High Aryl Hydrocarbon Hydroxylase Activity Research Articles

Mitochondrial Targeting of Cytochrome P450 (CYP) 1B1 and Its Role in Polycyclic Aromatic Hydrocarbon-induced Mitochondrial Dysfunction

We report that polycyclic aromatic hydrocarbon (PAH)-inducible CYP1B1 is targeted to mitochondria by sequence-specific cleavage at the N terminus by a cytosolic Ser protease (polyserase 1) to activate the cryptic internal signal. Site-directed mutagenesis, COS-7 cell transfection, and in vitro import studies in isolated mitochondria showed that a positively charged domain at residues 41-48 of human CYP1B1 is part of the mitochondrial (mt) import signal. Ala scanning mutations showed that the Ser protease cleavage site resides between residues 37 and 41 of human CYP1B1. Benzo[a]pyrene (BaP) treatment induced oxidative stress, mitochondrial respiratory defects, and mtDNA damage that was attenuated by a CYP1B1-specific inhibitor, 2,3,4,5-tetramethoxystilbene. In support, the mitochondrial CYP1B1 supported by mitochondrial ferredoxin (adrenodoxin) and ferredoxin reductase showed high aryl hydrocarbon hydroxylase activity. Administration of benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzodioxin induced similar mitochondrial functional abnormalities and oxidative stress in the lungs of wild-type mice and Cyp1a1/1a2-null mice, but the effects were markedly blunted in Cyp1b1-null mice. These results confirm a role for CYP1B1 in inducing PAH-mediated mitochondrial dysfunction. The role of mitochondrial CYP1B1 was assessed using A549 lung epithelial cells stably expressing shRNA against NADPH-cytochrome P450 oxidoreductase or mitochondrial adrenodoxin. Our results not only show conservation of the endoprotease cleavage mechanism for mitochondrial import of family 1 CYPs but also reveal a direct role for mitochondrial CYP1B1 in PAH-mediated oxidative and chemical damage to mitochondria.

Purification and properties of a new β-naphthoflavone inducible cytochrome P-450, aryl hydrocarbon hydroxylase from rat kidney

In rat kidney, β-naphthoflavone induced 53 kDa and 55 kDa proteins, which were both recognized by the antibodies against rat liver cytochrome P-450 1A1(55 kDa). The major inducible 53 kDa protein was purified from the β-naphthoflavone-treated rat kidney and shown to be a new cytochrome P-450 having a high aryl hydrocarbon hydroxylase activity. Purified cytochrome P-450, named P-450KAh, was homogeneous on SDS-polyacrylamide gel electrophoresis, and the apparent molecular weight was estimated to be 53 kDa. The absorption spectra of the oxidized form of P-450KAh showed a Soret peak at 416 nm, a characteristic of low-spin hemoprotein, and the Soret peak of the reduced cytochrome P-450-CO complex was at 446 nm. In the reconstituted system, purified P-450KAh showed high catalytic activity for benzo[a]pyrene hydroxylation and 7-ethoxycoumarin O-deethylation. P-450KAh could activate genotoxicities of not only B[a]P, but also 2-acetylaminofluorene and aflatoxin B1 on the umu test. These catalytic properties of P-450KAh were almost same as that of P-4501A1, a major P-450 form having arylhydrocarbon hydroxylase in liver microsomes of 3-methylcholanthrene-treated rats, and P-450KAh could not be distinguished from P-4501A1 even by immunochemical analysis. However, the electrophoretic peptide patterns after α-chymotrypsin or trypsin treatment of P-450KAh were different from those of P-4501A1, and the NH2-terminal 11 amino acid sequence of the P-450 was also different from those of P-4501A1 and any other P-450s of rat.

