Analysis of resveratrol as a lung cancer chemopreventive agent in A/J mice exposed to benzo[a]pyrene

Resveratrol inhibits PAH bioactivation through reduced expression of the CYP1A1 and CYP1B1 genes in human bronchial epithelial cells. Ad libitum access to a diet containing resveratrol showed no effect on benzo[a]pyrene-induced lung tumorigenesis in A/J mice. Also, resveratrol did not change CYP1A1 and CYP1B1 gene expression or benzo[a]pyrene protein adduct levels in the lung tissue. The lack of chemopreventive activity may have been caused by insufficient concentrations or nonreactive forms of resveratrol in the lungs.     

Inhibition of benzo[a]pyrene-activating enzymes and DNA binding in human bronchial epithelial BEAS-2B cells by methoxylated flavonoids

Cigarette smoking is a major risk factor in lung carcinogenesis via carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and nitrosamines. In this study, we used benzo[a]pyrene (BaP) as the classic PAH compound and BEAS-2B cells, a model of normal human bronchial epithelial cells, to investigate whether 5,7-dimethoxyflavone (5,7-DMF) and 3',4'-DMF compared with resveratrol (RV) have chemopreventive properties in this cancer. Exposure of BEAS-2B cells to [(3)H]BaP (1 microM) showed increasing binding to DNA up to 72 h of exposure, about 20-fold higher than that at 0.5 h exposure. BaP exposure also increased both CYP1A1/1B1 and microsomal epoxide hydrolase (mEH) enzyme activities with a maximum 10-fold increase at 48 h. BaP induced CYP1A1 protein and mRNA levels maximally after 48 h. In contrast, although CYP1B1 mRNA was rapidly induced, its protein expression showed a very poor response. Simultaneous treatment with BaP and 5,7-DMF, 3',4'-DMF or RV for 48 h inhibited BaP-DNA binding by > or =75%, with 3',4'-DMF being the most effective. 5,7-DMF affected CYP1A1 mRNA levels only modestly, whereas 3',4'-DMF was a potent inhibitor. The catalytic activity of CYP1A1/1B1 was reduced over 95% after exposure to 5,7-DMF, 3',4'-DMF or RV, most effectively by 3',4'-DMF. BaP-induced mEH activity was not affected by treatment with 5,7-DMF, but was significantly inhibited by 3',4'-DMF. In contrast, mEH activity was notably increased by RV. Most importantly, western blotting showed all three polyphenols dramatically reducing BaP-induced CYP1A1 protein expression. Both 5,7-DMF and 3',4'-DMF demonstrated very high, about 40-fold, accumulation in BEAS-2B cells. In summary, BaP exposure results in a high level of DNA binding in BEAS-2B cells, which is mainly mediated by induction of CYP1A1 protein, just as in the human lung. Two methoxylated dietary flavonoids with highly specific effects on BaP bioactivation block this DNA binding and CYP1A1 protein expression as effectively as RV, thus making them potential chemopreventive agents for BaP-induced lung carcinogenesis.     

Role of estrogen receptor in regulation of polycyclic aromatic hydrocarbon metabolic activation in lung

Epidemiological and biochemical studies have indicated that females may be at greater risk of smoking associated lung cancer compared with males. Among lung cancer patients, female smokers have been found to have higher levels of PAH-related DNA adducts and CYP1A1 gene expression in their normal lung tissue compared to male smokers. A possible role of steroid hormones in these sex differences via interactions between aryl hydrocarbon receptor and estrogen receptor mediated cellular effects has been suggested. In the present study the impact of the estrogen receptor (ERalpha) on CYP1A1 and CYP1B1 gene expression was studied in vitro in human bronchial epithelial cells. Transient transfection of the BEP2D cell line with ERalpha influenced neither constitutive expression of CYP1A1 or CYP1B1 nor induction of these genes by TCDD as measured by real-time RT-PCR. ERalpha had no effect on the constitutive or TCDD-induced enzymatic activity of CYP1A1 (EROD). We also studied the effect of steroid hormones on lung PAH metabolic activation in A/J mice. Intact and ovariectomized female mice were orally exposed to a single dose of benzo[a]pyrene. Ovariectomy did not influence the levels of either benzo[a]pyrene-derived protein or DNA adducts in the lung tissue measured by HPLC and 32P-postlabeling, respectively. In conclusion, the present data do not support the hypothesis of a role of estrogen or the ERalpha in regulating the metabolic activation of polycyclic aromatic hydrocarbons in lung.     

