CYP1A1) Activities: Impact of Smoking Status and CYP1A1, Aryl Hydrocarbon Receptor, and Glutathione S-Transferase M1 Genetic Polymorphisms1

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Vol. 10, 839–853, August 2001 Cancer Epidemiology, Biomarkers & Prevention 839

Human Lung Microsomal Cytochrome P4501A1 (CYP1A1) Activities: Impact of Smoking Status and CYP1A1, Aryl Hydrocarbon Receptor, and Glutathione S-Transferase M1 Genetic Polymorphisms1

Graeme B. J. Smith, Patricia A. Harper, pmol/min/mg protein, respectively; P > 0.05). Judy M. Y. Wong, Maria S. M. Lam, Ken R. Reid, Furthermore, microsomal EROD activities from (28 ؍ Dimitri Petsikas, and Thomas E. Massey2 current smokers with the GSTM1-null genotype (n ؍ Departments of Pharmacology and Toxicology [G. B. J. S., T. E. M.], Surgery were not significantly different from those (n 18) K. R. R., D. P.], and Medicine [T. E. M.], and School of Environmental carrying at least one copy of GSTM1 (12.61 ؎ 14.24 and] ;Studies [T. E. M.], Queen’s University, Kingston, Ontario K7L 3N6; 11.34 ؎ 12.53 pmol/min/mg protein, respectively Department of Pharmacology, University of Toronto, Toronto, Ontario M5S P 0.05). Additionally, when genotypic combinations of 1A8 [P. A. H., J. M. Y. W., M. S. M. L.]; and Division of Clinical > Pharmacology and Toxicology, The Hospital for Sick Children, Toronto, CYP1A1, AHR, and GSTM1 were assessed, there were no Ontario M5G 1X8 [P. A. H.], Canada significant effects on EROD activity. On the basis of microsomal enzyme activities from heterozygotes, CYP1A1*1/2A, CYP1A1*1/2B, CYP1A1*1/4, and AHR554 Abstract Arg/Lys variants do not appear to significantly affect There are numerous conflicting epidemiological studies CYP1A1 activities in human lung, and we observed no addressing correlations between cytochrome P450 1A1 association between CYP1A1 activity and the GSTM1-null (CYP1A1) genetic polymorphisms and lung cancer polymorphism. susceptibility, with associations plausibly linked to alterations in carcinogen bioactivation. Similarly, Introduction correlations between aryl hydrocarbon receptor gene Most chemicals that initiate lung cancer, including those found (AHR) codon 554 genotype and CYP1A1 inducibility in tobacco smoke, require bioactivation in the lung to their are controversial. The objective of this study was to “ultimate” genotoxic metabolites that interact with DNA. Indi- determine whether smoking status, and CYP1A1, AHR, vidual differences in the ability to bioactivate carcinogens may and glutathione S-transferase M1 gene (GSTM1) contribute to host susceptibility and, thus, may play a role in polymorphisms correlate with altered CYP1A1 activities. lung cancer risk (1–3). Lung microsomal CYP1A1-catalyzed 7-ethoxyresorufin In 1973, Kellerman et al. (4) first described an association O-dealkylation (EROD) activities were much higher in 3 between high AHH inducibility in cultured lymphocytes and ؍ tissues from current smokers (n 46) than in those from bronchogenic carcinoma. Subsequently, numerous studies es- ؎ and 0.77 13.46 ؎ 12.11 ;24 ؍ non-/former smokers (n tablished a link between AHH inducibility and lung cancer risk ؎ 1.74 pmol/min/mg protein, respectively, mean SD; P < (reviewed in Ref. 1). Much of human lung microsomal AHH 0.05). However, EROD activities in lung microsomes from activity has been attributed to a single member of the P450 ؍ current smokers CYP1A1*1/1 (n 33) and heterozygous multigene superfamily, CYP1A1, and cigarette smoking is con- were not (10 ؍ MspI variant CYP1A1*1/2A (n sidered to be the most important factor related to the pulmonary ؎ ؎ significantly different (12.23 13.48 and 8.23 9.76 expression of this enzyme (5–10). CYP1A1 bioactivates PAHs, pmol/min/mg protein, respectively, P > 0.05). Three a major class of tobacco procarcinogens (3). The CYP1A1 gene current smokers were heterozygous variant CYP1A1*1/2B exhibits inducibility in human lung through ligand binding to (possessing both *2A and *2C alleles), and exhibited the AHR, and CYP1A1 can be induced via an AHR-dependent activities similar to individuals CYP1A*1/1. One current mechanism by PAHs and other similar planar compounds smoker was heterozygous variant CYP1A1*4 and (Refs. 5, 11–13 and references therein). A positive correlation exhibited activities comparable with individuals exists between CYP1A1 activity and pulmonary-PAH-associ- CYP1A1*1/1 at that locus. EROD activities in microsomes ated DNA adduction, implicating this enzyme as an important ؍ 554 from current smokers AHR Arg/Arg (n 41) and factor in the etiology of lung cancer (reviewed in Refs. 14, 15). were not (5 ؍ heterozygous variant AHR554Arg/Lys (n In recent years, the genetic determinants of individual ؎ ؎ significantly different (12.13 13.56 and 12.01 14.23 differences in CYP1A1 expression and their association with lung carcinogenesis have been examined. CYP1A1 contains two prominent polymorphic sites associated with lung cancer: a 3Ј Received 2/1/01; revised 5/17/01; accepted 5/31/01. flank T to C transition known as the MspI mutation The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Canadian Institutes of Health Research (CIHR) Grant MT10382. 3 The abbreviations used are: AHH, aryl hydrocarbon hydroxylase; CYP1A1, 2 To whom requests for reprints should be addressed, at Department of Pharma- cytochrome P450 1A1; AHR, aryl hydrocarbon receptor; PAH, polycyclic aro- cology and Toxicology, Queen’s University, Kingston, ON K7L 3N6, Canada. matic hydrocarbon; GST, glutathione S-transferase; 7-ERF, 7-ethoxyresorufin; Phone: (613) 533-6115; Fax: (613) 533-6412; E-mail: [email protected]. SSCP, single-strand conformation polymorphism; EROD, 7-erf O-dealkylation.

