Harry V. Gelboin

The in vivo and in vitro induction of aryl hydrocarbon hydroxylase in mammalian cells of different species, tissues, strains, and developmental and hormonal states.

Abstract Aryl hydrocarbon hydroxylase from rat liver metabolizes a variety of polycyclic hydrocarbons. This membrane-bound enzyme system is present in various tissues of the monkey, hamster, and rat. In vivo , the prior administration of a polycyclic hydrocarbon induces the hydroxylase to higher levels in the liver, lung, gastrointestinal tract, and kidney of each of these mammals. The enzyme is also present in various tissues of six strains of mice; the hepatic enzyme is inducible in the Swiss, strains C-57, C3H, and A, but not in strains AKR/N or DBA, of the mouse. Thus, there are genetic differences in the regulation of enzyme induction as well as in the control levels of enzyme. The enzyme system is also induced transplacentally: treatment of the pregnant hamster or rat, with either a polycyclic hydrocarbon or phenobarbital, induces the enzyme system in specific fetal tissues as well as in the placenta. The extent of enzyme induction is greatest in adult, less in neonatal tissues, and least in fetal tissues. Induction of hydroxylase activity by 3-methylcholanthrene occurs in the liver, lung, and kidney of the adrenalectomized or hypophysectomized male rat, and the hepatic enzyme is induced by phenobarbital in the adrenalectomized male rat. The magnitude of aryl hydrocarbon hydroxylase induction varies greatly with the tissue and species—from no induction to more than 100-fold increases in enzyme activity. The enzyme system is also present and inducible in fetal cell cultures derived from whole hamster, mouse, rat, and chick. Further, hydroxylase activity is inducible in cell cultures derived from hamster fetal liver, lung, small intestine, and limbs, and in mouse 3T3 culture, an established cell line.

Aryl hydrocarbon (benzo(a)pyrene) hydroxylase in microsomes from rat tissues: differential inhibition and stimulation by benzoflavones and organic solvents.

Abstract The in vitro addition of 7,8-benzoflavone inhibits the aryl hydrocarbon (benzo[ a ]pyrene) hydroxylase 2 (AHH) 3 in hepatic microsomes from methylcholanthrene treated male rats, but not in hepatic microsomes from control or phenobarbital-treated animals. On the other hand, 7,8-benzoflavone inhibits microsomal hydroxylase from lung and kidney of both control and methylcholanthrene-treated rats; the inhibition is somewhat less in preparations from control animals. 7,8-Benzoflavone also inhibits the AHH from skin of control and benzanthracene-treated mice and from control and benzanthracene-treated hamster embryo cell cultures. In contrast to 7,8-benzoflavone, organic solvents such as monovalent alcohols or dimethylsulfoxide inhibit AHH in hepatic microsomes from control and phenobarbital treated rats, but have little inhibitory effect on methylcholanthrene-induced hepatic hydroxylase. The data indicate the existence of at least two forms of benzo[ a ]pyrene hydroxylating enzyme systems in rat tissues which can be differentially induced in vivo by administration of phenobarbital or methylcholanthrene.

Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome P450s

Steroid hydroxylation specificities were determined for 11 forms of human cytochrome P450, representing four gene families and eight subfamilies, that were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. Microsomes isolated from the P450-expressing Hep G2 cells were isolated and then assayed for their regioselectivity of hydroxylation toward testosterone, androstenedione, and progesterone. Four of the eleven P450s exhibited high steroid hydroxylase activity (150-900 pmol hydroxysteroid/min/mg Hep G2 microsomal protein), one was moderately active (30-50 pmol/min/mg) and six were inactive. In contrast, 10 of the P450s effectively catalyzed O-deethylation of 7-ethoxycoumarin, a model drug substrate, while only one (P450 2A6) catalyzed significant coumarin 7-hydroxylation. Human P450 4B1, which is expressed in lung but not liver, catalyzed the 6 beta-hydroxylation of all three steroids at similar rates and with only minor formation of other hydroxylated products. Three members of human P450 family 3A, which are expressed in liver and other tissues, also catalyzed steroid 6 beta-hydroxylation as their major activity but, additionally, formed several minor products that include 2 beta-hydroxy and 15 beta-hydroxy derivatives in the case of testosterone. These patterns are similar to those exhibited by rat family 3A P450s. Although several rodent P450s belonging to subfamilies 2A, 2B, 2C, 2D are active steroid hydroxylases, four of five human P450s belonging to these subfamilies exhibited very low activity or were inactive, as were the human 1A and 2E P450s examined in the present study. These studies demonstrate that individual human cytochrome P450 enzymes can hydroxylate endogenous steroid hormones with a high degree of stereospecificity and regioselectivity, and that some, but not all of the human cytochromes exhibit metabolite profiles similar to their rodent counterparts.

