Jun-Yan Hong

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.

Mechanism of induction of cytochrome P-450ac (P-450j) in chemically induced and spontaneously diabetic rats☆

Previous studies demonstrated that a microsomal high-affinity N-nitrosodimethylamine demethylase activity and cytochrome P-450ac (an acetone/ethanol-inducible form) were induced by streptozotocin-induced diabetes in rats. In the present work, the induction was studied in detail in two chemically induced (by streptozotocin and alloxan) diabetic rat models and one spontaneously (BB/Wor) diabetic rat model. All the diabetic conditions caused increases in three parameters: (a) microsomal N-nitrosodimethylamine demethylase activity which is known to be a good indicator of the level of P-450ac; (b) the levels of P-450ac as determined by immunoblot analysis; and (c) the levels of mRNA of P-450ac as determined by hybridization assays with a cDNA probe for this enzyme. These increases were abolished by treatment of the diabetic rats with insulin. The results suggest that the pathophysiological condition of diabetes is responsible for the induction of P-450ac and elevation of mRNA is involved in all of the three diabetic models investigated.

Efficient activation of aflatoxin B1 by cytochrome P450 2A13, an enzyme predominantly expressed in human respiratory tract†

The worldwide human exposure to aflatoxin B1 (AFB1), particularly in developing countries, remains to be a serious public health concern. Although AFB1 is best known as a hepatocarcinogen, epidemiological studies have shown a positive association between human lung cancer occurrence and inhalation exposure to AFB1. Cytochrome P450 (CYP)‐catalyzed metabolic activation is required for AFB1 to exert its carcinogenicity. Previous studies have identified CYP1A2 and CYP3A4 as the major enzymes for AFB1 activation in human liver. However, the key CYP enzymes in human lung that can efficiently activate AFB1in situ are unknown. In the present study, we demonstrate that CYP2A13, an enzyme predominantly expressed in human respiratory tract, has a significant activity in metabolizing AFB1 to its carcinogenic/toxic AFB1‐8,9‐epoxide and AFM1‐8,9‐epoxide at both low (15 μM) and high (150 μM) substrate concentrations. Under the same conditions, there was no detectable AFB1 epoxide formation by CYP2A6, which was also reported to be involved in the metabolic activation of AFB1. Consistent with the activity data, there was an ∼800‐fold difference in LC50 values of AFB1 (48‐hr treatment) between Chinese hamster ovary (CHO) cells expressing CYP2A13 and CYP2A6 (50 nM versus 39 μM). We further demonstrate that amino acid residues Ala117 and His372 in CYP2A13 protein are important for AFB1 epoxidation and its related cytotoxicity. Our results suggest that CYP2A13‐catalyzed metabolic activation in situ may play a critical role in human lung carcinogenesis related to inhalation exposure to AFB1.

IDENTIFICATION OF CRITICAL AMINO ACID RESIDUES OF HUMAN CYP2A13 FOR THE METABOLIC ACTIVATION OF 4-(METHYLNITROSAMINO)-1-(3-PYRIDYL)-1-BUTANONE, A TOBACCO-SPECIFIC CARCINOGEN

Among all the known human cytochrome P450 enzymes, CYP2A13 has the highest efficiency in catalyzing the metabolic activation (keto aldehyde and keto alcohol formation) of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent lung carcinogen in animals and a suspected human lung carcinogen. As part of the structure-activity relationship (SAR) study, the present work was done to identify the key amino acid residues in CYP2A13 that are responsible for this high catalytic efficiency by using a series of mutants (Ala117Val, His164Gly, Ser208Ile, His372Arg, and Pro465Ser). In these CYP2A13 mutants, the amino acid residues were substituted by the residues at the corresponding positions of CYP2A6, which shares 93.5% amino acid sequence identity with CYP2A13 but is significantly less active (<5%) than CYP2A13 in NNK α-hydroxylation. We demonstrated that, except for the His164Gly mutant, all the CYP2A13 mutant proteins showed a significant decrease in the catalytic efficiency (Vmax/Km) for NNK α-hydroxylation. The His372 to Arg substitution resulted in a 20-fold increase in the Km value and a 7-fold decrease in the Vmax value for keto aldehyde formation as well as a total loss of detectable keto alcohol formation. The Ala117 to Val substitution, however, only caused a selective decrease in the Vmax value for keto aldehyde formation. The role of these amino acid residues in CYP2A13-catalyzed reactions is clearly substrate-dependent, since the same Ala117Val and His372Arg mutants showed a 9-fold increase in the catalytic efficiency for coumarin 7-hydroxylation. Together with the computational substrate docking, our study provides new SAR in formation of human CYP2A13.