(CYP1A1*2A; Ref. 16); and an exon 7 heme binding region A We have previously demonstrated that the GSTM1 gene dele- to G transition resulting in an Ile to Val substitution tion results in a functional deficiency in GSTM1–1 activity in (CYP1A1*2C; Ref. 17). Individuals possessing both mutations human lung cytosols (86). Vaury et al. (87) suggested that high are denoted as CYP1A1*2B (Ref. 18 and CYP1A1 allele no- inducibility of CYP1A1 is associated with the GSTM1-null menclature website).4 genotype in cultured cell lines, possibly because of persistence Numerous epidemiological studies have addressed corre- of CYP1A1 inducers attributable to lowered GST-catalyzed lations between CYP1A1 genotype and lung cancer suscepti- metabolism. However, these results were not corroborated in bility, yielding conflicting results (Refs. 16, 19–51; reviewed in two other studies using cultured lymphocytes for in vitro phe- Table 1). Early studies first pointed toward an association notyping (64, 88). between variant genotype and lung cancer risk in Japanese What the majority of previous studies have failed to ad- populations (16, 19, 21, 25), with no associations found in dress is whether polymorphisms actually translate into signif- Caucasian populations (20, 22), conceivably because of rela- icant alterations in CYP1A1 enzyme activity/induction in the tively low allele frequencies in the latter. A number of studies target tissue for pulmonary carcinogens. Such knowledge is have found associations with lung cancers in individuals car- necessary to validate the presumed basis for reported associa- rying at least one copy of either variant allele (27, 28, 31, 35, tions between genotype and lung cancer susceptibility and to 39, 40, 42, 49), a more likely scenario in Caucasian popula- explain conflicting data in the literature. As such, objectives of tions, in which variant allele frequencies are low (52). The this study were to determine whether CYP1A1 and/or AHR assumption is that genetically determined alterations (polymor- genotypes correlate with CYP1A1-related activities in human phisms) in the expression of CYP1A1 affect related enzyme lung microsomes, and to determine the impact of the GSTM1 activities and, thus, the manner in which carcinogens are me- gene deletion polymorphism and smoking status on these ac- tabolized. tivities. Studies examining the effects of the CYP1A1*2A and CYP1A1*2C mutations on enzyme activity in various models Materials and Methods have also produced conflicting results (Refs. 53–66; Table 1). Multiple studies also have examined the association of CYP1A1 Chemicals. Chemicals were obtained as follows: Taq poly- genetic polymorphisms with biomarkers or biological outcomes merase, deoxynucleoside triphosphates, MgCl2, and PCR relevant to lung cancer and lung cancer risk. Association of buffer from Life Technologies, Inc., Gaithersburg, MD,; MspI CYP1A1 variants with DNA-adduct levels in lung tissues (67– and BsaI from New England Biolabs, Mississauga, ON, Can- 72) and circulating lymphocytes (73) from smokers, pulmonary ada; MeaIII from Boehringer Mannheim, Dorval, PQ, Canada; CYP1A1 expression (74), lung cancer prognosis (75), and 7-ERF and resorufin from Molecular Probes, Eugene, OR; PCR p53-tumor suppressor gene mutations (76–80) have all been primers from Cortec DNA Services Laboratories, Kingston, investigated, yielding conflicting results (Table 1). A second ON, Canada; Metaphor agarose from FMC Bioproducts, Rock- exon 7 heme binding region polymorphism,aCtoAtransver- land, ME. All other chemicals were reagent grade or higher and sion resulting in a Thr to Asn substitution, in close proximity to were obtained from common commercial suppliers. CYP1A1*2C has been identified (81). Although not associated Tissue Procurement. Human lung tissue, devoid of macro- with lung cancer susceptibility (81), recent reports from ex- scopically visible tumors, was obtained from Kingston General pression systems (65, 66) suggest differences in substrate af- Hospital, in accordance with procedures approved by the finities and catalytic activities associated with the CYP1A1*4 Queen’s University Research Ethics Board. After informed variant protein CYP1A1.4 relative to CYP1A1.1 (Table 1). consent, sections of peripheral lung (20–100 g) were removed Possible associations between the AHR gene polymor- during clinically indicated lobectomy. Immediately after re- phisms and CYP1A1 inducibility also have proven to be con- moval, the tissue was placed in 0.9% NaCl solution and kept on troversial (Refs. 64, 82–85; Table 2). Kawajiri et al. (83) found ice. Elapsed time between surgical resection and tissue manip- 3 that the AHR codon 554 Arg to Lys polymorphism (AHR554)in ulation was ϳ15 min. Initially, 0.5 cm was removed from the Ϫ exon 10 (receptor transactivation domain) was not associated tissue, snap-frozen in liquid N2, and stored at 80°C for DNA 3 with either CYP1A1 inducibility in cultured lymphocytes or isolation, and 1.5 cm sections were removed from the cut with lung cancer incidence in a Japanese population. Contrary specimen surface and placed in 10% neutral buffered formalin. to this observation, Smart and Daly (64) found that cultured The fixed tissue was dehydrated and embedded in paraffin, and lymphocytes from individuals carrying at least one AHR554 5-␮m sections were stained with H&E (89). They were then Lys-variant allele had significantly elevated CYP1A1 activity examined by light microscopy to confirm the absence of mi- and expression. However, a recent study by Wong et al. (85) croscopic tumors. Patients were characterized with respect to suggested no evidence for altered biological function or age, gender, surgical diagnosis, possible occupational exposure CYP1A1 induction associated with AHR554 Arg to Lys. to carcinogens, drug treatment 1 month prior to surgery, and Pulmonary glutathione S-transferases (GSTs) play a major self-reported smoking history (Table 3). Patients were classi- role in detoxification of reactive electrophiles via conjugation fied as former smokers if smoking cessation was greater than 2 with glutathione and, thus, are integral components in the months before surgery. This time interval was chosen to elim- balance between carcinogen activation and detoxification in the inate the inductive effects of cigarette smoke on CYP1A1 (5). lung. Numerous studies have assessed the combined effects of DNA Isolation and Genotyping. Genomic DNA was isolated CYP1A1 genetic polymorphisms and the GSTM1 deletion poly- by protease digestion followed by standard phenol:chloroform morphism in relation to lung cancer (21, 25, 27, 33, 39, 41, 42, extraction and ethanol precipitation (90). Patients were geno- 45, 47, 49, 51, 75, 76, 78, 80), the majority of which suggest typed for the CYP1A1*2A(MspI) polymorphism by PCR- increased risk when variant genotypes are combined (Table 1). RFLP (28). Restriction digest products were resolved in an ethidium bromide-stained 1.8% agarose gel. Genotyping for the CYP1A1*2C (Ile to Val) polymorphism was performed using a PCR-based designed-RFLP method (91). Restriction digest 4 Internet address: http://www.imm.ki.se/CYPalleles/cyp1a1.htm. 157- and 85-bp fragments for CYP1A1*1 homozygotes and

Table 1 CYP1A1 studies with relevance to lung cancer susceptibility and functionality of genetic polymorphisms

Study Population (region) Mutation(s) examined Findings and correlations

Kawajiri et al., 1990 Japanese Msp1 CYP1A1*2A/2A associated with lung cancer (especially SCCa); AHH inducibility (16) may be attributed to Msp1 genotypes Hayashi et al., 1991 (17) Japanese Msp1, Ile3Val Discovery of heme binding region (exon 7) polymorphism. Msp1 and Ile3Val mutations are genetically linked3CYP1A1*2B (new nomenclature) Nakachi et al., 1991 (19) Japanese Msp1 Patients CYP1A1*2A/2A contracted carcinoma after fewer cigarettes; individuals with CYP1A1*2A/2A genotype at high risk for SCC at low-dose levels of cigarette smoking; difference in susceptibility between genotypes reduced at high-dose levels Petersen et al., 1991 (53) Mediterranean family Msp1 Cosegregation of CYP1A1 inducibility phenotype and Msp1 (CYP1A1*1/2A) mutations (cultured lymphocyte AHH activity) Tefre et al., 1991 (20) Norwegian Msp1 No association with lung cancer risk: histological types, smoking history, or occupational exposure to asbestos investigated Clark et al., 1992 (54) North American Msp1 CYP1A1*1/2A mutation associated with inducibility phenotype measured by EROD in lymphocytes induced by TCDD (no homozygote variants detected) Hayashi et al., 1992 (21) Japanese Ile3Val, GSTM1 CYP1A1*2C/2C variants associated with SCLC and not AC; increased risk with GSTM1 null genotype Hirvonen et al., 1992 Finnish Msp1, Ile3Val No association of CYP1A1*2A with lung cancer risk: various histological subtypes (22) examined. Linkage or CYP1A1*2B allele established [overlapping with (119, 120)] Shields et al., 1992 (23) North American Msp1 No association of CYP1A1*2A with lung cancer risk; a trend indicating more CYP1A1*2A alleles in individuals with SCC was observed (not statistically significant) Cosma et al., 1993 (55) European-, Asian-, Msp1 No correlations between CYP1A1 gene inducibility (mRNA quantitation) and African American CYP1A1*2A variants (mitogen-stimulated lymphocytes). Apparent racial difference in expression levels observed with African Americans having lower induction values Cosma et al., 1993 (56) European-, Asian-, Msp1, Ile3Val CYP1A1*2C (even one allele) associated with inducibility phenotype; lymphocyte African American ERODs revealed significantly higher levels of inducible enzyme activity Kawajiri et al., 1993 None; yeast cell Ile3Val CYP1A1*2C mutation causes a 2-fold increase in CYP1A1 activity and (57) expression mutagenicity measured in the Ames test [benzo-(a)-pyrene as substrate] Kawajiri et al., 1993 Japanese Ile3Val Both CYP1A1*2C (*2A not examined) and p53 codon 72 Arg to Pro (24) polymorphism modified lung cancer risk, but worked independently; no etiological association between the two susceptibility genes Nakachi et al., 1993 (25) Japanese Msp1, Ile3Val, Both CYP1A1*2A and *2C polymorphisms associated with lung cancer risk, GSTM1 increased risk with GSTM1-null (stronger at low cigarette dose) Shields et al., 1993 (26) North American Msp1 No association of CYP1A1*2A alleles with lung cancer risk even when examined by histological type or cigarette consumption Shields et al., 1993 (67) North American Ile3Val, GSTM1 No association of CYP1A1*2C variants with PAH-adduct formation in human lung samples, GSTM1-null genotype associated with higher levels of DNA adduction Alexandrie et al., 1994 Scandinavian Msp1, Ile3Val, No association with lung cancer overall, but increased frequency of CYP1A1*1/2A (27) GSTM1 in patients diagnosed with SCC at a younger age (Ͻ66 yr). Significant association with SCC when combined with GSTM1-null genotypeb Crofts et al., 1994 (58) Heterogeneous Msp1, Ile3Val Both CYP1A1*2C heterozygotes and homozygotes exhibited increased CYP1A1 American induction and CYP1A1 activity; no effect of CYP1A1*2A on CYP1A1 induction in mitogen-stimulated lymphocytes. Increased inducibility and activity when Msp1 and Ile3Val combined (CYP1A1*2B) Drakoulis et al., 1994 German Msp1, Ile3Val CYP1A1*2C, (mostly heterozygotes) showed a marginally significant association (28) with lung cancer risk (especially SCC); no association CYP1A1*2A, with lung cancer risk (mostly heterozygotes) Landi et al., 1994 (59) Caucasian Msp1 CYP1A1*2A variants (one or both alleles) had significantly higher ERODs and CYP1A1 mRNA levels (lymphocytes whether induced by TCDD or not) Okada et al., 1994 (29) Japanese Msp1 CYP1A1*2A/*2A genotype is associated with lung cancer and metastasis particularly in light smokers with SCC Sugimura et al., 1994 South American Msp1 No association with CYP1A1*2A/*2A variants and lung cancer, even after (30) (Brazil) breakdown for race and tobacco consumption Wedlund et al., 1994 Mediterranean family Msp1, Ile3Val No association of CYP1A1*2A and CYP1A1*2C variants with AHH inducibility in (60) [same as (53)] lymphocytes (one patient CYP1A1*1/2A, exhibited high activity. Disputes Petersen et al. (53) Msp1 cosegregation datab Hamada et al., 1995 (31) South American Ile3Val CYP1A1*2C (at least one copy) associated with lung cancer risk (stronger in (Brazil) individuals with fewer pack-years) [same population as (30)] Ikawa et al., 1995 (32) Japanese (Miyagi) Msp1 No association of CYP1A1*2A variants with lung cancer; CYP1A1*1/*1 highly prevalent in cases Kato et al., 1995 (68) North American Ile3Val No statistical association of CYP1A1*2C variants with PAH adducts in human lung samples Kihara et al., 1995 (33) Japanese Msp1, Ile3Val, CYP1A1*2A/2A highly susceptible when combined with GSTM1-null genotype, GSTM1 but resistant to tobacco-related lung cancers when combined with GSTM1 positive genotype Nakachi et al., 1995 (34) Japanese Msp1, Ile3Val CYP1A1*2A/2A and CYP1A1*2C/2C variants associated with AC of the lung by grades of differentiation (cigarette dose examined) Sugimura et al., 1995 South American Msp1, Ile3Val, Both CYP1A1*1/2C and CYP1A1*2C/2C variants associated with lung variant (35) (Brazil) CYP2E1 cancer (especially with SCC); no association CYP1A1*2A or CYP2E1 alleles [same population as (30, 31)]