The induction of a specific form of cytochrome P-450 (P-450j) by fasting

In previous work we have demonstrated that liver microsomal N-nitrosodimethylamine demethylase (NDMAd) activity is increased in rats by fasting, and we have postulated that this is due to the induction of a specific form of cytochrome P-450. This communication provides evidence for such a hypothesis. Fasting for 24 and 48 h caused 59 and 116% increases, respectively, in NDMAd activity in male rats, and fasting for 48 h caused a 63% increase in female rats. These increases were accompanied by corresponding increases of cytochrome P-450j (P-450ac) determined by immunoblotting. Fasting for 24 and 48 h also increased the mRNA for P-450j by 153 to 250%, as determined by hybridization with a cDNA probe of this cytochrome. The results suggest that fasting affects the gene expression of P-450j.

Benzo(a)pyrene metabolites: efficient and rapid separation by high-pressure liquid chromatography.

High-pressure liquid chromatography can separate eight metabolites of benzo[a] pyrene formed by rat liver microsomes. This method offers major advantages over previous techniques used for the separation of oxygenated polycyclic aromatic hydrocarbons.

Monoclonal antibodies to liver microsomal cytochrome P-450E of the marine fish Stenotomus chrysops (scup): Cross reactivity with 3-methylcholanthrene induced rat cytochrome P-450☆

Hybridomas were prepared from myeloma cells and spleen cells of BALB/c female mice immunized with hepatic cytochrome P-450E purified from the marine fish, Stenotomus chrysops (scup). Nine independent hybrid clones produced MAbs, either IgG1, IgG2b, or IgM, that bound to purified cytochrome P-450E in radioimmunoassay. Antibodies from one clone MAb (1-12-3), also strongly recognized rat cytochrome P-450MC-B (P-450BNF-B; P-450c). The nine antibodies inhibited reconstituted aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin O-deethylase of scup cytochrome P-450E to varying degrees, and inhibited AHH activity of beta-naphthoflavone-induced scup liver microsomes in a pattern similar to that in reconstitutions, indicating that cytochrome P-450E is identical to the AHH catalyst induced in this fish by beta-naphthoflavone. MAb 1-12-3 also inhibited the reconstituted AHH activity of the major BNF-induced rat isozyme. Conversely, MAb 1-7-1 to rat cytochrome P-450MC-B had little effect on AHH activity of scup cytochrome P-450E, and did not recognize cytochrome P-450E in radioimmunoassay nor in an immunoblot. Scup cytochrome P-450E and rat cytochrome P-450MC-B thus have at least one common epitope recognized by MAb 1-12-3, but the epitope recognized by Mab 1-7-1 is absent or recognized with low affinity in cytochrome P-450E. The various assays indicate that the nine MAbs against cytochrome P-450E are directed to different epitopes of the molecule. These MAbs should be useful in determining phylogenetic relationships of the BNF- or MC-inducible isozymes and their regulation by other environmental factors.

Targeted Disruption of the Microsomal Epoxide Hydrolase Gene MICROSOMAL EPOXIDE HYDROLASE IS REQUIRED FOR THE CARCINOGENIC ACTIVITY OF 7,12-DIMETHYLBENZ[a]ANTHRACENE

Microsomal epoxide hydrolase (mEH) is a conserved enzyme that is known to hydrolyze many drugs and carcinogens, and a few endogenous steroids and bile acids. mEH-null mice were produced and found to be fertile and have no phenotypic abnormalities thus indicating that mEH is not critical for reproduction and physiological homeostasis. mEH has also been implicated in participating in the metabolic activation of polycyclic aromatic hydrocarbon carcinogens. Embryonic fibroblast derived from the mEH-null mice were unable to produce the proximate carcinogenic metabolite of 7,12-dimethylbenz[a]anthracene (DMBA), a widely studied experimental prototype for the polycylic aromatic hydrocarbon class of chemical carcinogens. They were also resistant to DMBA-mediated toxicity. Using the two-stage initiation-promotion skin cancer bioassay, the mEH-null mice were found to be highly resistant to DMBA-induced carcinogenesis. In a complete carcinogenesis bioassay, the mEH mice were totally resistant to tumorigenesis. These data establish in an intact animal model that mEH is a key genetic determinant in DMBA carcinogenesis through its role in production of the ultimate carcinogenic metabolite of DMBA, the 3,4-diol-1,2-epoxide.

