Expression of cytochromes P450 1A1 and 1B1 in human lung from smokers, non-smokers, and ex-smokers

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Abstract

Cytochromes P450 1A1 and 1B1 are known to bioactivate procarcinogens such as polycyclic aromatic hydrocarbons (PAHs) found in cigarette smoke and are inducible via an Ah receptor-mediated mechanism. The aim of this study was to examine the levels of expression of CYP1A1 and CYP1B1 in samples of lung from smokers (n = 18), non-smokers (n = 7), and ex-smokers (n = 7). Using immunoglobulin preparations of highly specific polyclonal antibodies and immunoblot analysis of microsomes from lung tissues, we determined the specific content for CYP1A1 and CYP1B1. For CYP1A1, we found median expression levels of 15.5 pmol/mg microsomal protein in smokers, 6.0 pmol/mg microsomal protein in non-smokers, and 19.0 pmol/mg microsomal protein in ex-smokers. The difference in median expression levels of smokers and ex-smokers compared to non-smokers was statistically significant. For CYP1B1, we found median expression levels of 1.8 pmol/mg microsomal protein in smokers, 1.0 pmol/mg microsomal protein in non-smokers, and 4.4 pmol/mg microsomal protein in ex-smokers. The difference in median expression levels between ex-smokers and non-smokers was statistically significant. These results suggest that levels of expression of CYP1A1 and CYP1B1 protein in lung tissues from smokers and ex-smokers are quantitatively greater than in non-smokers. By immunohistochemical analysis, we demonstrated the expression of CYP1A1 and CYP1B1 in normal human alveolar type I and II cells, ciliated columnar epithelial cells lining bronchoalveolar airways, and alveolar macrophages. These results confirm that CYP1A1 is expressed in normal human lung, appears to be induced in smokers, and show interindividual variation; the similar characteristics of CYP1B1 are demonstrated.

Introduction

Many carcinogens require bioactivation by endogenous enzymes to achieve their mutagenic and carcinogenic forms. Numerous carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and nitrosamines, are metabolized by cytochrome P-450 (CYP) to both mutagenic and non-mutagenic metabolites. Studies with benzo[a]pyrene (B[a]P) have shown that CYP enzymes are critical to the bioactivation of this procarcinogen to the postulated ultimate carcinogens, the diol-epoxides Conney et al., 1994, Gelboin, 1980. Additionally, B[a]P is capable of inducing the expression of CYP through an Ah receptor-dependent mechanism, thereby increasing the rate of its own metabolism Hankinson, 1995, Nebert and Gelboin, 1968, Whitlock, 1989.

CYP1 family enzymes are inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin and other PAHs through high affinity binding of the Ah receptor Rendic and Di Carlo, 1997, Whitlock, 1999. The CYP1A subfamily consists of two members, of which CYP1A1 catalyzes the metabolism of B[a]P and many other PAHs, and CYP1A2 that shares some substrate overlap (Rendic and Di Carlo, 1997). CYP1B1 is the only member of the 1B subfamily and has been shown to metabolize a large number of tobacco, environmental and dietary carcinogens, including B[a]P Hayes et al., 1996, Kim et al., 1998, Shimada et al., 1996a, Shimada et al., 1996b, Sutter et al., 1994. Dibenzo[a,l]pyrene (DB[a,l]P) and 7,12-dimethylbenz[a]anthracene (DMBA) are sterically hindered PAHs bioactivated preferentially by CYP1B1 to ultimate carcinogenic forms Buters et al., 1999, Luch et al., 1998, Otto et al., 1992, Shimada et al., 1996a. Both of these enzymes, CYP1A1 and CYP1B1, may play important roles in the bioactivation of tobacco smoke carcinogens.

Extra-hepatic expression of cytochromes P450 in humans is of importance in determining the metabolic activation of procarcinogens and xenobiotics. Unlike CYP1A2, which is primarily a hepatic enzyme, CYP1A1 has been found in many extra-hepatic tissues at low basal levels (Rendic and Di Carlo, 1997). In addition, CYP1A1 is highly inducible, leading to speculation that CYP1A1 may be the primary enzyme responsible for procarcinogen activation in extra-hepatic tissues (Okey, 1990). However, mRNA analysis has found CYP1B1 to be more widely distributed among human extra-hepatic tissues compared to CYP1A1 (Shimada et al., 1996a). In addition, CYP1B1 is also inducible through a similar Ah receptor-mediated mechanism as CYP1A1 Spink et al., 1997, Sutter et al., 1991, Sutter et al., 1994, Tang et al., 1996. Taken together, these data suggest that CYP1B1 may also play a major role in extra-hepatic procarcinogen metabolism along with CYP1A1.