Rat Liver Cytochrome P450IA2 Synthesized by Transfected COS-1 Cells Efficiently Activates Food-Derived Promutagens

A standard calcium phosphate technique was used to obtain transient expression of cDNAs for rat liver cytochrome P450s in COS-1 cells. Cells transfected with a pMT2-based vector expressing P450IA2 cDNA (pMT2-IA2) had high acetanilide-4-hydroxylase activity and very low aryl hydrocarbon hydroxylase (AHH) activity. Cells transfected with a hybrid expression vector, pMT2-IA2/IA1, coding for a P450IA2/IA1 fusion protein (consisting of the amino-terminal region of P450IA2 and the central and carboxy-terminal regions of P450IA1) had high AHH activity. This result and other data indicate that the P450IA2/IA1 fusion protein has the substrate specificity of P450IA1. Extracts of cells transfected with pMT2-IA2 readily converted 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and related food-derived promutagens into mutagenic forms. Extracts of cells transfected with pMT2-IA2/IA1 showed efficient activation of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp P-2). To facilitate comparison of activities of P450s synthesized from cDNA expression vectors, the promutagen activation assays were carried out with limiting enzyme and saturating or nearly saturating substrate concentrations. The transient expression system described here uses a standard expression vector and requires only microgram quantities of cell extract protein for activation of food-derived promutagens such as MeIQ and Trp P-2. It will be useful for identifying P450s active in promutagen activation and for analyzing structure-function relationships of different P450 molecules.

2‐(4′‐Chlorophenyl)benzothiazole is a potent inducer of cytochrome P450IA1 in a human and a mouse cell line

Two benzothiazole derivatives, 2-(4'-chlorophenyl)benzothiazole (CPBT) and 2-(4'-formylphenyl)benzothiazole (FPBT) were studied for their ability to induce aryl hydrocarbon hydroxylase activity in a mouse and a human cell line. In both the mouse hepatoma cell line, Hepa-1, and the human choriocarcinoma cell line, JEG-3, a high aryl hydrocarbon hydroxylase activity was observed after treatment with CPBT. In contrast, FPBT had a very weak inducing capacity in both cell lines. The maximal induction by CPBT was several times (e.g. in Hepa-1 about fourfold on average) greater than that observed with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A specific cDNA probe for mouse cytochrome P4501A1 gene was used to quantify mRNA levels in Hepa-1 cells. CPBT increased cytochrome P450IA1 mRNA to a level of 88% of that induced by TCDD. Immunoblot analysis with monoclonal antibody 1-7-1, directed against rat liver cytochrome P450IA1 and P450IA2, showed that the amount of P450IA1 is substantially increased in Hepa-1 cells after treatment with CPBT. The observation that CPBT competed TCDD off its specific cytosolic binding site suggests a receptor-mediated induction of cytochrome P450IA1 mRNA. An in vitro activation effect did not explain the exceptionally high hydroxylase activity. The results show that CPBT is a more efficient inducer of aryl hydrocarbon hydroxylase than TCDD in Hepa-1 and JEG-3 cells and that the induction is supported by P450IA1. The discordant effect of CPBT on mRNA and aryl hydrocarbon hydroxylase activity suggests that post-translational modifications of P450IA1 account for a major part of the increased enzyme activity.

Enzymatic activities of human lung tissue: relationship with smoking habits.

Twenty-two S12 preparations of surgical lung specimens obtained from smoker and non-smoker cancer patients were assayed to detect aryl hydrocarbon hydroxylase (AHH), dimethylnitrosamine demethylase (DMND), and glutathione-S-transferase (GST) activities, in both normal and neoplastic lung tissue from the same patients. Pulmonary fractions were also tested for their ability to activate some precarcinogens into mutagenic metabolites in the Ames test. Statistically significant differences were found for AHH and DMND activities between normal and neoplastic tissue of smoker patients. In addition, higher AHH activity in the neoplastic tissue of the smoker group was observed compared with that found in the non-smoker group. No differences were found for GST activity. All the lung S12 preparations were able to metabolize water-soluble bases and water-insoluble bases, derived from main-stream cigarette smoke condensate, into mutagenic agents in the Salmonella test system. However, S12 preparations from smoker group neoplastic tissues were more effective.

The effect of cigarette smoke on aryl-hydrocarbon hydroxylase (AHH) activity of the human kidney

AHH activity was measured in kidney cortex biopsies obtained during surgery from patients with hydronephrosis and from unaffected portions of kidneys with renal cell carcinomas. AHH activity was slightly, but not significantly, higher in the group of non-smoking cancer patients when compared to non-smoking hydronephrotic subjects. Smoking induced a significant increase of AHH activity in the hydronephrotic kidneys but not in cancer kidneys, probably due to the higher baseline values and scatter in the cancer group. Pooling cancer and non-cancer kidneys, smokers had a higher AHH activity than non-smokers, this increase being dose-related.