Table 1 Continued

Study Population (region) Mutation(s) examined Findings and correlations Goto et al., 1996 (75) Japanese Msp1, GSTM1 CYP1A1*2A (even one allele) associated with decreased survival, particularly when combined with GSTM1-null genotype (prognostic significance) Jacquet et al., 1996 (36) European Msp1 No association of CYP1A1*2A variants with susceptibility, inducibility only slightly higher for both CYP1A1*1/2A and CYP1A1*2A/2A; no correlation between CYP1A1*2A lung cancer and inducibility Kawajiri et al., 1996 Japanese Msp1, Ile3Val, CYP1A1*2A/2A and CYP1A1*2C/2C significantly associated with p53 mutations (76) GSTM1 in lung cancer; synergism when combined with GSTM1-null Kiyohara et al., 1996 Japanese Msp1, Ile3Val Cosegregation of CYP1A1*2A/2A genotype with AHH inducibility (noninduced (61) lymphocytes induced with 3-methylcholanthrene); CYP1A1*2C/2C mutants significantly higher AHH activity compared with heterozygotes and wild typeb Xu et al., 1996 (37) North American Msp1 CYP1A1*2A (even one allele) is associated with increased lung cancer risk (smoking dose accounted for) Zhang et al., 1996 (62) None; Escherichia Ile3Val CYP1A1*2C variant had slight but significantly higher EROD activities. Similar coli expression benzo(a)pyrene oxidation activities: suggesting no causal effect on susceptibilityb Bouchardy et al., 1997 Caucasian (France) Msp1, Ile3Val No association of CYP1A1*2A and/or CYP1A1*2C variants with lung cancer risk (38) (heterozygotes included in the at-risk grouping). Associated risk not increased after adjustment for tobacco and asbestos exposure, nor when stratified on smoking status, daily consumption, smoking duration, and histological type Garcia-Closas et al., Mostly Caucasian Msp1, GSTM1 CYP1A1*2A (even one allele) is associated with increased lung cancer risk, 1997 (39) (North American) evident after adjusting for pack-years; additionally GSTM1 deletion may contribute to lung cancer risk in combination with CYP1A1*2A heterozygous variant [expansion of study by Xu et al. (37)] Ishibe et al., 1997 (40) Mexican- and African Msp1, Ile3Val, CYP1A1*2A or CYP1A1*2C (one or more alleles) associated with an approximate Americans CYP1A1*3c 2-fold increase in lung cancer risk among light smokers Mooney et al., 1997 (73) North American Msp1, Ile3Val, Smokers with heterozygote or homozygote CYP1A1*2C genotypes had GSTM1 significantly higher levels of DNA damage (PAH adducts) in circulating lymphocytes Ohshima and Xu, 1997 Japanese Ile3Val No significant relation between p53 mutations and CYP1A1*2C and/or GSTM1 (77) genotypes in lung cancer patients 3 Persson et al., 1997 (63) None; yeast Ile Val No differences in KM or Vmax for EROD activities between CYP1A1*1 and expression CYP1A1*2C variants: not functionally important Hong et al., 1998 (41) Korean Msp1, Ile3Val, No association with lung cancer; CYP1A1*2C alleles more prevalent among GSTM1 controls than in lung cancer patients, particularly SCC (protective-like effect); CYP1A1*2A variants, no difference in genotypic frequencies between lung cancer and controls, similar results when combined with GSTM1-null genotype Le Marchand et al., Caucasian, Japanese, Msp1, GSTM1 CYP1A1*2A (at least one variant allele) associated with increased risk of SCC, 1998 (42) Hawaiian increased risk when combined with GSTM1 (no association with overall lung cancer riskb) Przygodzki et al., 1998 Caucasians (North Msp1, Ile3Val, CYP1A1*2C (mostly heterozygote variants) found in excess among lung cancer (78) American) GSTM1, CYP2E1 patients with p53 mutations, no significant increase in mutations when combined with GSTM1-null genotype Rojas et al., 1998 (69) Russian Msp1, Ile3Val, Individuals with CYP1A1*1/*2A or *1/*2B combined with GSTM1-null genotypes GSTM1 showed low levels of BPDE DNA adducts (lung tissue), three individuals with rare combinations CYP1A1*2A/*2A or CYP1A1*2A/*2B and GSTM1-null genotypes showed significantly higher DNA adduction Saarikoski et al., 1998 Finnish Msp1, Ile3Val CYP1A1*2A heterozygote exhibited the most intense results in both in situ (74) hybridization and immunohistochemical analyses for CYP1A1 in human lung tissue (one patientb) Schoket et al., 1998 (70) Hungarian Msp1 No statistically significant correlation between CYP1A1*2A genotypes and bronchial tissue DNA adducts after adjustment for either smoking status or malignancy Sugimura et al., 1998 Japanese (Okinawa) Ile3Val CYP1A1*2C/2C genotype was associated with a significantly higher lung cancer (43) risk, particularly SCC Taioli et al., 1998 (44) African-Americans Msp1, Ile3Val, No association with lung cancer when examined individually, but composite CYP1A1*3 genotype of CYP1A1*2A/2B was associated with overall lung cancer risk Bennett et al., 1999 (45) Caucasians (Females, Ile3Val, GSTM1, No association between CYP1A1*2C (*2A not examined) variant and lung cancer North American) GSTT1 risk attributable to environmental tobacco smoke exposure in never-smoking women, association for GSTM1-null alone was found Butkiewicz et al., 1999 Polish Ile3Val, GSTM1, CYP1A1*1/2C (*2A not examined) significantly associated with high DNA-adduct (71) GSTP1, CYP2D6 levels in lung tissues from individuals diagnosed with SCC when combined with GSTM1-null; a significant prevalence of this combined genotype was also found in patients with high adduct levels diagnosed with AC; relationship not observed among GSTM1-positive individuals Kim et al., 1999 (46) Korean Msp1, Ile3Val, No association of CYP1A1*2A or *2B variants with lung cancer in Koreans: CYP1A1*3, identification of a solitary *2C allele (i.e.,no*2A variants detected); CYP1A1*4d CYP1A1*3 and *4 variants not detected in this population Persson et al., 1999 (47) Chinese Msp1, Ile3Val, No evidence that carriers of certain alleles have an increased risk of lung cancer. GSTM1, CYP2E1, Frequency of CYP1A1*2A and *2B alleles among lung cancer patients and HYLe controls not significantly different Rusin et al., 1999 (79) Polish Ile3Val No statistically significant association found between p53 mutations and CYP1A1*2C, may be attributable to lack of statistical power