Interest in human lung expression of CYP stems from the cause–effect relationship between tobacco smoking and lung cancer (Samet, 1994). It has been hypothesized that genetic variances in CYP expression, inducibility, or activity are responsible for individual susceptibility to cancer Alexandrie et al., 1994, Nebert, 1991. CYP1A1 expression and activity has been extensively characterized in human tissues. However, most studies have focused on mRNA expression Czerwinski et al., 1994, Willey et al., 1997. Because of minimal availability of reagents and high quality normal tissue samples, only a few studies have reported on positive CYP1A1 protein expression in human lung by immunoblot and immunohistochemical techniques Anttila et al., 1991, Anttila et al., 1992, Anttila et al., 1997, Kivisto et al., 1995, Saarikoski et al., 1998.

CYP1B1 in humans has not been as well studied. In one immunochemical study, CYP1B1 protein was detected in human tumors but not in corresponding normal tissues (Murray et al., 1997). This conflicts with current knowledge about CYP1B1 mRNA expression, which has been found extensively in extra-hepatic human tissues Shimada et al., 1996a, Sutter et al., 1994. In this report, we have obtained normal human lung tissues from donors representing smokers, non-smokers, and ex-smokers to characterize the protein expression and cellular localization of CYP1B1 and CYP1A1. The results described in this manuscript are a direct extension of the three decades of pioneering research conducted by Dr. Bresnick in the area of cytochrome P450 regulation in response to PAHs. His work contributed greatly to our current understanding of the mechanisms by which PAHs affect aryl hydrocarbon hydroxylase activity and the CYP1 enzymes that catalyze this reaction.

Section snippets

Isolation of human lung microsomes

Frozen tissue samples were obtained either as anatomical gifts from the International Institute for the advancement of Medicine (Scranton, PA) or as discarded normal tissue adjacent to tissue surgically removed at the Vanderbilt University Medical Center. All samples were collected under protocols approved by the Committee on Human Research at the respective institutions. Sample information is provided in Table 1. In the case of surgical samples, smoking status was self-reported. In the case of

Results

Expression of CYP1A1 and CYP1B1 was analyzed quantitatively by immunoblot. A representative immunoblot showing high and low CYP1A1 and CYP1B1 is presented in Fig. 1 from each of the three groups: smokers, non-smokers, and ex-smokers. CYP was detected in all microsomal samples analyzed. A summary of the individual data and smoking history is presented in Table 1. Levels of CYP1B1 protein (presented as picomole CYP1B1 protein per milligram microsomal protein) ranged from 0.4 to 12.7 in smokers;

Discussion

Measurements of the amount of CYP1A1 and CYP1B1 protein in human lung were performed on isolated microsomes and presented as picomole CYP per milligram microsomal protein. Isolation of the endoplasmic reticular fraction aids in immunoblotting analysis of CYP by enriching CYP content and allows the isolation of active enzymes Guengerich, 1990, Prough et al., 1977. CYP enzymes have been characterized historically by their content in the microsomal fraction and are sometimes difficult to detect if

Acknowledgements

This research was supported by grants from the US Public Service ES08148, NIEHS Center Grant ES03819, and Training Grant ES07141.

References (80)

  • N.J. Walker et al.

    Characterization of the dose–response of CYP1B1, CYP1A1, and CYP1A2 in the liver of female Sprague–Dawley rats following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin

    Toxicol. Appl. Pharmacol

    (1999)
  • C. Wei et al.

    CYP1A2 is expressed along with CYP1A1 in the human lung

    Cancer Lett

    (2001)
  • J.P. Whitlock

    The control of cytochrome P-450 gene expression by dioxin

    Trends Pharmacol. Sci

    (1989)
  • A.K. Alexandrie et al.

    Genetic susceptibility to lung cancer with special emphasis on CYP1A1 and GSTM1: a study on host factors in relation to age at onset, gender and histological cancer types

    Carcinogenesis

    (1994)
  • S. Anttila et al.

    Smoking and peripheral type of cancer are related to high levels of pulmonary cytochrome P450IA in lung cancer patients

    Int. J. Cancer

    (1991)
  • S. Anttila et al.

