Comparisons of (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol activation by human cytochrome P450 and aldo-keto reductase enzymes: effect of redox state and expression levels.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that are metabolically activated to proximate carcinogenic trans-dihydrodiols. PAH trans-dihydrodiols are further activated in humans by cytochrome P450 (P450) 1A1 and 1B1 to yield diol-epoxides or by aldo-keto reductases (AKR) 1A1 and 1C1-1C4 to yield reactive and redox-active o-quinones. Reconstituted in vitro systems were used to compare the steady-state kinetic constants for human P450 (P450 1A1 and 1B1) and AKR (AKR1A1, AKR1C1-1C4) mediated metabolism of (+/-)- trans-7,8-dihydroxy-7,8-dihydrobenzo[ a]pyrene ((+/-)-B[ a]P-7,8-diol) at physiological pH. It was found that P450 isoforms yielded much greater k cat/ K m values than AKR enzymes. Initial rates of (+/-)-B[ a]P-7,8-diol oxidation were measured for AKR1A1, AKR1C2, P450 1A1, and P450 1B1 as the ratio of NADPH/NAD (+) cofactors was varied to determine the redox state necessary for AKRs to successfully compete for trans-dihydrodiols. P450 and AKR enzymes equally competed for (+/-)-B[ a]P-7,8-diol substrate at an NADPH/NAD (+) ratio equal to 0.001. The resting NADPH/NAD (+) ratio was determined in A549 human lung adenocarcinoma cells to be 0.28. These data suggest that the P450 pathway would be favored over the AKR pathway if the enzymes were equally expressed. Basal mRNA transcript levels of AKR1C1-1C3 exceed those of both basal and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced P450 1A1 and 1B1 by up to 90-fold in A549 cells as measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR) methods. AKR expression levels were comparable to TCDD-induced P450 1A1 and 1B1 in HBEC-KT immortalized normal human bronchial epithelial cells. Functional assays of both A549 and HBEC-KT cell lysates demonstrated a lack of TCDD-inducible P450 1A1/1B1 activity but robust basal expression of AKR1A1 and AKR1C activities, where the functional assay for P450 detection is 300-fold more sensitive than the functional assay for AKR isoforms. These data suggest that AKR enzymes may effectively compete with P450 1A1/1B1 for PAH trans-dihydrodiol activation in human lung cells.

[1]  H. Krebs,et al.  The redox state of free nicotinamide-adenine dinucleotide phosphate in the cytoplasm of rat liver. , 1969, The Biochemical journal.

[2]  K. Nakanishi,et al.  BENZO(A)PYRENE-NUCLEIC ACID DERIVATIVE FOUND IN VIVO- STRUCTURE OF A BENZO(A)PYRENETETRAHYDRODIOL EPOXIDE-GUANOSINE ADDUCT , 1976 .

[3]  Barry H. Smith,et al.  A continuous tumor‐cell line from a human lung carcinoma with properties of type II alveolar epithelial cells , 1976, International journal of cancer.

[4]  E. Jacobson,et al.  Pyridine nucleotide levels as a function of growth in normal and transformed 3T3 cells. , 1976, Archives of biochemistry and biophysics.

[5]  D. Jerina,et al.  Changes of mechanisms and product distributions in the hydrolysis of benzo[a]pyrene-7,8-diol 9,10-epoxide metabolites induced by changes in pH. , 1977, Journal of the American Chemical Society.

[6]  D. Jerina,et al.  Binding of benzo[a]pyrene 7,8-diol-9,10-epoxides to DNA, RNA, and protein of mouse skin occurs with high stereoselectivity. , 1978, Science.

[7]  A. Conney,et al.  Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: G. H. A. Clowes Memorial Lecture. , 1982, Cancer research.

[8]  R. Veech,et al.  Measurement of tissue purine, pyrimidine, and other nucleotides by radial compression high-performance liquid chromatography. , 1984, Analytical biochemistry.