Pharmacologically active metabolites of drugs and other foreign compounds. Clinical, pharmacological, therapeutic and toxicological considerations.

Drugs and other foreign compounds administered to man may be biotransformed into metabolites that are pharmacologically active. These metabolites can either be beneficial at the right concentrations in the body or pharmacodynamically toxic, and in some instances they can be responsible for the pharmacological activity seen after parent drug administration. When urinary excretion is the major pathway of elimination of the active metabolite, it will accumulate in patients with renal failure given the parent drug. If the metabolite has the same pattern of pharmacological response as the parent drug, the patient will appear to have an enhanced intensity of effect. If the metabolite has a pattern of pharmacological activity different from that of the parent drug, the effects observed may differ qualitatively as well as quantitatively from what was expected. A few examples of this have been demonstrated in man: (a) the therapeutic (abolition of premature ventricular contractions and prevention of paroxysmal atrial tachycardia) and toxic effects of procainamide in some cardiac patients with poor renal function are associated with high levels of N-acetylprocainamide; (b) the severe irritability and twitching seen in uraemic patients treated with pethidine (meperidine) are associated with high levels of its norpethidine metabolite; (c) the severe muscle weakness and tenderness seen in patients with renal failure receiving cloflbrate are associated with excessive accumulation of the free acid metabolite of cloflbrate; (d) patients with severe renal insufficiency taking allopurinol appear to experience a higher incidence of side effects, possibly due to the accumulation of oxipurinol; (e) hydroxyamylobarbitone has been incriminated as the cause of impaired cognitive function in uraemic patients given amylobarbitone (amobarbital); and (f) methyldopamine may be responsible for the enhanced sensitivity to methyldopa seen in patients with impaired renal function. Chemically highly reactive (mainly oxidised) metabolites of ingested foreign compounds are thought to mediate many different types of serious toxicity including carcinogenesis, mutagenesis, dysmorphogenesis and cellular necrosis. The chemical instability of this type of metabolite and its covalent binding to cellular macromolecules is suspected as initiating these conditions. 2-Aminonaphthylene, benzidine and 4-aminobiphenyl are aromatic amines that are known bladder carcinogens in man. The hydroxylamine metabolites are viewed as the first step in the metabolic activation of these aromatic amines. Genetic slow acetylators may be more at risk for bladder cancer from these amines than rapid acetylators. Carcinogenic polycyclic aromatic hydrocarbons, such as benzo(a)pyrene, require metabolic activation, probably to epoxide-diols, to elicit their pharmacological effects. Individuals with higher aryl hydrocarbon hydroxylase activity in their cultured, mitogen-activated peripheral lymphocytes may have an increased risk for certain cancers including bronchogenic carcinoma. Epoxide-diol metabolites of benzo(a)anthracene, but not the parent compound, are highly mutagenic in the Ames test without the need for an oxidising system. An epoxide metabolite of phenytoin, cyclophosphamide and benzo(a)pyrene is believed responsible for the dysmorphogenic effect of the parent compounds. Also, paracetamol (acetaminophen)-induced cellular necrosis in the liver is believed due to a highly reactive oxidised metabolite. Intravenous N-acetylcysteine is an effective agent against paracetamolinduced hepatic necrosis, provided treatment is begun within 8 hours of paracetamol overdosage. Finally, the major antigenic determinant in penicillin-induced allergy is a penicilloyl-protein conjugate. Monitoring plasma levels of drugs can be an important guide to therapy. However, if a drug has a chemically stable active metabolite, determination of the parent drug alone may cause misleading interpretations of blood level measurements. The plasma level of the active metabolite should also be determined and its concentration-response and time-action characteristics taken into account in any clinical decision based on drug level monitoring.

