The role of 12 cDNA-expressed human,rodent, and rabbit cytochromes P450 in the metabolism of benzo[a]pyrene and benzo[a]pyrene trans-7, 8-dihydrodiol

The potent carcinogen benzo[a]pyrene (B[a]P) and its metabolite B[a]P trans-7, 8-dihydrodiol (7, 8-diol) require metabolic activation by the microsomal cytochrome P450s (P450s) to exert several adverse biological effects, including binding to DNA, toxicity, mutagenicity, and carcinogenicity. In the study reported here, we defined the role of each of 12 individual cDNA-expressed cytochrome P450s in the metabolism of B[a]P and 7, 8-diol. Human P450s 1A1 and 1A2 were expressed in the absence or presence of epoxide hydrolase (EH) in a human lymphoblastoid cell line, and six human and five rodent and rabbit P450s were expressed from cDNA with vaccinia virus vectors in the hepatoma cell line Hep G2. B[a]P metabolism resulted in nine metabolites (three diols, three quinones, and three phenols), which were separated, identified, and quantitated by high-pressure liquid chromatography. In the human lymphoblastoid cells, human 1A1 metabolized B[a]P at a rate 4.5 times greater than that for 1A2. EH was shown to be directly involved in B[a]P activation, since increasing the amount of EH resulted in less 7-hydroxybenzo[a]pyrene and more 7, 8-diol formation. Of the human P450s expressed with the vaccinia virus vectors in Hep G2 cells, 1A2 and 2C9 showed the highest activity and 2B6 showed moderate activity for B[a]P metabolism. Mouse 1A1 had activity 40 times higher than any human, rabbit, or rodent P450s, indicating the potential pitfalls of extrapolating P450 activity across species. Metabolism of the 7, 8-diol resulted in six metabolites (four tetrols and two triols). In the lymphoblastoid cells, human 1A1 was shown to be 4.2 times more active than 1A2 for 7, 8-diol metabolism. Among human P450s expressed from vaccinia virus, 1A2, 2E1, and 2C9 gave the highest activity, and 2C8 and 3A4 showed moderate activity for 7, 8-diol metabolism to the diol epoxides. Again, mouse 1A1 was much more active than any other P450. These studies, in which we determined the capacity of individual P450 in the metabolism and activation of B[a]P and 7, 8-diol, may thus lead to a better understanding of how P450s control the detoxification and activation of polycyclic aromatic hydrocarbons. © 1994 Wiley-Liss, Inc.     

Induction of CYP1A1 gene expression in H4-II-E rat hepatoma cells by benzo[e]pyrene.

In the rat, expression of the CYP1A1 gene is closely associated with arylhydrocarbon hydroxylase (AHH) enzyme activity. AHH is an inducile enzyme activity known to play an important role in the bioactivation of polycyclic aromatic hydrocarbons (PAHs) to mutagenic and carcinogenic metabolites. PAH-induced expression of the CYP1A1 gene appears to be regulated by several trans-acting factors, including the Ah receptor and the 4S PAH-binding protein. In this study, we used the PAH isomers benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP) to further evaluate the role of the 4S PAH-binding protein in induction of the CYP1A1 gene in H4-II-E rat hepatoma cells. Although BaP is believed to bind to both the Ah receptor and the 4S protein, BeP has been reported to bind exclusively to the 4S protein. The results of the study presented here indicate that BaP and BeP induce the expression of the CYP1A1 gene, as measured by ethoxyresorufin O-deethylase (EROD) activity, in a concentration-dependent manner. However, BaP is about 25 times as potent as BeP in inducing EROD activity in these cells. Slot-blot analysis of total RNA isolated from these cells indicated that BeP, BaP, and 3-methylcholanthrene increased the level of CYP1A1 mRNA expression. Sucrose-gradient analysis of BeP binding activity indicated that BeP bound with high affinity to the 4S PAH-binding protein, but not to the Ah receptor. These results suggest that the 4S protein may play a role in the PAH-induced expression of the CYP1A1 gene in rat H4-II-E cells.     