[9]  L. Cucchiarini,et al.  Simultaneous extraction and reverse-phase high-performance liquid chromatographic determination of adenine and pyridine nucleotides in human red blood cells. , 1985, Analytical biochemistry.

[10]  L. Cucchiarini,et al.  A very fast ion-pair reversed-phase HPLC method for the separation of the most significant nucleotides and their degradation products in human red blood cells. , 1987, Analytical biochemistry.

[11]  T. Penning,et al.  Cytotoxicity of polycyclic aromatic hydrocarbon o-quinones in rat and human hepatoma cells. , 1993, Chemical research in toxicology.

[12]  T. Penning,et al.  Reactivity of benzo[a]pyrene-7,8-dione with DNA. Evidence for the formation of deoxyguanosine adducts. , 1993, Carcinogenesis.

[13]  M. Rämet,et al.  p53Protein expression is correlated with benzo[a]pyrene-DNA adducts in carcinoma cell lines , 1995 .

[14]  E. Cavalieri,et al.  Central role of radical cations in metabolic activation of polycyclic aromatic hydrocarbons. , 1995, Xenobiotica; the fate of foreign compounds in biological systems.

[15]  H. Yamazaki,et al.  Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. , 1996, Cancer research.

[16]  T. Penning,et al.  Disposition and biological activity of benzo[a]pyrene-7,8-dione. A genotoxic metabolite generated by dihydrodiol dehydrogenase. , 1996, Biochemistry.

[17]  F. Guengerich,et al.  Drug metabolism by Escherichia coli expressing human cytochromes P450 , 1997, Nature Biotechnology.

[18]  T. Penning,et al.  DNA strand scission by polycyclic aromatic hydrocarbon o-quinones: role of reactive oxygen species, Cu(II)/Cu(I) redox cycling, and o-semiquinone anion radicals,. , 1997, Biochemistry.

[19]  N. Ledirac,et al.  Comparative study of CYP1A1 induction by 3-methylcholanthrene in various human hepatic and epidermal cell types. , 1997, Toxicology in vitro : an international journal published in association with BIBRA.

[20]  J. Abel,et al.  Transforming Growth Factor-β1Coregulates mRNA Expression of Aryl Hydrocarbon Receptor and Cell-Cycle-Regulating Genes in Human Cancer Cell Lines , 1997 .

[21]  W. Thilly,et al.  Quantitative RT-PCR measurement of cytochromes p450 1A1, 1B1, and 2B7, microsomal epoxide hydrolase, and NADPH oxidoreductase expression in lung cells of smokers and nonsmokers. , 1997, American journal of respiratory cell and molecular biology.

[22]  T. Shimada,et al.  Recombinant human cytochrome P450 1B1 expression in Escherichia coli. , 1998, Archives of biochemistry and biophysics.

[23]  T. Penning,et al.  Expression and characterization of four recombinant human dihydrodiol dehydrogenase isoforms: oxidation of trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene to the activated o-quinone metabolite benzo[a]pyrene-7,8-dione. , 1998, Biochemistry.

[24]  J. H. Kim,et al.  Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1. , 1998, Carcinogenesis.

[25]  D. Lin,et al.  Development of an antipeptide antibody that binds to the C-terminal region of human CYP1B1. , 1999, Drug metabolism and disposition: the biological fate of chemicals.

[26]  T. Penning,et al.  Isoform-specific induction of a human aldo-keto reductase by polycyclic aromatic hydrocarbons (PAHs), electrophiles, and oxidative stress: implications for the alternative pathway of PAH activation catalyzed by human dihydrodiol dehydrogenase. , 1999, Cancer research.

[27]  T. Penning,et al.  The arginine 276 anchor for NADP(H) dictates fluorescence kinetic transients in 3 alpha-hydroxysteroid dehydrogenase, a representative aldo-keto reductase. , 1999, Biochemistry.

[28]  J. Whitlock,et al.  Induction of cytochrome P4501A1. , 1999, Annual review of pharmacology and toxicology.