    Immunohistochemical detection of pulmonary cytochrome P450IA and metabolic activities associated with P450IA1 and P450IA2 isozymes in lung cancer patients

    Environ. Health Perspect

    (1992)
  • S. Anttila et al.

    Expression and localization of CYP3A4 and CYP3A5 in human lung

    Am. J. Respir. Cell Mol. Biol

    (1997)
  • E. Bauer et al.

    Oxidation of benzo[a]pyrene by recombinant human cytochrome P450 enzymes

    Chem. Res. Toxicol

    (1995)
  • J.T. Buters et al.

    Cytochrome P450 CYP1B1 determines susceptibility to 7, 12-dimethylbenz[a]anthracene-induced lymphomas

    Proc. Natl. Acad. Sci. U.S.A

    (1999)
  • E.L. Cavalieri et al.

    Comparative dose-response tumorigenicity studies of dibenzo[alpha,l]pyrene versus 7,12-dimethylbenz[alpha]anthracene, benzo[alpha]pyrene and two dibenzo[alpha,l]pyrene dihydrodiols in mouse skin and rat mammary gland

    Carcinogenesis

    (1991)
  • Cohen, J., 1988. Statistical Power Analysis for the Behavioral Sciences, 2nd ed. L. Erlbaum Associates, Hillsdale,...
  • A.H. Conney et al.

    Studies on the metabolism of benzo[a]pyrene and dose-dependent differences in the mutagenic profile of its ultimate carcinogenic metabolite

    Drug Metab. Rev

    (1994)
  • Conover, W.J., 1980. Practical Nonparametric Statistics, 2nd ed. Wiley, NY, p. xiv,...
  • M. Czerwinski et al.

    Quantification of CYP2B7, CYP4B1, and CYPOR messenger RNAs in normal human lung and lung tumors

    Cancer Res

    (1994)
  • T.R. Devereux

    Alveolar type II and Clara cells: isolation and xenobiotic metabolism

    Environ. Health Perspect

    (1984)
  • J.C. Gautier et al.

    Contribution of human cytochrome P450 to benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol metabolism, as predicted from heterologous expression in yeast

    Pharmacogenetics

    (1996)
  • H.V. Gelboin

    Benzo[a]pyrene metabolism, activation and carcinogenesis: role and regulation of mixed-function oxidases and related enzymes

    Physiol. Rev

    (1980)
  • F.P. Guengerich

    Purification and characterization of xenobiotic-metabolizing enzymes from lung tissue

    Pharmacol. Ther

    (1990)
  • J.M. Guidice et al.

    Evidence for CYP2D6 expression in human lung

    Biochem. Biophys. Res. Commun

    (1977)
  • J. Hakkola et al.

    Expression of CYP1B1 in human adult and fetal tissues and differential inducibility of CYP1B1 and CYP1A1 by Ah receptor ligands in human placenta and cultured cells

    Carcinogenesis

    (1997)
  • O. Hankinson

    The aryl hydrocarbon receptor complex

    Annu. Rev. Pharmacol. Toxicol

    (1995)
  • Hasleton, P.S., Spencer, H., 1996. Spencer's Pathology of the Lung, 5th ed. McGraw-Hill, NY,p. xii,...
  • C.L. Hayes et al.

    17 Beta-estradiol hydroxylation catalyzed by human cytochrome P450 1B1

    Proc. Natl. Acad. Sci. U.S.A

    (1996)
  • Hettmansperger, T.P., 1984. Statistical Inference Based on Ranks. Wiley, NY, p. xviii,...
  • S.W. Jakobsson et al.

    Monooxygenase activities of human liver, lung, and kidney microsomes—A study of 42 post mortem cases

    Acta Pharmacol. Toxicol. (Copenh.)

    (1982)
  • N.T. Karki et al.

    Aryl hydrocarbon hydroxylase in lymphocytes and lung tissue from lung cancer patients and controls

    Int. J. Cancer

    (1987)
  • G. Kellermann et al.

    Genetic variation of aryl hydrocarbon hydroxylase in human lymphocytes

    Am. J. Hum. Genet

    (1973)
  • G. Kellermann et al.

    Aryl hydrocarbon hydroxylase inducibility and bronchogenic carcinoma

    N. Engl. J. Med

    (1973)
  • G. Kellermann et al.

    Variation of microsomal mixed function oxidase(s) and human lung cancer

    Cancer

    (1980)
  • J.H. Kim et al.

    Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450

    Carcinogenesis

    (1998)
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