Table 1 Continued

Study Population (region) Mutation(s) examined Findings and correlations Wang et al., 1999 (80) Taiwanese Msp1, GSTM1 No association of CYP1A1*2A variants or GSTM1-null genotype with p53 tumor suppressor gene mutation, CYP1A1 and GSTM1-1 not indicated in metabolism of carcinogens responsible for the deletions in the immediate vicinity of repetitive sequences and/or tandem repeat sequences observed in p53 in Taiwanese lung cancer patients Anttilla et al., 2000 Finnish CYP1A1, AHR, ARNT No association of poorly induced CYP1A1 phenotype in human lung microsomes (114) with inactivating mutation in the structural or regulatory portions of CYP1A1, AHR,ortheAHR nuclear translocator gene (ARNT) Cheng et al., 2000 (72) Taiwanese Msp1, GSTM1 No association of CYP1A1*2A variant alone or in combination with GSTM1-null with DNA-adduct levels in human lung tissue; adduct levels did correlate with CYP1A1 expression by immunohistochemistry Dolzˇan et al., 2000 (48) Slovenian Msp1, Ile3Val No association of CYP1A1*2A or *2B alleles. Possible association between CYP1A1*2C/2C and SCC (no statistical power)b; population frequency too low to be a potentially useful marker Dresler et al., 2000 (49) Predominantly Ile3Val, GSTM1 CYP1A1*2C (*2A not examined) variants (mostly heterozygotes) associated with Caucasians (North increased risk of lung cancer for females particularly when combined with American) GSTM1-null Lin et al., 2000 (50) Taiwanese Msp1, HYL CYP1A1*2A/2A genotypes associated with SCC and risk increased when combined with high and normal HYL1 genotypes London et al., 2000 (51) Chinese (Men in Ile3Val, GSTM1 No association of CYP1A1*2C variant alleles with lung cancer overall; suggestion Shanghai) that individuals having at least one CYP1A1*2C allele might be related to lung cancer risk among low-level smokers, particularly those with GSTM1-null genotype Smart and Daly, 2000 Caucasians CYP1A1*1B,f No association of any of the variant alleles with differences in CYP1A1 activity (64) CYP1A1*1C,f (EROD) or immunoblotting in cultured lymphocytes, no correlation with Msp1, Ile3Val, GSTM1-null genotype; suggest an association with AHR polymorphism (see CYP1A1*3, Table 2) CYP1A1*4 Schwarz et al., 2000 None; insect cell Ile3Val, CYP1A1*4 CYP1A1*2 and *4 variant proteins CYP1A1.2 and CYP1A1.4 have slightly (65) expression increased KM for EROD, but have equivalent Vmax compared with CYP1A1.1. All three variants hydroxylate steroid hormones with varying efficiencies in stereo and regioselective manner 3 Schwarz et al., 2001 None; insect cell Ile Val, CYP1A1*4 CYP1A1*2 and *4 variant proteins CYP1A1.2 and CYP1A1.4 have lower KM for (66) expression all benzo(a)pyrene metabolites compare with CYP1A1.1 and exhibited significantly increased formation of diol epoxide 2, and exhibited only minor differences in kinetic behavior for EROD with slightly higher Vmax values a SCC, squamous cell carcinoma; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; SCLC, small cell lung cancer; AC, adenocarcinoma; CYP2E1, cytochrome P450 2E1 gene; CYP2D6, cytochrome P450 2D6 gene; GSTP1, glutathione S-transferase P1 gene; GSTT1, glutathione S-transferase T1 gene; BPDE, benzo(a)pyrene diol epoxide; ARNT, aryl hydrocarbon receptor nuclear translocator gene. b This point is especially pertinent to the study. c Race-specific African-American polymorphism. d Exon 7 Thr to Asn CYP1A1 genetic polymorphism. e Microsomal epoxide hydrolase gene. f Novel CYP1A1 genetic polymorphisms; CYP1A1*1A, wild type.

157-, 136-, and 85-bp fragments for CYP1A1*2C heterozygotes tients were genotyped for the AHR554 Arg to Lys polymorphism were resolved in a 2% agarose:2% Metaphor (1:1) agarose gel. by PCR-SSCP as described by Wong et al. (85). Given the close linkage of these mutations in Caucasians (22), Tissue Preparation and Enzyme Activity. EROD was used it is most likely that CYP1A1*2A and CYP1A1*2C variants are to assess human pulmonary CYP1A1 activities. Although occurring on the same allele. Thus, individuals were denoted as CYP1A2 and CYP1B1 can also catalyze the dealkylation of CYP1A1*2B when genotyping revealed alleles containing both 7-ERF, CYP1A2 expression in human lung remains controver- MspI and Ile to Val mutations. Genotyping for the CYP1A1*4 sial (74, 93), and the CYP1B1 protein is not significantly Thr to Asn polymorphism was modified from Cascorbi et al. expressed in normal human lung tissue (94, 95). Furthermore, (81). Briefly, the PCR product from the CYP1A1*2C genotyp- 7-ERF is a highly selective substrate for CYP1A1, with human ing protocol (242 bp) was digested with BsaI, yielding restric- recombinant CYP1B1 and CYP1A2-catalyzed oxidation activ- tion digest 217-bp fragments for CYP1A1*1 homozygotes and 242- and 217-bp fragments for CYP1A1*4 heterozygotes, and ities being one-tenth and one-forty-fifth those of CYP1A1, were resolved as described for CYP1A1*2C. DNA from an respectively (96). Thus, the EROD assay is highly selective for individual genotyped CYP1A1*1/4 and confirmed by direct CYP1A1 activity in human lung microsomes. sequencing (Cortec DNA Services Laboratories, Kingston, ON) Peripheral human lung microsomes were prepared from was used as a control in all subsequent CYP1A1*4 genotyping fresh or frozen lung tissues using standard subcellular fraction- assays. Patients were also genotyped for the GSTM1 gene ation techniques (97, 98). In the case of frozen tissues, lung 3 deletion polymorphism after resolving PCR products in a 3% tissue was initially cut into 1.5-cm sections, wrapped in alu- Ϫ agarose gel (92). Control DNA samples from individuals who minum foil and snap-frozen in liquid N2, and stored at 80°C were heterozygote variant for both CYP1A1*2A and *2C and until microsomal preparation. Protein concentration was deter- GSTM1-null for the gene deletion polymorphisms were gener- mined by the method of Lowry et al. (99). A modified version ously provided by Dr. D. A. Bell (National Institute of Envi- of the EROD assay (100) was used to assess CYP1A1 activity. ronmental Health Sciences, Research Triangle Park, NC). Pa- Briefly, the 3.0-ml EROD reaction mixture contained 0.1 M

Table 2 AHR studies with relevance to CYP1A1 inducibility and lung cancer susceptibility

Study Population (region) Mutation(s) examined Findings and correlations Catteau et al., 1995 (82) Caucasians (France) None, EROD (cultured Evidence for a major gene effect together with a polygenic lymphocytes) component [EROD measured in lymphocytes after induction by benz(a)anthracene] Kawajiri et al., 1995 Japanese AHR554: Arg3Lys No association of AHR554 polymorphism with CYP1A1 (AHH) (83) (AHR exon 10) inducibility in lymphocytes or with lung cancer incidence; expressed level of CYP1A1 mRNA associated with AHR and ARNTa mRNA levels Micka et al., 1997 (84) Caucasians: AHR exon 9 (amino CYP1A1 high-inducibility phenotype segregates with 3-generation acid sequence) chromosome 7p15 region (AHR localized to this region); no family association between exon 9 amino acid differences and inducibility (exon 9, region of murine AHR inducibility polymorphism) Smart and Daly, 2000 Caucasians AHR554: Arg3Lys, Individuals carrying at least one copy of AHR554Lys variant (64) G1768A(V570I) showed significantly higher levels of CYP1A1 activity and (AHR exon 10) protein expression Wong et al., 2000 (85) Various (ethnic AHR554:Arg3Lys Human AHR555Lys expressed in vitro: Arg and Lys-containing allele (AHR Exon 10) receptors had equivalent abilities to bind a dioxin-responsive distribution) element after TCDD treatment, and stimulate CYP1A1 mRNA expression in transfected receptor-deficient Hepa-1 cells a ARNT, AHR nuclear translocator; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