[29]  T. Shimada,et al.  Metabolism of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene by recombinant human cytochrome P450 1B1 and purified liver epoxide hydrolase. , 1999, Chemical research in toxicology.

[30]  D. Harrison,et al.  Major differences exist in the function and tissue-specific expression of human aflatoxin B1 aldehyde reductase and the principal human aldo-keto reductase AKR1 family members. , 1999, The Biochemical journal.

[31]  I. Blair,et al.  Synthesis and characterization of polycyclic aromatic hydrocarbon o-quinone depurinating N7-guanine adducts. , 1999, Chemical research in toxicology.

[32]  Michael E. Burczynski,et al.  Human 3α-hydroxysteroid dehydrogenase isoforms (AKR1C1–AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones , 2000 .

[33]  O Pelkonen,et al.  Induction and regulation of xenobiotic-metabolizing cytochrome P450s in the human A549 lung adenocarcinoma cell line. , 2000, American journal of respiratory cell and molecular biology.

[34]  T. Shimada,et al.  Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P450 1B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009. , 2001, Drug metabolism and disposition: the biological fate of chemicals.

[35]  N. Palackal,et al.  The ubiquitous aldehyde reductase (AKR1A1) oxidizes proximate carcinogen trans-dihydrodiols to o-quinones: potential role in polycyclic aromatic hydrocarbon activation. , 2001, Biochemistry.

[36]  I. Roots,et al.  Differential metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 variants. , 2001, Carcinogenesis.

[37]  P. Hainaut,et al.  Patterns of p53 G-->T transversions in lung cancers reflect the primary mutagenic signature of DNA-damage by tobacco smoke. , 2001, Carcinogenesis.

[38]  A. Haugen,et al.  Lung carcinogenesis: Resveratrol modulates the expression of genes involved in the metabolism of PAH in human bronchial epithelial cells , 2001, International journal of cancer.

[39]  Tzei-Yi Lin,et al.  Overexpression of dihydrodiol dehydrogenase as a prognostic marker of non-small cell lung cancer. , 2001, Cancer research.

[40]  P. Bushel,et al.  Differential toxicogenomic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin in malignant and nonmalignant human airway epithelial cells. , 2002, Toxicological sciences : an official journal of the Society of Toxicology.

[41]  N. Palackal,et al.  Activation of Polycyclic Aromatic Hydrocarbontrans-Dihydrodiol Proximate Carcinogens by Human Aldo-keto Reductase (AKR1C) Enzymes and Their Functional Overexpression in Human Lung Carcinoma (A549) Cells* , 2002, The Journal of Biological Chemistry.

[42]  Roger A. Jones,et al.  Formation of benzo[a]pyrene diol epoxide-DNA adducts at specific guanines within K-ras and p53 gene sequences: stable isotope-labeling mass spectrometry approach. , 2002, Biochemistry.

[43]  I. Roots,et al.  Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane. , 2002, European journal of biochemistry.

[44]  Yanbin Zhang,et al.  Two-step error-prone bypass of the (+)- and (−)-trans-anti-BPDE-N2-dG adducts by human DNA polymerases η and κ , 2002 .

[45]  Y. Li,et al.  Single amino acid mutations, but not common polymorphisms, decrease the activity of CYP1B1 against (-)benzo[a]pyrene-7R-trans-7,8-dihydrodiol. , 2003, Carcinogenesis.

[46]  S. Spivack,et al.  Phase I and II carcinogen metabolism gene expression in human lung tissue and tumors. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[47]  B. Maresca,et al.  Differential gene expression between squamous cell carcinoma of esophageus and its normal epithelium; altered pattern of mal, akr1c2, and rab11a expression. , 2004, World journal of gastroenterology.

[48]  F. Curriero,et al.  Expression of cytochromes P450 1A1 and 1B1 in human lung from smokers, non-smokers, and ex-smokers. , 2004, Toxicology and applied pharmacology.