Purification and characterization of the cytochrome P-448 component of a benzo(a)pyrene hydroxylase from Saccharomyces cerevisiae

Cytochrome P-448, a type of cytochrome P-450, from brewer's yeast ( Saccharomyces cerevisiae) grown under conditions of glucose repression was isolated and purified. Triton X-100 in very low concentration proved to be very effective in stabilizing P-448 in the microsomal fraction and later prevented its conversion to cytochrome P-420 through solubilization with various ionic and nonionic detergents. Highest yields were obtained with 1% sodium cholate, in the presence of 0.1% Triton X-100 and reduced glutathione. A novel combination of hydrophobic adsorption and other chromatographic techniques was used for the purification of cytochrome P-448. These involve the use of amino octyl-Sepharose 4B, instead of the low-yielding aminohexyl derivative, followed by the fast-running hydroxyapatite-cellulose column. Finally, the use of DEAE-Sephacel was found to increase greatly the purity of the cytochrome P-448 obtained. The molecular weight of this preparation was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( M r, 55,500). Using the known molar extinction coefficient of the carbon monoxide-difference spectrum the estimate of degree of purity of cytochrome P-448 obtained by this purification procedure was between 88 and 97%. Electrophoresis also showed that this preparation was completely homogeneous and assays showed that it was also completely free of cytochrome b s, cytochrome c reductase and cytochrome P-420. Purified cytochrome P-448 reconstituted with cytochrome P-450 (cytochrome c) reductase, isolated from yeast, showed 10-fold higher aryl hydrocarbon hydroxylase activity with benzo[ a]pyrene as a substrate than the corresponding microsomal fraction enzyme. Kinetics of benzo[ a]pyrene hydroxylation were determined: K m (33 μ m) was comparable with that reported for purified hepatic cytochrome P-448. The number of binding sites of microsomal and purified cytochromes P-450 (from liver of phenobarbital-induced rats) and yeast cytochrome P-448 with benzo[a]pyrene has been determined using and equilibrium gel filtration method. There is one binding site in each case (contrast with six sites for microsomal enzymes). The Scatchard plot gives number of binding sites, apparent association constants ( K), and the equivalent dissociation constants ( K s ). Comparison is made with spectral dissociation constants for these enzymes and benzo[ a]pyrene. Thus the proportion bound, dissociation constant ( K s ), and stoichiometry of rat liver (phenobarbital induced) and yeast cytochrome P-448 with benzo[ a]pyrene were compared with corresponding values for microsomal fractions of both systems. Purified enzymes had higher K s values in both cases, and the proportion of enzyme that bound benzo[ a]pyrene was high (53%) for liver and this value is 100% for purified enzyme from yeast, which is the same as the value obtained for the microsomal enzyme from yeast.

Induction of aryl hydrocarbon hydroxylase activity and pulmonary carcinoma

Aryl hydrocarbon hydroxylase activity in lymphoblasts from normal Finnish adults and from patients with pulmonary carcinomas and other types of malignancy has been studied by a modification of previously used techniques. High absolute induced aryl hydrocarbon hydroxylase activity was found in 39% of patients with untreated lung cancer but only in 15% of normal people. No increased frequency was found in the control group comprising other malignancies. The diagnosis of pulmonary carcinoma was made at a lower mean age (4.9 years younger) in the individuals with high aryl hydrocarbon hydroxylase activity than in those with low activity. High absolute aryl hydrocarbon hydroxylase activity was dominantly inherited in normal individuals, and the frequency of athe Ahb gene in the Finnish population was 8%.

Aniline hydroxylation in the human placenta—Mechanistic aspects

Attempts to demonstrate aniline p-hydroxylating activity in particulate fractions of human or rat placental homogenates yielded consistently negative results, even in subfractions with very high aryl hydrocarbon hydroxylase activity. p-Hydroxylation of aniline, however, was readily detected in 104,000 g supernatant fractions from both species. Characteristics of the placental reaction differed markedly from the reaction classically observed in hepatic microsomes. Ammonium sulfate fractionation and column chromatography analyses indicated that hemoglobin and/or methemoglobin were strongly associated with placental catalytic activity. Substitution of hemoglobin for placental tissues in reaction flasks resulted in an easily measurable reaction. Cytochrome c could substitute for hemoglobin but was a much less efficient catalyst under the reaction conditions utilized. Flavins were not required as components of these model system reactions, but FMN or FAD markedly accelerated the cytochrome c catalyzed reactions. Comparisons of tissue-free (model system) reactions with those catalyzed by components present in placental homogenates indicated that hemoglobin was a principal factor responsible for catalysis of p-hydroxylation of aniline in these placental preparations, thus suggesting that placental trophoblasts per se were either devoid of or extremely low in aniline p-hydroxylating activity. The results also indicated separate mechanisms for aniline and 3,4-benzpyrene hydroxylation processes.