Inhibitory effect of Phenethyl Isothiocyanate Against Benzo[a] Pyrene-Induced Rise in CYP1A1 mRNA and Apoprotein Levels as its Chemopreventive Properties

Background: Phenethyl isothiocyanate (PEITC), the most comprehensively studied aromatic isothiocyanate,has been shown to act as an anti-cancer agent mainly through modulation of biotransformation enzymesresponsible for metabolizing carcinogens in the human body. Humans are often exposed to carcinogenic factors,some of which through the diet, such as polycyclic aromatic hydrocarbon benzo[a]pyrene via the consumptionof over-cooked meats. Inhibition of the enzymes responsible for the bioactivation of this carcinogen, for exampleCYP1A1, the major enzyme required for polycyclic aromatic hydrocarbons (PAHs) bioactivation, is recognizedas a chemoprevention strategy. Objective: To evaluate the inhibitory effects of PEITC against benzo[a]pyreneinducedrise in rat liver CYP1A1 mRNA and apoprotein levels. Materials and Methods: Precision cut rat liverslices were treated with benzo[a]pyrene at 1 and 5 μM in the presence of PEITC (1-25 μM) for 24 hours, followedby determination of CYP1A1 mRNA and apoprotein levels using quantitative polymerase chain reaction andimmunoblotting. Results: Findings revealed that PEITC inhibited benzo[a]pyrene-induced rise in rat liverCYP1A1 mRNA in a dose-dependent manner as well as the apoprotein levels of CYP1A. Conclusions: It wasdemonstrated that PEITC can directly inhibit the bioactivation of benzo[a]pyrene, indicating chemopreventivepotential.     

Characterization of common CYP1B1 variants with different capacity for benzo[a]pyrene-7,8-dihydrodiol epoxide formation from benzo[a]pyrene

Cytochrome P450 1B1 (CYP1B1), an extrahepatic enzyme inducible by smoking, is overexpressed in many tumors and catalyzes the metabolic activation of procarcinogens such as polycyclic aromatic hydrocarbons. In human, CYP1B1 is genetically polymorphic and five common missense mutations causing amino acid substitution have been identified. In this study, we have investigated CYP1B1 haplotypes present in a Spanish population and carried out functional analyses of the corresponding enzymes in yeast using benzo[a]pyrene as a substrate. CYP1B1*1, CYP1B1*2, CYP1B1*3, CYP1B1*4, CYP1B1*6, and CYP1B1*7, encoding combinations of the Arg48Gly, Ala119Ser, Leu432Val, Asn453Ser, and Ala443Gly amino acid substitutions, were present at frequencies of 14.3%, 25.5%, 38.8%, 18.1%, 0.4%, and 2.6%, respectively. The variant CYP1B1 forms were heterologously expressed with human reductase in Saccharomyces cerevisiae and kinetic analyses of benzo[a]pyrene metabolism were carried out. CYP1B1.7, having the amino acid substitutions Arg48Gly, Ala119Ser, Leu432Val, and Ala443Gly, exhibited a significantly decreased capacity (P < 0.001) for the formation of (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol from benzo[a]pyrene as indicated by lower intrinsic clearance (Vmax/Km). A somewhat decreased clearance was observed for CYP1B1.4, whereas no significant differences in kinetic properties among the remaining variant enzymes were observed as compared with CYP1B1.1. Thus, genetic polymorphism in the CYP1B1 gene, as defined by the haplotypes investigated, might cause interindividual differences in susceptibility (e.g., to lung cancer induced by smoking). The results indicate the necessity to make molecular epidemiologic investigations regarding the association of the specific CYP1B1 haplotypes and cancer risk.     

