NADPH:cytochrome P-450(c) reductase: Biochemical characterization in rat brain and cultured neurons and evolution of activity during development

NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochromec as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's V_max values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.     

Functional Expression of the Alkane-Inducible Monooxygenase System of the Yeast: Candida tropicalis IN Saccharomyces cerevisiae

The genes for the alkane-inducible monooxygenase system of the yeast Candida tropicalis, namely a cytochrome P450_alk (P450_alk) and a NADPH cytochrome P450 oxidoreductase (NCPR) gene, were transferred in Saccharomyces cerevisiae. The P450_alk gene was expressed in this host with the help of the yeast alcohol dehydrogenase I (ADHI) promoter and terminator, whereas the NCPR gene could be expressed with its own structural elements. The presence of P450_alk in S. cerevisiae microsomal fractions resulted in a new acquired lauric acid terminal hydroxylation activity. Moreover, the same activity, coupled with the appearance of 12-hydroxylauric acid derivatives, could be obtained by the addition of lauric acid to intact cells expressing P450_alk. The coordinate expression of the P450_alk and NCPR genes in S. cerevisiae elevated the turnover rate of the P450_alk monooxygenase activity about 2-fold.     

Differences in induction of hepatic cytochrome p450 isozymes by mice in eight methylenedioxyphenyl compounds

Eight methylenedioxyphenyl (MDP) compounds were examined for their ability to induce cytochrome P450 (P450) in mouse liver. Induction by safrole, isosafrole, and dihydrosafrole was studied in both C57BL/6N (Ah-responsive) and DBA/2N (Ahnonresponsive) male mice after IP administration of 200 mg/kg/day MDP compound for 3 days. Hepatic P450 content, ethylmorphine N-demethylase, ethoxy-resorufin O-deethylase, and acetanilide hydroxylase activities were induced to the same extent in both strains of mice. Benzo(a)pyrene hydroxylase activity, however, was not induced in either C57 or DBA mice. The similarity of results in both strains of mice indicated induction of these P450 isozymes by these three MDP compounds is not mediated by the Ah receptor. Induction of P450 by butylbenzodioxole (n-butyl-BD), tertiarybutylbenzodioxole (t-butyl-BD), methylbenzodioxole (methyl-BD), nitrobenzodioxole (nitro-BD), and bromobenzodioxole (bromo-BD) was examined only in C57BL/6N mice. Methyl-BD, nitro-BD, and bromo-BD did not induce hepatic microsomal proteins or selected P450 monooxygenase activities. In contrast, n-butyl-BD, and t-butyl-BD induced P450 content, ethylmorphine N-demethylase, acetanilide hydroxylase, and ethoxyresorufin O-deethylase activities. Benzo(a)pyrene hydroxylase was not induced by any of the treatments. Induction of these P450 activities is consistent with induction of P450 IIB1 and P450 IA2, but not induction of P450 IA1. Western blot analysis with antibodies to P450 isozymes induced with either phenobarbital (Pb) or 3-methylcholanthrene (3-MC) confirmed that both IIB1 and IA2 were induced, but that IA1 was not induced.     

Radical Cations in Aromatic Hydrocarbon Carcinogenesis

Most carcinogens, including polycyclic aromatic hydrocarbons (PAH), require metabolic activation to produce the ultimate electrophilic species that bind covalently with cellular macromolecules to trigger the cancer process. Metabolic activation of PAH can be understood in terms of two main pathways: one-electron oxidation to yield reactive intermediate radical cations and monooxygenation to produce bay-region diol epoxides. The reason we have postulated that one-electron oxidation plays an important role in the activation of PAH derives from certain common characteristics of the radical cation chemistry of the most potent carcinogenic PAH. Two main features common to these PAH are: 1) a relatively low ionization potential, which allows easy metabolic removal of one electron, and 2) charge localization in the PAH radical cation that renders this intermediate specifically and efficiently reactive toward nucleophiles. Equally important, cytochrome P-450 and mammalian peroxidases catalyze one-electron oxidation. This mechanism plays a role in the binding of PAH to DNA. Chemical, biochemical and biological evidence will be presented supporting the important role of one-electron oxidation in the activation of PAH leading to initiation of cancer.     

Causal connection between detoxification enzyme activity and consumption of a toxic plant compound

Insect herbivores can increase their detoxification activities against a particular plant poison in response to prolonged ingestion of the same compound. For example, larval tobacco hornworms (Manduca sexta) experience a dramatic increase in cytochrome P450 activity against nicotine after ingesting nicotine. While it is generally assumed that this induction process permits increased consumption of toxic plant tissues, we are not aware of any direct experimental support for this assumption. Using a two-tiered approach, we examined the functional significance of P450 induction to M. sexta larvae ingesting a toxic but non-deterrent concentration of nicotine. First, we related the time-course of P450 induction in midgut microsomes to changes in nicotine consumption. When offered a nicotine diet, larvae failed to show a significant increase in consumption before 36 h, which was coincident with the time-course of the induction of midgut P450 activities against aldrin and nicotine. Second, we determined whether inhibiting the induced P450 activities affected nicotine consumption. We found that the increase in nicotine consumption following the induction of nicotine metabolism could be strongly inhibited by treatment with piperonyl butoxide, which by itself did not inhibit consumption. These results provide direct evidence for a causal connection between P450-mediated detoxification activity and consumption of a toxic plant compound.Abbreviation PB piperonyl butoxide     

