disappearance of the bacterial lawn. The colonies observed on mutagenicity test plates are not mutants but rather pin point survivors.

Brain cytochrome P450 and testosterone metabolism by rat brain subcellular fractions: presence of cytochrome P450 3A immunoreactive protein in rat brain mitochondria.

The hydroxylation of testosterone by rat brain subcellular fractions has been studied using an HPLC method with an enhanced resolution for the separation of testosterone and its monohydroxy derivatives. Although the analysis time is longer than that reported for earlier methods, a baseline separation was obtained between all hydroxytestosterones, excepting 6 alpha-hydroxytestosterone and 15 beta-hydroxytestosterone, which were separated using a second chromatography system. This separation was important as rat brain microsomes metabolized testosterone to 15 alpha-, 6 beta-, 15 beta-, 16 beta-, 2 beta-, 1 beta-hydroxytestosterone and androstenedione. Testosterone metabolism was found to be linear with time and protein concentration. The rat brain mitochondrial fraction metabolized testosterone to androstenedione. Small amounts of immunoreactive bands comigrating with purified cytochromes P450j, P450b, and P450p were detected by Western blot analysis in rat brain microsomes, while only an immunoreactive protein related to cytochrome P450p was found in the mitochondrial fractions. Immunoinhibition studies showed that BEA33, a monoclonal antibody to cytochrome P450b and simultaneously recognizing cytochromes P450e and P450a, was able to inhibit the metabolism of testosterone to the 1 beta-, 15 alpha-, 2 beta-, and 6 alpha-hydroxylated metabolites, whereas polyclonal anti-cytochrome P450p did not inhibit the formation of the 6 beta-hydroxytestosterone by rat brain microsomes. The metabolism of testosterone by rat brain microsomal or mitochondrial fractions was refractory to induction by 3-methylcholanthrene or pregnenolone-16 alpha-carbonitrile. Thus, in the brain multiple isozymes of cytochrome P450 are constitutively expressed in different subcellular fractions, which suggests that brain cytochrome P450 may play an important role in the metabolism of endogenous compounds. The significance and role of cytochrome P450p-related protein in the rat brain mitochondrial fraction are yet to be determined.

The effects of alpha, beta-unsaturated aldehydes on hepatic thiols and thiol-containing enzymes.

The effects of series of alpha, beta-unsaturated aldehydes on hepatic glutathione, cytochrome P450, and NADPH-cytochrome c reductase activity were compared with time. Male F-344 rats were dosed with muconaldehyde (36 mumol/kg), acrolein (89 mumol/kg), crotonaldehyde (450 mumol/kg), or the saturated aldehyde propionaldehyde (89 mumol/kg) and terminated 0.5, 4, or 24 hr later. Acrolein or muconaldehyde reduced glutathione to 51 and 75% of controls, respectively, at 4 hr; glutathione returned to control values at 24 hr. Only at 24 hr, acrolein, muconaldehyde, or crotonaldehyde decreased cytochrome P450 to 61, 71, and 67% of control values, respectively; ethylmorphine N-demethylation was decreased to a greater extent, i.e., to 35, 60, and 23% of controls. The reductase activity was unchanged at any time following the treatment with reactive aldehydes which were not hepatotoxic (as shown by glucose 6-phosphatase activity, histological changes, or serum enzymes). Propionaldehyde changed none of these activities. Acrolein (44.5 mumol/kg) given 4 hr prior to phenobarbital (50 mg/kg) for two consecutive days decreased the phenobarbital induction of cytochrome P450 to 45% of phenobarbital alone. This treatment also decreased the 2 alpha, 2 beta, 6 beta, 16 alpha, and 16 beta hydroxylation of testosterone as well as androstenedione formation showing effects on individual cytochrome P450 isozymes. NADPH-cytochrome c reductase induction was not decreased by this treatment, thus indicating that in vivo these changes are due to a mechanism other than generalized inhibition of protein synthesis.

Regulation of two members of the steroid-inducible cytochrome P450 subfamily (3A) in rats.

Monoclonal antibodies have been successfully isolated which are isozyme-specific for cytochrome P450p (3A1) or P4501 (3A2), two members of the steroid-inducible cytochrome P450 subfamily exhibiting 89% amino acid sequence homology, and these antibodies show less than 5% cross-reaction with 11 other cytochromes P450 (P450a-P450k). A library of 28 purified monoclonal antibodies was established and characterized as to epitope specificity. Appropriate antibodies were selected and utilized to investigate the regulation of expressed cytochrome P450p and P4501 proteins as a function of age, sex, and treatment of rats with various inducing agents. Cytochrome P450p is not detectable in hepatic microsomes from untreated immature or adult male and female rats. Following dexamethasone treatment, expression of cytochrome P450p is observed in all groups with the levels reaching 30-37% of total microsomal cytochrome P450. Administration of other inducers such as pregnenolone 16 alpha-carbonitrile also yield enhanced levels of cytochrome P450p. Measurable amounts of constitutive cytochrome P4501 were detected in hepatic microsomes from immature and adult males as well as immature females but not in adult females. Cytochrome P4501 expression is inducible by dexamethasone in immature rats of both sexes and adult males, although dexamethasone is more effective as an inducer of cytochrome P450p than cytochrome P4501. Hence, not only is cytochrome P4501 protein expressed in immature animals of both sexes, it is also inducible in both sexes. These studies show that constitutive expression and induction of steroid-inducible cytochrome P450s may vary as a function of age.