Benzo(a)pyrene diolepoxide (BPDE)-DNA adduct levels in leukocytes of smokers in relation to polymorphism of CYP1A1, GSTM1, GSTP1, GSTT1, and mEH.

Benzo(a)pyrene [B(a)P] diolepoxide (BPDE)-DNA adducts were measured in the leukocytes of 41 healthy smokers using high-performance liquid chromatography coupled with a fluorimetric detector. The correlation between exposure to B(a)P through smoking and BPDE-DNA adduct levels was poor (r = 0.31), although subjects in the high exposure group [B(a)P > 50 ng/d] had a slightly higher level of adducts compared with the less exposed group (mean +/- SE, 1.70 +/- 0.3 versus 1.09 +/- 0.1; P = 0.057). We studied the effect on BPDE-DNA adducts of individual variations in genes controlling B(a)P metabolism, classifying subjects in "low-risk" and "high-risk" genotypes for smoking-related B(a)P DNA damage. The high-risk group included subjects characterized by a combination of increased B(a)P activation [cytochrome P450 1A1 (CYP1A1) MspI and/or exon 7 Ile462Val allele variants and microsomal epoxide hydrolase (mEH) fast activity] and decreased deactivation ability [presence of glutathione S-transferase M1 (GSTM1) null allele and wild-type glutathione S-transferase P1 (GSTP1)]. The low-risk group included smokers with lower B(a)P activation (wild-type CYP1A1, low or intermediate mEH activity) and higher deactivation capacity (active GSTM1, GSTP1 Ile105Val allele). Subjects in the low-risk group had lower levels of BPDE-DNA adducts compared with subjects in the high-risk genotype group; this difference was significant using two markers (CYP1A1 and GSTM1, median +/- SD, 0.77 +/- 1.16 versus 1.89 +/- 0.39; P = 0.03) or three markers (CYP1A1, GSTM1, and GSTP1, median +/- SD, 0.66 +/- 0.93 versus 1.43 +/- 1.17; P = 0.013). The discrimination between groups was reduced when including mEH as an additional marker (P = 0.085). In conclusion, CYP1A1, GSTM1, and GSTP1 genotyping seems to be a risk predictor of BPDE-DNA adduct formation in leukocytes.     

Associations between smoking, polymorphisms in polycyclic aromatic hydrocarbon (PAH) metabolism and conjugation genes and PAH-DNA adducts in prostate tumors differ by race.

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts may induce mutations that contribute to carcinogenesis. We evaluated potential associations between smoking and polymorphisms in PAH metabolism [CYP1A1 Ile 462Val, CYP1B1 Ala 119Ser and Leu 432Val, microsomal epoxide hydrolase (mEH) Tyr 113His and His139Arg, CYP3A4 A(-392)G] and conjugation [glutathione S-transferase (GST) M1 null deletion, GSTP1 Ile 105Val] genes and PAH-DNA adduct levels (measured by immunohistochemistry) in tumor and nontumor prostate cells in 400 prostate cancer cases. Although no statistically significant associations were observed in the total sample, stratification by ethnicity revealed that Caucasian ever smokers compared with nonsmokers had higher adduct levels in tumor cells (mean staining intensity in absorbance units +/- SE, 0.1748 +/- 0.0052 versus 0.1507 +/- 0.0070; P = 0.006), and Caucasians carrying two mEH 139Arg compared with two 139His alleles had lower adducts in tumor (0.1320 +/- 0.0129 versus 0.1714 +/- 0.0059; P = 0.006) and nontumor (0.1856 +/- 0.0184 versus 0.2291 +/- 0.0085; P = 0.03) cells. African Americans with two CYP1B1 432Val compared with two 432Ile alleles had lower adducts in tumor cells (0.1600 +/- 0.0060 versus 0.1970 +/- 0.0153; P = 0.03). After adjusting for smoking status, carrying the putative "high-risk" genotype combination, the faster metabolism of PAH-epoxides to PAH-diol-epoxides (CYP1B1 432Val/Val and mEH 139Arg/Arg) with lower PAH-diol-epoxide conjugation (GSTP1 (105)Ile/Ile), was associated with increased adducts only in Caucasian nontumor cells (0.2363 +/- 0.0132 versus 0.1920 +/- 0.0157; P= 0.05). We present evidence, for the first time in human prostate that the association between smoking and PAH-DNA adducts differs by race and is modified by common genetic variants.     