␮ Na/KPO4 buffer (pH 7.6), 5.0 M 7-ERF (in DMSO), 0.25 mM lifetime cigarette consumption did not correlate with higher NADPH, and 0.5 mg microsomal protein/ml. Resorufin forma- EROD activities; Fig. 1a). However, there was a significant tion was monitored spectrofluorometrically over time. Initial negative correlation between patient age and EROD activity reaction velocity was estimated from the linear portion of the (Fig. 1b). product formation curve and was quantitated using a resorufin CYP1A1 Allele Frequencies and EROD Activities. PCR- standard curve. RFLP-based CYP1A1 genotyping methods revealed both Data Analysis. EROD results are based on duplicate values for CYP1A1*2A and CYP1A1*2B heterozygotyes and CYP1A1*4 each patient. Statistically significant differences between non-/ heterozygotes. None of the patients was homozygous for the former smokers and current smokers were determined by the CYP1A1*2A, *2B,*2C,or*4 mutations. Allele frequencies in Mann-Whitney U test, because of heterogeneity of variance. this predominantly Caucasian North American population were Otherwise, statistically significant differences were determined 0.864, 0.136, 0.036, and 0.014 for CYP1A1*1, CYP1A1*2A, using Student’s t test. Correlations were examined using Pear- CYP1A1*2B, and CYP1A1*4 respectively. CYP1A1 activities son linear correlation analysis. For combined genotype analy- from CYP1A1*1/1 current smokers (n ϭ 33) and heterozygous sis, one-way ANOVA was used, followed by the Newman- variant (CYP1A1*1/2A) smokers (n ϭ 10) were not signifi- Keuls post hoc test, unless Bartlett’s test revealed heterogeneity cantly different (Fig. 2a; Table 4). Only three current smokers of variance, when a nonparametric ANOVA (Kruskal-Wallis) were heterozygous CYP1A1*1/2B and exhibited CYP1A1 ac- was used to analyze the data. In all cases, significance was tivities similar to individuals CYP1A1*1/1 (Fig. 2a; Table 4). assigned at P Ͻ 0.05. None of the patients’ genotypes exhibited CYP1A1*2C alleles (i.e., exon 7 Ile to Val alone). One current smoker was het- Results erozygous variant CYP1A1*1/4 and exhibited relatively high, Demographics. Patient demographics were obtained and re- but comparable, CYP1A1 activities to individuals genotyped corded preoperatively (Table 3). EROD activities were assessed homozygous CYP1A1*1 at this locus (patient 6JM, Table 3). in human lung microsomes made from peripheral lung speci- AHR Allele Frequencies and EROD Activities. SSCP anal- mens obtained from 45 male and 25 female individuals under- ysis for AHR554 Arg to Lys polymorphism revealed seven Ϯ going lobectomy (average age, 62 9.9 years; range, 40Ð82 AHR554Arg/Lys heterozygotes, giving allele frequencies of years; Table 3). Although racial demographics were not ob- 0.050 and 0.950 for Lys554 and Arg554 respectively. None of tained from individual patients, the population in the catchment the patients was homozygous AHR554Lys/Lys. There was no area for Kingston General Hospital is predominantly Cauca- significant difference in CYP1A1 activity in lung microsomes sian. from current smokers AHR554Arg/Lys (n ϭ 5) compared with EROD Activities. Mean EROD activities in microsomes from microsomes from individuals genotyped AHR554Arg/Arg (n ϭ Ϯ ϭ current smokers (12.11 13.46 pmol/min/mg; n 46) were 41; Fig. 2b). approximately 15-fold higher than those in microsomes from non-/former smokers (0.77 Ϯ 1.74 pmol/min/mg; n ϭ 24). In GSTM1 Effects on EROD Activities. Mean CYP1A1 activities in microsomes from current smokers with GSTM1 null fact, in 16 of 24 non-/former smoker tissues, EROD activity ϭ was nondetectable (i.e., Ͻ0.10 pmol/min/mg; Table 3). Mean genotype (n 28) and those in microsomes from individuals ϭ EROD activities were not significantly different between tis- carrying at least one copy of the GSTM1 gene (n 18) were sues from female (8.72 Ϯ 9.75 pmol/min/mg; n ϭ 16) and male very similar (Fig. 2c). (13.93 Ϯ 14.81 pmol/min/mg; n ϭ 30) current smokers. In Combined Genotypes and EROD Activities. There were no current smokers for whom self-reported complete smoking significant differences in EROD activities with various histories were available (n ϭ 36), there was no correlation combinations of the CYP1A1, AHR554, and GSTM1 genotypes between EROD activity and pack-year consumption (i.e., high (Table 4).

Table 3 Patient demographics, genotypes, and EROD activities

Smoking history AHR codon GSTM1 (time post- Drugc treatment/possible occupational Patienta 554 genotype EROD smoking exposure to carcinogens and/or Pathology CYP1A1 genotype (age,sex) Arg3Lys (gene activityd cessation); no. of enzyme inducers prior to surgery genotype deletion) pack-yearsb 9BM (70,M) Current; N/Ae Acetominophen 3 (Tylenol 3), Small cell carcinoma CYP1A1*1/1 Arg/Arg Positive 15.65 (life-long) tetracycline 1CM (65,M) Former (7 yr); N/ None Small cell carcinoma CYP1A1*1/2A Arg/Arg Positive ND A (life-long) 2CM (65,F) Former (3 yr); 44 Nifedipine (Adalat), gemfibrozil Adenocarcinoma CYP1A1*1/1 Arg/Arg Null ND (Lopid), ASA, trimipramine, glyceryl trinitrate 3CM (66,M) Current; 45 Spironolactone-hydrochlorothiazide Large cell bronchogenic CYP1A1*1/1 Arg/Lys Positive 6.24 (Aldactazide), lovastatin (Mevacor) carcinoma 4CM (81,M) Former (8 yr); N/ None Small cell carcinoma CYP1A1*1/2A Arg/Arg Positive ND A (life-long) 5CM (41,M) Former (4 mo); 25 Acetominophen (Tylenol 3) Granulomatosis CYP1A1*1/1,f *1/4 Arg/Arg Null ND 6CM (63,M) Current; 20 N/A Large cell carcinoma CYP1A1*1/2B Arg/Arg Positive 22.41 7CM (63,F) Current; 45 Propanolol hydrochloride (Inderol), Adenocarcinoma CYP1A1*1/1 Arg/Arg Null 20.03 oxazepam (Serax) 8CM (66,F) Former (N/A); N/A Adenocarcinoma CYP1A1*1/1 Arg/Arg Null ND N/A 9CM (49,M) Former (2 yr); 40 N/A Granulomatosis CYP1A1*1/1 Arg/Arg Positive ND 1DM (75,M) Current; (very Isoniazid, acetominophen (Tylenol), Cavitary lesion, TB CYP1A1*1/1 Arg/Arg Null 46.68 heavy) amitriptyline hydrochloride, positive (skin test), oxazepam (Serax), glyburide, aspergillosis diclfenac sodium (Voltaren) granulomatosis, acute pneumonia 2DM (63,M) Current; 40 Ipratropium bromide (Atravent) N/A CYP1A1*1/2A Arg/Arg Positive 22.71 4DM (65,F) Former (2 mo); 46 Acetominophen (Tylenol 2), Nonmalignant node CYP1A1*1/1 Arg/Arg Positive 5.11 lorezepam (Ativan) 5DM (48,M) Current; 25 None Hemoptysis CYP1A1*1/1 Arg/Arg Null 30.52 6DM (65,M) Current; 80 None Large cell carcinoma CYP1A1*1/2A Arg/Arg Positive ND 7DM (80,M) Current; 70 Beclomethasone dipropionate Squamous cell CYP1A1*1/2A Arg/Arg Null ND (Beclovent), isosorbide dinitrate carcinoma (Isordil), diltiazam hydrochloride (Cardizem), glyburide, salbutamol sulfate (Novosalmol) 8DM (56,M) Current; 40 Ibuprofen (Motrin)//bull-dozer Squamous cell CYP1A1*1/1 Arg/Lys Null 36.53 operator carcinoma 9DM (60,F) Current; N/A None Hemoptysis/carcinoid CYP1A1*1/1 Arg/Arg Null ND (life-long) tumour 1EM (77,M) Former (20 yr); 40 Salbutamol (Ventolin) Bronchogenic CYP1A1*1/1 Arg/Arg Null ND adenocarcinoma 2EM (74,M) Current; 45 Magnesium salicytate (Dodd’s back Non-small cell CYP1A1*1/1 Arg/Arg Positive 1.53 pills) carcinoma 3EM (63,F) Current; 50 Sodium levothyroxine (Eltroxin) Granulomatous lesion CYP1A1*1/2A Arg/Arg Null 6.44 4EM (58,M) Current; 20 Acetominophen 3 (Tylenol 3), Bronchogenic CYP1A1*1/1 Arg/Arg Positive ND Metformin hydrochloride, obstruction/nodules nifedipine 5EM (82,M) Current; 35 None Large cell carcinoma CYP1A1*1/1 Arg/Lys Null ND 6EM (43,F) Current; 35 Prednisone, salbutamol (Ventolin), Carcinoma, previously CYP1A1*1/1 Arg/Arg Null 26.19 ipratropium bromide (Atrovent)// TB-positive positive TB exposure in past 1FM (50,M) Current; 34 None Adenocarcinoma CYP1A1*1/1 Arg/Arg Positive 6.13 2FM (52,M) Current; 40 No drugs//fireman Squamous cell CYP1A1*1/2A Arg/Arg Null ND bronchogenic carcinoma 3FM (57,F) Current; 40 Diazepam (Valium), fentanyl, Large cell carcinoma CYP1A1*1/2B Arg/Lys Null 6.65 atropine sulphate, lidocaine hydrochloride (Xylocaine) 4FM (59,F) Nonsmoker None Adenocarcinoma CYP1A1*1/1 Arg/Arg Null ND (smoker in house); N/A 5FM (55,M) Current; N/A (very None Squamous cell CYP1A1*1/1 Arg/Arg Null 29.57 heavy) carcinoma 6FM (65,M) Current; N/A None Squamous cell CYP1A1*1/1 Arg/Arg Positive 7.91 carcinoma 7FM (61,M) Current; 45 None Squamous cell CYP1A1*1/1 Arg/Arg Null 15.24 carcinoma 8FM (69,F) Current; N/A None Carcinoma CYP1A1*1/1 Arg/Arg Null 2.63 9FM (50,M) Current; 50 None Large cell carcinoma CYP1A1*1/1 Arg/Arg Null 8.69 1GM (76,F) Current; 54 Sodium levothyroxine Large cell carcinoma CYP1A1*1/1 Arg/Arg Null 4.19