Genetic variation of human cytochrome P450 reductase as a potential biomarker for mitomycin C-induced cytotoxicity

The importance of genetic variation in clinical response to various drugs is now well recognized. Identification of genetic biomarkers that can predict efficacy and toxicity of chemotherapeutic drugs in cancer patients holds great promise in treatment improvement and cost reduction. Mitomycin C (MMC) is a common anticancer drug used for the treatment of numerous types of tumors. Metabolism-mediated activation, by either one-electron or two-electron reduction, plays a critical role in the chemotherapeutic action of MMC. NADPH-cytochrome P450 (oxido)reductase (POR) is a major enzyme responsible for MMC activation through the one-electron reductive pathway, which leads to the production of semiquinone anion radicals and subsequent DNA damage in the cells. Recently, a total of six naturally occurring human POR variants with single amino acid changes (Y181D, A287P, R457H, V492E, C569Y, and V608F) have been identified. Although the catalytic efficiency of these variants in reduction of cytochrome c was reported to be altered, their capability in activating MMC, a direct substrate of POR, has not been examined. In the present study, we demonstrated that except for the C569Y variant, MMC-induced toxicity assayed as cell viability and proliferative capability was significantly decreased in the Flp-In Chinese hamster ovary cells stably expressing all the other POR variants in comparison with the cells expressing wild-type human POR. Cells expressing the V608F and Y181D variants had a complete loss of the capability to activate MMC. Our finding suggests that these functional POR genetic variations may serve as a potential biomarker to predict the chemotherapeutic response to MMC.

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by human CYP1B1 genetic variants.

Human cytochrome P450 1B1 (CYP1B1) plays a critical role in the metabolic activation of a variety of procarcinogens, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The existence of human CYP1B1 missense genetic variants has been demonstrated, but their activities in metabolizing PhIP are unknown. In this study, we expressed 15 naturally occurring CYP1B1 variants (with either single or multiple amino acid substitutions) and determined their activity changes in metabolizing PhIP to its two major metabolites, 2-hydroxyamino-PhIP and 4′-hydroxy-PhIP. Although the PhIP-metabolizing activities of four variants (Ala119Ser, Pro379Leu, Ala443Gly, Arg48Gly/Leu432Val) were comparable with that of the expressed wild-type CYP1B1, five variants (Trp57Cys, Gly61Glu, Arg48Gly/Ala119Ser, Arg48Gly/Ala119Ser/Leu432Val, Arg48Gly/Ala119Ser/Leu432Val/Ala443Gly) exhibited more than 2-fold decrease in activity and a reduction in the catalytic efficiency (Vmax/Km) for both N- and 4-hydroxylation of PhIP. Six variants (Gly365Trp, Glu387Lys, Arg390His, Pro437Leu, Asn453Ser, Arg469Trp) showed little activity in PhIP metabolism, but the molecular mechanisms involved are apparently different. The microsomal CYP1B1 protein level was significantly decreased for the Trp365, Lys387, and His390 variants and was not detectable for the Ser453 variant. In contrast, there was no difference between the Trp469 variant and the wild-type in the microsomal CYP1B1 protein level and P450 content but the Trp469 variant totally lost its metabolic activity toward PhIP. The Leu437 variant also had a substantial amount of CYP1B1 protein in the microsomes, but there was a lack of detectable P450 peak and activity. Our results should be useful in selecting appropriate CYP1B1 variants as cancer susceptibility biomarkers for human population studies related to PhIP exposure.

Critical Amino Acid Residues for Nicotine 5′-Hydroxylation in Human CYP2A Enzymes

Abstract Objective We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-(3-pyridyl)-1-butanone(NNK) and aflatoxin B1(AFB1) carcinogenic activation. The present study was designed to identify other potential amino acid residues that contribute to the different catalytic characteristics of two CYP2A enzymes, CYP2A6 and CYP2A13, in nicotine metabolism and provide insights of the substrate and related amino acid residues interactions. Methods A series of reciprocally substituted mutants of CYP2A6Ile 300 → Phe, CYP2A6Gly 301 Ala, CYP2A6Ser 369 → Gly, CYP2A13Phe 300 → Ile, CYP2A13Ala 301 → Gly and CYP2A13Gly 369 → Ser were generated by site-directed mutagenesis/baculovirus-Sf9 insect cells expression. Comparative kinetic analysis of nicotine 5′hydroxylatin by wild type and mutant CYP2A proteins was performed. Results All amino acid residue substitutions at 300, 301 and 369 caused significant kinetic property changes in nicotine metabolism. While CYP2A6Ile 300 → Phe and CYP2A6Gly 301 →Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe 300 →Ile and CYP2A13Ala 301 →Gly replacement introduced remarkable catalytic efficiency decreases. In addition, all these catalytic efficiency alterations were caused by V max variations rather than K m changes. Substitution of #369 residue significantly affected both K m and V max values. CYP2A6Ser 369 → Gly increase the catalytic efficiency via a significant K m decrease versus V max enhancement, while the opposite effects were seen with CYP2A13Gly 369 → Ser. Conclusion #300, #301 and #369 residues in human CYP2A6/13 play important roles in nicotine 5′-oxidation. Switching #300 or #301 residues did not affect the CYP2A protein affinities toward nicotine, although these amino acids are located in the active center. Ser 369 to Gly substitution indirectly affected nicotine binding by creating more space and conformational flexibility for the nearby residues, such as Leu 370 which is crucial for many hydroxylations.