[49]  D. Phillips,et al.  Role of estrogen receptor in regulation of polycyclic aromatic hydrocarbon metabolic activation in lung. , 2004, Lung cancer.

[50]  S. Nesnow,et al.  Identification and characterization of novel stable deoxyguanosine and deoxyadenosine adducts of benzo[a]pyrene-7,8-quinone from reactions at physiological pH. , 2004, Chemical research in toxicology.

[51]  J. Pollack,et al.  Immortalization of Human Bronchial Epithelial Cells in the Absence of Viral Oncoproteins , 2004, Cancer Research.

[52]  Trevor M Penning,et al.  Competing roles of cytochrome P450 1A1/1B1 and aldo-keto reductase 1A1 in the metabolic activation of (+/-)-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene in human bronchoalveolar cell extracts. , 2005, Chemical research in toxicology.

[53]  Hiroyuki Aburatani,et al.  Overexpression of the Aldo-Keto Reductase Family Protein AKR1B10 Is Highly Correlated with Smokers' Non–Small Cell Lung Carcinomas , 2005, Clinical Cancer Research.

[54]  A. Troxel,et al.  Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) by PAH o-quinones: involvement of reactive oxygen species and copper(II)/copper(I) redox cycling. , 2005, Chemical research in toxicology.

[55]  K. Straif,et al.  Carcinogenicity of polycyclic aromatic hydrocarbons. , 2005, The Lancet. Oncology.

[56]  S. Nesnow,et al.  Benzo[a]pyrene-7,8-quinone-3'-mononucleotide adduct standards for 32P postlabeling analyses: detection of benzo[a]pyrene-7,8-quinone-calf thymus DNA adducts. , 2006, Analytical biochemistry.

[57]  Yu-Min Shen,et al.  Comparison of p53 mutations induced by PAH o-quinones with those caused by anti-benzo[a]pyrene diol epoxide in vitro: role of reactive oxygen and biological selection. , 2006, Chemical research in toxicology.

[58]  A. Troxel,et al.  Polycyclic aromatic hydrocarbon (PAH) o-quinones produced by the aldo-keto-reductases (AKRs) generate abasic sites, oxidized pyrimidines, and 8-oxo-dGuo via reactive oxygen species. , 2006, Chemical research in toxicology.

[59]  A. Hartmann,et al.  (www.interscience.wiley.com) DOI: 10.1002/path.2039 , 2006 .

[60]  D. Peehl,et al.  Transcript profiling of the androgen signal in normal prostate, benign prostatic hyperplasia, and prostate cancer. , 2006, Endocrinology.

[61]  C Roland Wolf,et al.  Generation of a stable antioxidant response element-driven reporter gene cell line and its use to show redox-dependent activation of nrf2 by cancer chemotherapeutic agents. , 2006, Cancer research.

[62]  Stacy L. Gelhaus,et al.  Metabolism of benzo[a]pyrene in human bronchoalveolar H358 cells using liquid chromatography-mass spectrometry. , 2007, Chemical research in toxicology.

[63]  Stacy L. Gelhaus,et al.  Aldo-keto reductase- and cytochrome P450-dependent formation of benzo[a]pyrene-derived DNA adducts in human bronchoalveolar cells. , 2007, Chemical research in toxicology.

[64]  K. Gates.  Abstracts, American Chemical Society Division of Chemical Toxicology, 234th ACS National Meeting, Boston, MA, August 19–23, 2007 , 2007 .

[65]  R. Lea,et al.  Identification of a novel co-transcription of P450/1A1 with telomerase in A549. , 2007, Gene.

[66]  J. Rokach,et al.  Regulation of 5-hydroxyeicosanoid dehydrogenase activity in monocytic cells. , 2007, The Biochemical journal.

[67]  Trevor M Penning,et al.  Human aldo-keto reductases: Function, gene regulation, and single nucleotide polymorphisms. , 2007, Archives of biochemistry and biophysics.