The other kind of biological NMR—Studies of enzyme-substrate interactions

NMR spectroscopy has proved to be a valuable tool in the study of the interactions between enzymes and their substrates. The kinds of structural and dynamic information which can be obtained are illustrated by studies of three enzymes involved in drug metabolism. Cytochromes P₄₅₀ play a crucial role in metabolism of a wide range of exogenous chemicals. NMR has been used to measure distances from the haem iron of the cytochrome to protons of the bound substrate, leading to detailed structural models for the enzyme-substrate complexes. The other two enzymes, chloramphenicol acetyltransferase and β-lactamase, are responsible for bacterial resistance to specific antibiotics. In chloramphenicol acetyltransferase, NMR has been used to determine the conformation of coenzyme A bound to the enzyme, while in the case of β-lactamase the pK of a specific lysine residue at the active site has been determined, providing valuable information on the catalytic mechanism. Special issue dedicated to Dr. Herman Bachelard.     

Anatomical distribution of NADPH-cytochrome P450 reductase and cytochrome P4502D forms in rat brain: Effects of xenobiotics and sex steroids

Since the brain is not a homogeneous organ, but one dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monoxygenase system in brain, as well as the regulation of its expression, is important in elucidating its function in that organ. In order to study these issues, female rats-both ovariectomized and not-were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and P450 reductase determined using polymerase chain reaction. Results of this investigation indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR was utilized to demonstrate the effects of xenobiotics (phenobarbital, β-naphthoflavone, imipramine) and sex hormones (testosterone, estrogen) on the levels of these messages in the female rat brain. Significant induction of message for P4502D forms was noted with testosterone in the absence of estrogen. The level of mRNA for reductase was not significantly influenced by any of the treatments, however. These results raise the issue of a sexual dimorphism in the rat regarding P4502D expression in brain.     

Distribution and activity of microsomal NADPH-dependent monooxygenases and amino acid decarboxylases in cruciferous and non-cruciferous plants, and their relationship to foliar glucosinolate content

The NADPH-dependent conversion of amino acids to their aldoximes is an initial step in glucosinolate biosynthesis. A number of microsomal aldoxime-forming monooxygenase activities were detected in leaves from a variety of glucosi-nolate-containing species, whereas barley, bean and tobacco leaves did not contain any such activities. The substrates for these monooxygenases in each species largely correlated with the spectrum of glucosinolates found in that species. No activity was detected that metabolized homomethionine (supposed precursor of 2-propenylglucosinolate [sinigrin]), even in species where sinigrin was the major glucosinolate. In Sinapis species containing hydroxybenzylglucosinolate (sinalbin), activity with L-Tyr was detected, whereas Brassica species containing sinalbin had no such activity. However, these Brassicas did contain an L-Phe monooxygenase activity. Partial characterization of the monooxygenases indicated that in Brassica species, Nasturtium officinalis and Raphanus sativus these resembled the flavin-linked monooxygenases previously found in oilseed rape (Brassica napus) and Chinese cabbage (Brassica campestris). The L-Tyr-dependent activity in Sinapis species, and the L-Phe-dependent activity in Tropacolum majus, had characteristics of cytochrome P450-type enzymes. No similarity was found with any other known amino acid metabolizing enzymes (including decarboxylases, amino acid oxidases and diamine/polyamine oxidases).     

The anticarcinogen 3,3′-diindolylmethane is an inhibitor of cytochrome P-450

Dietary indole-3-carbinol inhibits carcinogenesis in rodents and trout. Several mechanisms of inhibition may exist. We reported previously that 3,3′-diindolylmethane, an in vivo derivative of indole-3-carbinol, is a potent noncompetitive inhibitor of trout cytochrome P450 (CYP) 1A-dependent ethoxyresorufin O-deethylase with K_i values in the low micromolar range. We now report a similar potent inhibition by 3,3′-diindolylmethane of rat and human CYP1A1, human CYP1A2, and rat CYP2B1 using various CYP-specific or preferential activity assays. 3,3′-Diindolylmethane also inhibited in vitro CYP-mediated metabolism of the ubiquitous food contaminant and potent hepatocarcinogen, aflatoxin B₁. There was no inhibition of cytochrome c reductase. In addition, we found 3,3′-diindolylmethane to be a substrate for rat hepatic microsomal monooxygenase(s) and tentatively identified a monohydroxylated metabolite. These observations indicate that 3,3′-diindolylmethane can inhibit the catalytic activities of a range of CYP isoforms from lower and higher vertebrates in vitro. This broadly based inhibition of CYP-mediated activation of procarcinogens may be an indole-3-carbinol anticarcinogenic mechanism applicable to all species, including humans. © 1995 John Wiley & Sons, Inc.