Table 3 Continued

Smoking history AHR codon GSTM1 (time post- Drugc treatment/possible occupational Patienta 554 genotype EROD smoking exposure to carcinogens and/or Pathology CYP1A1 genotype (age,sex) Arg3Lys (gene activityd cessation); no. of enzyme inducers prior to surgery genotype deletion) pack-yearsb 2GM (57,F) Former (8 yr); 40 None Carcinoma CYP1A1*1/1 Arg/Arg Null ND 3GM (67,M) Current; N/A (very None Squamous cell CYP1A1*1/2A Arg/Arg Null 28.34 heavy) carcinoma 4GM (76,M) Former (16 yr); 35 None Squamous cell CYP1A1*1/1 Arg/Arg Positive 0.59 carcinoma 5GM (69,M) Former (19 yr); 50 None Large cell carcinoma CYP1A1*1/1 Arg/Arg Null ND 6GM (65,M) Former (N/A); None Carcinoma CYP1A1*1/2A Arg/Arg Null ND N/A 7GM (58,F) Current; 39 ASA/butabital/caffeine (Fiorinal), Small cell carcinoma CYP1A1*1/1 Arg/Lys Positive 10.64 oxazepam, chloral hydrate, folic acid, B12 injections 8GM (72,M) Current ASA Non-small cell CYP1A1*1/1 Arg/Arg Null ND cigar/former carcinoma cigarette (9 yr); 110 9GM (74,F) Current; 60 Perphenazine, trazodone Adenocarcinoma CYP1A1*1/2A Arg/Arg Null 6.42 hydrochloride, chloral hydrate, ciprofloxacin, acetominophen (Tylenol) 1HM (64,F) Former (2.5 yr); None Large cell carcinoma CYP1A1*1/1 Arg/Arg Positive ND N/A 2HM (60,M) Current; 50 Atropine sulfate, fentanyl, diazepam Squamous cell CYP1A1*1/1 Arg/Arg Null ND (Valium), penicillin, tetracycline, carcinoma/pneumonia salbutamol (Ventolin), sodium cefuroxime, ranitidine (Zantac), acetominophen (Tylenol 2) 3HM (59,M) Current; 59 Finasteride (Proscar) Large cell carcinoma/ CYP1A1*1/2A Arg/Arg Positive 8.3 obstructive pneumonitis 4HM (55,F) Former (6 mo); 35 None Large cell carcinoma CYP1A1*1/1 Arg/Arg Positive 6.62 5HM (61,F) Current; 55 None Squamous cell CYP1A1*1/1 Arg/Arg Positive 2.72 carcinoma/lipoid pneumonia 6HM (68,F) Current; 40 Levothyroxin, oxazepam, ascorbic Adenocarcinoma CYP1A1*1/1 Arg/Arg Positive ND acid & vitamin B compound (possibly (Beminal) bronchogenic) 7HM (65,M) Former (12 yr); Indomethacin (Indocid), diclofenac Squamous cell CYP1A1*1/2B Arg/Arg Positive 0.38 120 sodium (Voltaren), famotidine carcinoma (Pepcid), enalapril maleate (Vasotec), ASA (Aspirin)//alcoholic (6 beers/day), liver cirrhosis, gout 8HM (72,M) Former (9 yr); 80 Enalapril maleate (Vasotec) Squamous cell CYP1A1*1/2B Arg/Arg Null 0.19 carcinoma (obstructive) 9HM (67,F) Current; 70.5 Radioactive iodine, ipratropium Squamous cell CYP1A1*1/1 Arg/Arg Null ND bromide (Atrovent), carcinoma (poorly beclamethasone dipropionate differentiated) (Beclaforte), amiloride hydrochloride/hydrochlorothiazide (Moduret) 1IM (66,M) Current; 40 Diltiazem hydrochloride (Cardizem), Squamous cell CYP1A1*1/1 Arg/Arg Null 3.62 salbutamol (Ventolin), budesonide carcinoma (Pulmicort), ranitidine (Zantac), ASA (Aspirin) 2IM (76,F) Former (4 yr); 50 Ranitidine (Zantac) Large cell carcinoma CYP1A1*1/2A Arg/Arg Null 0.69 (adenocarcinoma, metastatic) 3IM (66,M) Former (6 yr); 50 None Squamous cell CYP1A1*1/1 Arg/Arg Positive ND carcinoma/emphysema 4IM (49,M) Current; 80 None Large cell carcinoma/ CYP1A1*1/1 Arg/Arg Positive 14.49 adenosquamous carcinoma/emphysema 5IM (68,M) Former (12 yr); Budesonide (Pulmicort), salbutamol Squamous cell CYP1A1*1/1 Arg/Arg Positive 1.77 N/A (Ventolin), omeprazole magnesium carcinoma (poorly (Losec), cisapride monohydrate differentiated) (Prepulsid), levothyroxine (Synthroid), diltiazem hydrochloride (Cardizem), ASA (Aspirin)