ERCC2/XPD基因缺失对苯并[a]芘所致DNA损伤修复的影响

目的： 探讨核苷酸切除修复交叉互补基因2 (excision repair cross complementation group 2/Xeroderma pigmentosum D,ERCC2/XPD)在苯并[a]芘所诱导的细胞DNA损伤与修复过程中的作用。方法：应用中国仓鼠卵巢细胞系CHO野生型AA8和ERCC2表达缺失型UV5作为细胞对照模型,MTT法比较两种细胞经苯并[a]芘处理后细胞抑制率的差别;彗星试验和Rad51免疫荧光试验检测不同浓度苯并[a]芘处理及修复24 h后细胞DNA损伤修复的情况。结果：与野生型AA8细胞相比,UV5细胞对苯并[a]芘所致损伤更加敏感,细胞存活率降低 (P<0.05)。彗星试验和Rad51免疫荧光试验结果显示,UV5细胞由于缺失ERCC2/XPD基因,修复苯并[a]芘所致DNA损伤能力降低 (P<0.05)。 结论：ERCC2/XPD蛋白在核苷酸切除修复中发挥解旋作用,对苯并[a]芘所致DNA损伤修复至关重要。     

Induction of epithelial-mesenchymal transition-related genes by benzo[a]pyrene in lung cancer cells

BACKGROUND: It is believed that epithelial-mesenchymal transition (EMT) occurs during the development and progression of cancer; however, the correlation between tobacco smoking and EMT remains to be elucidated. METHODS: Cells from the bronchioloalveolar carcinoma cell line A549 were exposed to benzo(a)pyrene (B[a]P) for 24 weeks, and morphology, proliferative activity, and gene expression profiles were analyzed. RESULTS: Although no apparent morphologic changes were observed, the B[a]P-exposed A549 cells exhibited enhanced proliferative activity in 1% bovine serum that contained medium, and dramatic changes in expression levels were observed in a large number of genes. Of those, the expression of EMT-related genes, such as migration-stimulating factor, plasminogen activator inhibitor-1, fibronectin, twist, transforming growth factor-beta2, basic fibroblast growth factor, and electron transport system, were up-regulated; whereas gene expression of E-cadherin was decreased. Most enhanced expression levels remained 8 weeks after the retrieval of B[a]P in culture. CONCLUSIONS: The current results indicated that B[a]P seems to induce EMT in lung cancer cells, and it also may drive disease progression in patients with lung cancer.     

Frameshift mutation in codon 176 of the p53 gene in rat esophageal epithelial cells transformed by benzo[a]pyrene dihydrodiol

Mutations in the p53 tumor suppressor gene have been associated with exposure to environmental chemical carcinogens. Cultured rat esophageal epithelial cells were transformed in vitro by treatment with benzo[a]pyrene dihydrodiol (BP-DHD). A BP-DHD-transformed cell line and control cell lines were analyzed for mutations in the p53 gene and in the Ha-ras gene by single-strand conformation polymorphism analysis of polymerase chain reaction-amplified products and direct DNA sequencing. The deletion of one cytosine in codons 174–176 (TGCCCCCAC → TGCCCCAC) of the p53 gene was found only in the BP-DHD-transformed cell line. The BP-DHD-transformed cells were highly invasive and tumorigenic when transplanted into syngeneic rats, whereas control lines either were nontumorigenic or formed epithelial cysts. BP-DHD-transformed cells and control lines were negative for mutations in the Ha-ras gene. Our results suggest that the tumorigenic potential of the BP-DHD-transformed cell line is associated with a frameshift mutation in codon 176 of the p53 gene but not with mutations in the Ha-ras gene. The G/C-rich codons 174–176 in the ratp53 gene may be specific targets for BP-DHD. ©1995 Wiley-Liss, Inc.     