Functional evaluation of novel single nucleotide polymorphisms and haplotypes in the promoter regions of CYP1B1 and CYP1A1 genes

Interindividual variation in the expression of the carcinogen- and estrogen-metabolizing enzymes cytochrome P4501B1 and 1A1 (CYP1B1 and CYP1A1) has been detected in human lung. To search for polymorphisms with functional consequences for CYP1B1 and CYP1A1 gene expression, we examined 1.5 kb of the promoter region of each gene. Genomic DNA from 21 Caucasian individuals was amplified by polymerase chain reaction (PCR) for direct cycle sequencing. Eight single nucleotide polymorphisms (SNPs) for CYP1B1 and 13 SNPs for CYP1A1 were found. The majority of polymorphisms occurred as multiSNP combinations for individual subjects. The wild-type sequences were cloned into a luciferase reporter construct. The most frequent polymorphisms were then recreated by iterative site-directed mutagenesis, replicating single polymorphisms and multiSNP combinations. These wild-type and variant constructs were functionally evaluated in transient transfection experiments employing exposures to either the index polycyclic aromatic hydrocarbon (PAH) inducer benzo[a]pyrene (B[a]P), a composite mixture of cigarette smoke extract (CSE), or the repressor chemopreventive agent trans-3,4,5-trihydroxystilbene (reseveratrol). Results indicated that all wild-type and variant constructs responded in qualitatively concordant fashion to the inducers and to the repressor. The CYP1B1 haplotypes and the majority of CYP1A1 haplotypes were shown to have no functional consequence, as compared to those of the wild-type promoter sequences. Two constructs of composite polymorphisms of CYP1A1 appeared to result in a statistically significant increase in basal promoter activity (1.38- and 1.50-fold, respectively), but the degree of functional impact was judged unlikely to be biologically important in vivo. We conclude that the observed promoter region polymorphisms in these genes are common, but are of unclear functional consequence.     

Tumorigenic activity of non-alternant polynuclear aromatic hydrocarbons in newborn mice

The tumorigenic activity of benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene, and indeno-[1,2,3-cd]pyrene was evaluated in newborn CD-1 mice. The total doses of these non-alternant polycyclic aromatic hydrocarbons employed in this study ranged from 0.5 to 2.1 mumol per mouse. The results of this assay indicate that both benzo[b]fluoranthene and benzo[j]fluoranthene exhibit significant tumorigenic activity. In contrast to these results, neither benzo[k]fluoranthene nor indeno[1,2,3-cd]pyrene were tumorigenic under these assay conditions.     

Glutathione transferase pi plays a critical role in the development of lung carcinogenesis following exposure to tobacco-related carcinogens and urethane

Human cancer is controlled by a complex interaction between genetic and environmental factors. Such environmental factors are well defined for smoking-induced lung cancer; however, the roles of specific genes have still to be elucidated. Glutathione transferase pi (GSTP) catalyzes the detoxification of electrophilic diol epoxides produced by the metabolism of polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP), a common constituent of tobacco smoke. Activity-altering polymorphisms in Gstp have therefore been speculated to be potential risk modifiers in lung cancer development. To clearly establish a role for GSTP in lung tumorigenesis, we investigated whether deletion of the murine Gstp genes (Gstp1 and Gstp2) alters susceptibility to chemically induced lung tumors following exposure to BaP, 3-methylcholanthrene (3-MC), and urethane. Gstp-null mice were found to have substantially increased numbers of adenomas relative to wild-type mice following exposure to all three compounds (8.3-, 4.3-, and 8.7-fold increase for BaP, 3-MC, and urethane, respectively). In Gstp-null mice, the capacity of pulmonary cytosol to catalyze conjugation of the BaP diol epoxide was significantly reduced. Concomitant with this, a significant increase in the level of BaP DNA adducts was measured in the lungs of null animals; however, no increase in DNA adducts was measured in the case of 3-MC exposure, suggesting that an alternative protective pathway exists. Indeed, significant differences in pulmonary gene expression profiles were also noted between wild-type and null mice. This is the first report to establish a clear correlation between Gstp status and lung cancer in vivo.     