Table 3 Continued

Smoking history AHR codon GSTM1 (time post- Drugc treatment/possible occupational Patienta 554 genotype EROD smoking exposure to carcinogens and/or Pathology CYP1A1 genotype (age,sex) Arg3Lys (gene activityd cessation); no. of enzyme inducers prior to surgery genotype deletion) pack-yearsb 6IM (40,F) Current; 25 Piroxicam (Feldene), amitriptyline Adenocarcinoma CYP1A1*1/1 Arg/Arg Null ND hydrochloride (fibromyagia) 7IM (60,M) Current; (long ASA, blood pressure pills Squamous cell CYP1A1*1/2A Arg/Arg Null 2.56 time) carcinoma 8IM (65,M) Current; 50 ASA/caffeine/codeine phosphate Adenocarcinoma CYP1A1*1/1 Arg/Arg Positive ND (282s), metoprolol (Betaloc), (poorly salbutamol (Ventolin), ipratropium differentiated)/ bromide (Atrovent), TUMS asthma 9IM (71,M) Former (2 yr); 60 Glyceryl trinitrate, nifedipine Squamous cell CYP1A1*1/1 Arg/Lys Null ND (Adolat), enalapril maleate carcinoma (Vasotec), ASA (Aspirin), (moderate pentoxifylline (Trental) differentiation) 1JM (55,F) Current; N/A Terbutaline sulfate (Bricanyl), Adenocarcinoma CYP1A1*1/1 Arg/Arg Null 2.79 salbutamol (Ventolin), ipratropium bromide (Atrovent), steroids, glyburide, metformin, cisapride monohydrate (Prepulsid), iansoprazole (Prevacid), prednisone, fluoxitine hydrochloride (Prozac), budesonide (Pulmicort), trazodone hydrochloride 2JM (60,F) Current; 40 Conjugated estrongens (Premorin), Adenocarcinoma CYP1A1*1/1 Arg/Arg Null 18.94 oxybutinin chloride (Ditropan), medroxyprogestarone acetate (Provera), acetaminophen (Tylenol) 3JM (46,M) Current; 49.5 Diazepam (Valium), fluvoxamine Adenocarcinoma CYP1A1*1/1 Arg/Arg Null 17.56 maleate (Luvox), warfarin sodium (Coumadin) 4JM (55,M) Current; 50 ASA//3 glasses wine/day Non-small cell CYP1A1*1/2B Arg/Arg Positive 45.98 carcinoma/ emphysema 5JM (69,M) Former (1 yr); 48 Diazepam, ranitidine Squamous cell CYP1A1*1/1 Arg/Arg Null ND carcinoma 6JM (46,M) Current; 31 Bupropion hydrochloride (Zyban), Squamous cell CYP1A1*1/1,f *1/4 Arg/Arg Null 39.57 codeine syrup carcinoma 7JM (57,F) Former (3 mo); 40 None Adenocarcinoma CYP1A1*1/1 Arg/Lys Null 3.07 8JM (42,F) Current; 24 None Adenocarcinoma CYP1A1*1/1 Arg/Arg Positive 31.77 (poorly differentiated) 9JM (58,M) Current; 69 Chlodiazepoxide hydrochloride Adenocarcinoma CYP1A1*1/2A Arg/Arg Positive 7.52 (Librium), carbamazapine, (poorly lamitrogine (Lamictal), ASA differentiated) (Novasen) 1KM (75,M) Nonsmoker (life Digoxin (Lanoxin), warfarin sodium Adenocarcinoma CYP1A1*1/1 Arg/Arg Null ND time) (Coumadin), ranitidine, lorazepam, (previous acetaminophen/oxycodone abdominal hydrochloride (Oxycocet) malignancy) a Patients are assigned codes for confidentiality. b Pack-years (no. of packs smoked per day ϫ no. of years; 1 pack-year ϭ smoking 1 pack per day for 1 year). c Generic drug name (proprietary name). d EROD activity in pmol/min/mg protein. e N/A, not available; ND, not detectable; TB, tuberculosis; ASA, acetylsalicylic acid. f Patients 5CM and 6JM CYP1A1*1/4 heterozygous mutants, all other patients genotyped CYP1A1*1/1 at this locus.

Atypical CYP1A1 Phenotypes, Corresponding Genotypes, 9HM, Table 3) which has been shown to inhibit induction of and Demographics. EROD activities were not detectable in AHH activity in lung tissue from mice treated with the PAH microsomes from 12 of 46 current smokers (Table 3). Three of benzanthracene (101). the 12 patients were CYP1A1*1/2A and the remaining were Furthermore, EROD activities were detected in micro- CYP1A1*1/1. One of the twelve patients was AHR554Arg/Lys, somes from 8 of 24 non-/former smokers (ranging from 0.19 to and 4 of the 12 patients carried at least one copy of GSTM1. All 6.62 pmol/min/mg), with the highest activities in microsomes of the 12 patients were current long-term smokers with at least from the three patients who reported cessation of smoking 20 pack-years of exposure. A number of these individuals also within the previous six months. All three patients were were receiving pharmacotherapy (Table 3), although most of CYP1A1*1/1, with only one individual AHR554Arg/Lys. Micro- the drugs apparently do not interact with CYP1A1. A possible somal EROD activities from the remaining five patients exception is beclomethasone dipropionate (patients 7DM and (CYP1A1*1/1, n ϭ 2; CYP1A1*1/2A, n ϭ 1; CYP1A1*1/2B,

Fig. 1. Correlation analysis of lung microsomal EROD activities and (a) pack- year consumption [only from current smokers with complete smoking histories (n ϭ 36)]; (b) age [same patients as in (a)].

n ϭ 2; and AHR554Arg/Arg, n ϭ 5) were very low with the exception of one patient coded 5 IM, who had EROD activities of 1.77 pmol/min/mg. Among other pharmacotherapies, this individual was being treated for gastric reflux disease with omeprazole, a know inducer of CYP1A1 (Table 3; Refs. 102, 103). Lung disease may have contributed to EROD activities. Pulmonary microsomes from patient 1DM, a longtime current smoker, exhibited the highest EROD activities in our patient population. This individual, genotyped CYP1A1*1/1, AHR554Arg/Arg, and GSTM1-null, showed a positive tuberculosis skin test at the time of surgery, and presented with an active pulmonary Aspergillus fumigatus infection. Initially, lo- Fig. 2. Lung microsomal EROD activities (Ϯ SD) in current smokers with (a) bectomy was indicated for a cavitary lesion believed to be different CYP1A1 genotypes; (b) different AHR554 genotypes; and (c) different caused by tuberculosis. However, postoperative pathology cul- GSTM1 genotypes. tures revealed no sign of Mycobacterium, and did reveal an Aspirigillus fumigatus infection, resulting in a diagnosis of aspirigillosis granulomatosis combined with acute pneumonia. Numerous epidemiological studies have suggested increased risk for lung cancer in individuals carrying even one Discussion copy the CYP1A1*2A variant allele in North Americans (pre- The association between human CYP1A1 genetic polymor- dominantly Caucasian; Refs. 37, 39, 42), in Scandinavians (27), phisms and lung cancer remains controversial despite a rela- and in other ethnic populations (40, 42). Ishibi et al. (40) also tively large number of epidemiological studies in various pop- demonstrated an association of heterozygosity for the ulations (Table 1). Similarly, the existence of a human CYP1A1*2B variant with a 2-fold increase in lung cancer risk polymorphism in AHR affecting induction of CYP1A1 remains in Mexican- and African-Americans, and Dresler et al. (49) unclear. The majority of studies have failed to address whether found an increased risk for lung cancer in a group of females, such polymorphisms actually translate into significant func- the vast majority of which were heterozygous for CYP1A1*2C; tional alterations in CYP1A1 enzyme activity/induction in the the increased risk was particularly apparent when combined human lung. The principle aim of the present study was to with GSTM1-null. In contrast, a recent meta-analysis by Houl- determine whether a correlation exists between genotype and ston (105) found little evidence to support an association of biotransformation phenotype in human lung microsomes. We CYP1A1*2A or *2C variants with lung cancer risk. Several have previously demonstrated observable functional differ- studies examining the functionality of the CYP1A1 genetic ences in polymorphic forms of GST enzymes in human lung polymorphisms in cultured lymphocytes have suggested differ- cytosols using selective substrates (86, 104). ences in inducibility/activity measured by AHH or EROD to be