Alpha-Naphthoflavone: An Inhibitor of Hydrocarbon Cytotoxicity and Microsomal Hydroxylase

Alpha-naphthoflavone inhibits the metabolism of 3,4-benzopyrene and 7,12-dimethylbenz(a)anthracene in hamster enlbryo cell cultures and protects the cells against the inhibition of cell multiplication by these carcinogens. Alphla-nalphthoflavone also inhibits the aryl hydrocarbon hydroxylase activity in homogenates of induced hamster embryo cells and in liver microsomes from rats previously treated with polycyclic aromatic hydrocarbons, but not in microsomes from control rats.

Role of human cytochrome P450 3a4 and 3a5 in the metabolism of taxotere and its derivatives: enzyme specificity, interindividual distribution and metabolic contribution in human liver

Taxotere, a promising anticancer agent, is metabolized almost exclusively in liver and excreted from bile in all species. To determine which cytochrome P450 is involved in taxotere biotransformation, 11 cDNA-expressed human cytochrome P450s were examined for their activity in the metabolism of taxotere and its derivatives. Of all P450s, cytochrome P450 3A4 and 3A5 were the most active for the oxidation of taxotere to the primary metabolite RPR104952 and for subsequent oxidation of RPR104952 to RPR111059 and RPR111026. RP70617, an epimer of taxotere was also metabolized by both P450 3A enzymes to form metabolite XII. The activity of 3A4/5 enzymes for these substrates was 4-50-fold greater than the other P450s examined. The Kms of 3A4 and 3A5 for taxotere were 0.91 and 9.28 microM, and Vmax for the formation of RPR104952 were 1.17 and 1.36 m(-1), respectively. The contribution of the 3A enzyme complex to the metabolism of taxotere in human livers from 21 individuals was assessed with the inhibitory monoclonal antibody and ranged from 64-93%. The primary oxidative metabolism of taxotere by human liver microsomes was well correlated with 3A4-dependent reactions for testosterone 6beta-hydroxylation (r2 = 0.84), taxol aromatic hydroxylation (r2 = 0.67) and aflatoxin B1 3alpha-hydroxylation (r2 = 0.63); whereas a poor correlation was found for reactions specifically catalysed by other P450s (all r2 < or =O.17). The extent of taxotere metabolism also closely correlated with levels of 3A4 enzyme in human livers quantified with immunoblot monoclonal antibody (r2 = 0.61). These results demonstrate that the P450 3A4 and 3A5 enzymes are major determinants in taxotere oxidation and suggest that care must be taken when administering this drug with other drugs that are also substrates for these enzymes.

Aryl hydrocarbon (benzo[a]pyrene) hydroxylases in liver from rats of different age, sex and nutritional status: Distinction of two types by 7,8-benzoflavone

Abstract Two types of aryl hydrocarbon (benzo[a]pyrene) hydroxylase have been distinguished in liver from rats of different sex and age by their sensitivity to the synthetic flavonoid, 7,8-benzoflavone. One type, which is stimulated by the 7,8-benzoflavone, is found in newborn rats and predominates in the liver of adult male rats. This type is inducible by phenobarbital. A second type, which is inhibited by 7,8-benzoflavone, comprises a larger fraction in the liver of adult female rats and is inducible by polycyclic hydrocarbons in immature and mature animals of either sex. The presence of this form in adult female liver is also indicated by the kinetics of the hydroxylase reaction. Removal of solid food for 18 hr not only decreases hepatic aryl hydrocarbon hydroxylase activity in female rats, but also lowers the degree of inhibition by 7,8-benzoflavone. Kinetic data suggest that at low concentrations 7,8-benzoflavone acts as a competitive inhibitor but at higher concentrations inhibits the hydroxylation reaction by a more complex mechanism.