Arylhydrocarbon receptor-dependent induction of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in genetically engineered C57BL/6J mice

Arylhydrocarbon receptor knock-out, AhR(-/-), mice have recently been shown to be rather resistant to benzo[a]pyrene (B[a]P)-induced tumor formation, probably reflecting the inability of these mice to express significant levels of cytochrome P450 (P450 or CYP) 1A1 that activates B[a]P to reactive metabolites (Y. Shimizu, Y. Nakatsuru, M. Ichinose, Y. Takahashi, H. Kume, J. Mimura, Y. Fujii-Kuriyama and T. Ishikawa (2000) PROC: Natl Acad. Sci. USA, 97, 779-782). However, it is not precisely determined whether CYP1B1, another enzyme that is also active in activating B[a]P, plays a role in the B[a]P carcinogenesis in mice. To understand the basis of roles of CYP1A1 and CYP1B1 in the activation of chemical carcinogens, we compared levels of induction of liver and lung CYP1A1, 1A2, and 1B1 by various polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls in AhR(+/+) and AhR(-/-) mice. Liver and lung CYP1A1 and 1B1 mRNAs were highly induced in AhR(+/+) mice by a single intraperitoneal injection of each of the carcinogenic PAHs, such as B[a]P, 7,12-dimethylbenz[a]anthracene, dibenz[a,l]pyrene, 3-methylcholanthrene, 1,2,5,6-dibenzanthracene, benzo[b]fluoranthene, and benzo[a]anthracene and by a co-planar PCB congener 3,4,3',4'-tetrachlorobiphenyl. We also found that 6-aminochrysene, chrysene, benzo[e]pyrene, and 1-nitropyrene weakly induced the mRNA expression of CYP1A1 and 1B1, whereas anthracene, pyrene, and fluoranthene that have been reported to be non-carcinogenic in rodents, were very low or inactive in inducing these P450s. The extents of induction of liver CYP1A2 by these chemicals were less than those of CYP1A1 and 1B1 in AhR(+/-/+/-) mice. In AhR(-/-) mice, there was no induction of these P450s by PAHs and polychlorinated biphenyls. Liver microsomal activities of 7-ethoxyresorufin and 7-ethoxycoumarin O-deethylations and of mutagenic activation of (+/-)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P to DNA-damaging products were found to correlate with levels of CYP1A1 and 1B1 mRNAs in the liver. Our results suggest that carcinogenicity potencies of PAHs may relate to the potencies of these compounds to induce CYP1A1 and 1B1 through AhR-dependent manner and that these induced P450s participate in the activation of B[a]P and related carcinogens causing initiation of cancers in mice.     

Detoxification of optically active bay- and fjord-region polycyclic aromatic hydrocarbon dihydrodiol epoxides by human glutathione transferase P1-1 expressed in Chinese hamster V79 cells