Table 4 CYP1A1, AHR, and GSTM1 genotypes and lung microsomal EROD results of Wong et al. (85), we did not see an association activities from current smokersa between AHR genotype and CYP1A1 activities. Our GSTM1 results differ from those of an earlier report of CYP1A1 genotype enhanced CYP1A1 inducibility with the GSTM1 null genotype CYP1A1*1/1 CYP1A1*1/2A CYP1A1*1/2B in human lymphoblastoid B cells (87), but agree with subse- Total 12.23 Ϯ 13.48 (33)b 8.23 Ϯ 9.76 (10) 25.01 Ϯ 19.79 (3) quent studies that did not find a role for GSTM1 genotype in CYP1A1 inducibility (64, 88). Thus, association of the GSTM1 554 Ϯ Ϯ Ϯ AHR Arg/Arg 11.95 13.49 (29) 8.23 9.76 (10) 34.20 16.67 (2) gene deletion polymorphism with lung cancer susceptibility GSTM1- 8.03 Ϯ 10.17 (10) 9.63 Ϯ 9.49 (4) 34.20 Ϯ 16.67 (2) positive (reviewed in Ref. 2) more likely relates to the demonstrated GSTM1-null 14.01 Ϯ 14.77 (19) 7.29 Ϯ 10.71 (6) decreased conjugation of electrophilic substrates (86, 106). AHR554Arg/Lys 13.35 Ϯ 16.06 (4) 6.65 (1) Combinations of the CYP1A1 and AHR genetic and GSTM1 GSTM1- 8.44 Ϯ 3.11 (2) polymorphisms also do not appear to affect CYP1A1 related positive activities in peripheral human lung. Ϯ GSTM1-null 18.27 25.83 (2) 6.65 (1) In the present study, human lung microsomes from 70 a Mean EROD activities (pmol/min/mg protein) Ϯ SD (number of individuals). surgical specimens obtained from a predominantly Caucasian b Patient 6JM (only current smoker with CYP1A1*1/4 genotype) is included North American population produced EROD activities similar within this group. to those reported by Wheeler et al. (6). Consistent with the concept of smoking-mediated CYP1A1 induction in human lung (5Ð10), EROD activities were detectable in microsomes associated with heterozygotes possessing either a CYP1A1*2A from patients who were recorded as current smokers, but were (36, 53, 54, 59) or a CYP1A1*2C allele (56, 58). However, low or undetectable in most of those recorded as former smok- Cosma et al. (55) found no correlation between CYP1A1*2A ers/nonsmokers (Table 3). The detectable levels of CYP1A1 variants and gene inducibility measured by mRNA quantitation, activity in microsomes from a small number of patients who and Wedlund et al. (60) found no association between were classified as former smokers could conceivably be attrib- CYP1A1*2A or *2B variants and AHH inducibility in cultured uted to inaccuracies in individual patient smoking status self- lymphocytes in the same Mediterranean family examined by reporting and/or to induction by therapeutic agents. In the case Petersen et al. (Ref. 53; reviewed in Table 1). Contrary to of the former, results demonstrating CYP1A1 induction in results of Rojas et al. (69) in a Russian population, Butkiewicz current smokers compared with non-/former smokers suggested et al. (71) found significantly higher PAH DNA-adduct levels accurate self-reporting on the whole. For the latter, we did in lung tissues from Polish individuals genotyped observe CYP1A1 activity in microsomes from a longtime for- CYP1A1*1/2C combined with GSTM1-null genotypes, which mer smoker (12 years) treated with omeprazole, a compound suggested that heterozygous variant forms of CYP1A1 may known to induce CYP1A1 in cell lines and human tissues (102, contribute to high levels of carcinogen activation and DNA- 103). Also of potential significance was our observation of very adduction. Interestingly, Saarikoski et al. (74) noted that lung high CYP1A1 activity in lung microsomes from a reported tissue from a CYP1A1*1/2A individual demonstrated greater current smoker with asperigillosis. This points to the potential expression of CYP1A1 by immunohistochemical and in situ role of inflammatory mediators in altering pulmonary CYP hybridization analysis, compared with tissues from five activities. For example, Ohnhaus and Bluhm (107) found high CYP1A1*1/1 individuals. activities for P450 enzyme non-selective 7-ethoxycoumarin-O- Our examination of the effects of the CYP1A1 genetic deethylase activities in pulmonary microsomes from tubercu- polymorphisms on related enzyme activities in human lung losis-positive patients. At present, the contribution that elevated suggested that CYP1A1 heterozygous genotypes (CYP1A1*1/ CYP1A1 activity and, hence, altered carcinogen metabolism 2A, CYP1A1*1/2B, and CYP1A1*1/4) do not affect CYP1A1 might make to the observed link between lung disease and lung activities in peripheral human lung. The lack of observed con- cancer (108), is not known. tributions of genotype on EROD activities could have been Consistent with other studies (10, 109, 109Ð112), we affected by interindividual variability in microsomal activities found marked interindividual variability in pulmonary or by the absence of homozygous variant microsomal samples CYP1A1 activities in microsomes from current smokers. Inter- and, in the case of the CYP1A1*4 allele, could be limited by the estingly, peripheral lung microsomes from some (ϳ26%) cur- fact that only one current smoker carried one copy of this allele. rent smokers exhibited nondetectable EROD activities, and this It is important to note that associations between CYP1A1 vari- was compatible with reports distinguishing a subset of individ- ants and lung cancer require high n values because of the uals with no detectable CYP1A1 induction by immunochem- multifactorial processes involved in the development of the end istry or by measuring AHH or EROD (7, 74, 113). However, point. However, CYP1A1 enzyme activity is much more prox- consistent with the results of Anttilla et al. (114), a variant imate to CYP1A1 genotype, so relevant differences in pheno- CYP1A1 or AHR554 genotype did not account for the lack of type, if present, should be revealed more readily. detectable CYP1A1 activity as this group comprised individu- Observed allele frequencies for the CYP1A1*1,*2A, als CYP1A1*1/1, CYP1A1*1/2A, AHR554Arg/Arg, and AHR554 and*2B alleles from our patients were similar to those reported Arg/Lys. in the literature for healthy Caucasians (52) and were lower for In contrast to Mollerup et al. (115), who reported that the CYP1A1*4 allele (81) and, therefore, do not suggest an females had significantly higher levels of pulmonary mRNA association with lung cancer risk. Allele frequencies for CYP1A1 expression, we found no significant differences in AHR554Arg and Lys were not higher than expected and were EROD activities between lung microsomes from males and similar to those reported in other mixed North American pop- females. Our results suggest that the association of CYP1A1*2C ulations (85). and increased lung cancer risk in females (49) may not be Mutation of AHR might affect ligand binding and hence attributable to higher CYP1A1 activities. The lack of signifi- CYP1A1 inducibility. However, on the basis of the limited cant correlation between EROD activity and pack-year con- number of Lys554 heterozygotes analyzed and consistent with sumption in current smokers, but a significant decline in EROD

activity associated with aging, suggests that, as the body ages, for altered gene regulation in primary pulmonary carcinomas. J. Natl. Cancer the inducibility/activity of CYP1A1 decreases (Fig. 1, a and b). Inst., 82: 1333Ð1339, 1990. The lack of correlation of EROD and pack-year consumption is 6. Wheeler, C. W., Park, S. S., and Guenthner, T. M. Immunochemical analysis of a cytochrome P-450IA1 homologue in human lung microsomes. Mol. Phar- probably affected by the age-related decline in EROD activi- macol., 38: 634Ð643, 1990. ties, because pack-year values were generally higher in older 7. Anttila, S., Hietanen, E., Vainio, H., Camus, A. M., Gelboin, H. V., Park, S. S., patients. Similar findings demonstrating a negative correlation Heikkilae, L., Karjalainen, A., and Bartsch, H. Smoking and peripheral type of between age and CYP activity in human liver have been re- cancer are related to high levels of pulmonary cytochrome P450IA in lung cancer ported previously (116Ð118). On the basis of patient self- patients. Int. J. Cancer, 47: 681Ð685, 1991. reporting, none of our patients recorded as current smokers 8. Shimada, T., Yun, C. H., Yamazaki, H., Gautier, J. C., Beaune, P. H., and Guengerich, F. P. Characterization of human lung microsomal cytochrome P-450 ceased smoking within the time interval between the day of 1A1 and its role in the oxidation of chemical carcinogens. Mol. Pharmacol., 41: surgery and the two-month-prior-to-surgery cutoff designated 856Ð864, 1992. for classification as a “former smoker.” Thus all CYP1A1 9. Anttila, S., Vainio, H., Hietanen, E., Camus, A. M., Malaveille, C., Brun, G., activities from current smokers are believed to be representative Husgafvel-Pursiainen, K., Heikkila, L., Karjalainen, A., and Bartsch, H. Immu- of tobacco smoke induction up to the day prior to surgery. nohistochemical detection of pulmonary cytochrome P450IA and metabolic activities associated with P450IA1 and P450IA2 isozymes in lung cancer patients. 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