Dihydrodiol epoxides (DEs) are important carcinogenic metabolites of polycyclic aromatic hydrocarbons (PAHs). The metabolic formation of four stereoisomeric DEs (a pair of optically active diastereomers termed as syn- and anti-form) is possible. Glutathione tranferases (GSTs) have been demonstrated to catalyze the detoxification of DEs. Purified GSTs display remarkable differences in catalytic efficiencies towards bay- and fjord-region DEs along with a high degree of regio- and stereoselectivity. Here we determined to which extent heterologously expressed human GSTP1-1, a major GST isoform in lung, affects the mutagenicity of stereoisomeric bay-region DEs of benzo[a]pyrene in Chinese hamster V79 cells. To evaluate the influence of sterical crowding in the substrate on the activity of GSTP-1, the study was extended to the strongly mutagenic fjord-region (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene. GSTP1-1,reduced preferentially the mutagenicity (studied at the hprt locus) of (+)-anti and (+)-syn-DEs of benzo[a]pyrene (by 66 and 67%) as compared with the corresponding (-)-anti- and (-)-syn-enantiomers (by 15 and 13%). These results are in line with previous studies on the enantioselectivity of purified GSTP1-1 towards the DE isomers of benzo[a]pyrene and benzo[c]phenanthrene showing that enantiomers with (R)-configuration at the benzylic oxiranyl carbon are better substrates than those with (S)- configuration. Interestingly, the (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene were efficiently detoxified by GSTP-1-1 in the constructed cell line (reduction of mutagenicity by 66 and 64%). This study demonstrates that differences in the caalytic activity seen for purified GST towards individual mutagens do not necessarily reflect the detoxification of DEs by the same enzyme in a living cell and provides further evidence that specific human GSTs play a role in the detoxification of DEs of PAHs.      

Patterns of benzo[alpha]pyrene metabolism in normal human pulmonary alveolar macrophages

Pulmonary alveolar macrophages (PAMs) obtained by bronchopulmonary lavage from 6 normal non-smoking volunteers were incubated with [3H]-benzo[alpha]pyrene to ascertain the normal metabolism and conjugation of polycyclic aromatic hydrocarbons. Through the use of a crude glucuronidase preparation, both glucuronic acid and sulfate conjugates were examined. Phenols and quinones were identified by high-pressure liquid chromatography as the principal free metabolites formed during 1 h incubation with benzanthracene induced PAMs. In addition, phenols and quinones were major substrates utilized by these cells for conjugation during the incubation period. The ranges of benzo[alpha]pyrene metabolites produced by PAMs from non-smokers were compiled and the variation in production as well as detoxification of proximate carcinogenic benzo[alpha]pyrene metabolites are presented.     

Epoxide hydratase and glutathione S-transferase activities in human lungs

It has been reported human lung has cytochrome P-450 dependent mono-oxygenase activity which is necessary for the formation of mutagneic and carcinogenic metabolities of polycyclic aromatic hydrocarbons, such as benzo[a]pyrene. We now report epoxide hydratase and glutathione S-transferase, enzymes important in the further metabolism of certain benzo[a]-pyrene metabolites formed by the monooxygenase system, have been detected in human lung tissues from patients undergoing surgery for lung cancer.     

A pilot study of detection of DNA adducts in white blood cells of roofers by 32P-postlabelling

To assess the utility of DNA adducts as biomarkers of exposure to carcinogens in an industrial population, a pilot study of roofers occupationally exposed to a mixture of polycyclic aromatic hydrocarbons was conducted. DNA was isolated from peripheral white blood cells of roofers and non-occupationally exposed subjects matched for age, sex and smoking status. Occupational exposures to anthracene, fluoranthene, pyrene, benzanthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[g,h,i]perylene and benzo[k]fluoranthene were assessed by personal breathing zone air sampling and skin wipes. Exposures to benzo[a]pyrene in air of exposed subjects ranged from 0.60 microgram/m3 to 1.39 micrograms/m3, and exposures to total polycyclic aromatic hydrocarbon (the sum of eight hydrocarbons) ranged from 6.0 micrograms/m3 to 13.8 micrograms/m3 on the day before blood collection. In the biomarker studies 10 of 12 roofers, but only 2 of 12 comparison subjects, had detectable levels of aromatic DNA adducts by 32P-postlabelling assay (p less than 0.01). The two non-roofers with detectable adducts had levels at or near the detection limit of 2 adducts per 10(9) nucleotides. In two roofer samples which were studied in a mixing experiment, the major adduct spots did not co-migrate with the guanosine N2 adduct of benzo[a]pyrene diol epoxide. These results suggest that the 32P-postlabelling assay may be useful for monitoring exposures to complex mixtures of aromatic hydrocarbons in industrial populations.