Combined subcarcinogenic benzo[a]pyrene and UVA synergistically caused high tumor incidence and mutations in H-ras gene, but not p53, in SKH-1 hairless mouse skin

Combined subcarcinogenic doses of benzo[a]pyrene (BaP) and UVA induced H-ras, but not p53, gene mutations 8 weeks before tumor emergence in SKH-1 mice. Neither UVA (40 kJ/m2) nor BaP (8 nmol) induced any tumors after mice were topically treated 3 times/week for 25 weeks. However, combined BaP-UVA treatment synergistically increased tumor incidence and multiplicity. All tumors induced by BaP-UVA were malignant. The epidermis was collected from mice treated for 2, 6 and 10 weeks. DNA from UVB- (0.3 kJ/m2) or BaP-UVA-(8 nmol and 40 kJ/m2-induced tumors was isolated and screened for H-ras and p53 mutations. Four types of point mutation, GGC-->GAC, GCC, GTC and CGC, occurred in UVB-induced tumors at H-ras codon 13; and one type of point mutation, GGA-->GAA, at codon 12. Treatment with either BaP alone or BaP-UVA for 10 weeks caused GGA-->GAA mutation at codon 12 or GGC-->GAC mutation at codon 13 in nontumor skin, respectively, as well as in tumors induced by BaP-UVA. All of the 10-week samples treated with either BaP or BaP-UVA showed detectable mutations at codons 12 and 13, but the genetic load was significantly higher in BaP-UVA-treated mice than in those exposed only to BaP. UVA alone induced mutations at codon 12 in only one-third of samples. G-->A mutations induced by BaP or BaP-UVA at position 38 of codon 13 have not been reported previously. C-->T transitions were detected in p53 hot spots of exon 8 in 2 of 19 BaP-UVA-induced tumors but were not found in nontumor skin.     

In vitro benzo[a]pyrene diol epoxide-induced DNA damage and chromosomal aberrations in primary lymphocytes, smoking, and risk of squamous cell carcinoma of the head and neck

Cigarette smoking is a major risk factor for squamous cell carcinoma of the head and neck (SCCHN), but only a fraction of those exposed to cigarette smoke develops SCCHN, suggesting variation in individual susceptibility. Tobacco smoke contains a number of carcinogens that cause various kinds of damage to DNA. In this study, we simultaneously measured benzo[a]pyrene diol epoxide-induced DNA damage and chromosomal aberrations by the comet assay and the mutagen sensitivity assay, respectively, in cultured primary lymphocytes from newly recruited 123 patients with SCCHN and 136 age- and sex-matched controls. Using the control median as the cut-off, the elevated risk of SCCHN was 2.35 (95% CI, 1.37-4.03), 2.28 (95% CI, 1.34-3.98) and 3.25 (95% CI, 1.85-5.07) for high levels of tail extension, tail length and oliver tail moment of the comet assay, respectively, and 1.75 (95% CI, 1.04-2.94) for high levels of chromosomal aberrations of the mutagen sensitivity assay. The effects of these 2 types of measurements were additive; subjects with high levels of both DNA damage and chromosomal aberrations had a 4.77-fold increased risk (95% CI, 2.73-8.36) of SCCHN. Cigarette smoking further elevated this risk to more than 20-fold (OR 23.6; 95% CI, 8.92-62.3). These data support our previous finding that suboptimal repair contributed to susceptibility to SCCHN and the new data further suggests a possible gene-environment interaction that may play an important role in the etiology of SCCHN. Further validation studies are warranted.     

Exceptional activity of murine glutathione transferase A1-1 against (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-induced DNA damage in stably transfected cells

We have shown previously that the alpha class murine glutathione transferase (GST) isoenzyme mGSTA1-1, unlike other mammalian class alpha GSTs, is highly efficient in catalyzing the glutathione (GSH) conjugation of (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogenic metabolite of benzo[a]pyrene. The present studies were undertaken to determine the efficacy of mGSTA1-1 in cellular protection against (+)-anti-BPDE-induced DNA damage in HepG2 cells stably transfected with mGSTA1 cDNA. Untransfected HepG2 cells, vector-transfected HepG2 cells (HepG2-vector), and cells transfected with mGSTA4 cDNA (HepG2-mGSTA4), an alpha class murine GST isoenzyme with low (+)-anti-BPDE-GSH conjugating activity, were used as controls for comparison. Intracellular GSH conjugation of (+)-anti-BPDE was significantly higher in mGSTA1-1-overexpressing HepG2 cells (HepG2-mGSTA1) than in HepG2-vector or HepG2-mGSTA4 cells. The formation of DNA-adducts of (+)-anti-BPDE, following a 10-, 20-, or 30-min exposure to 0.1, 0.5, or 1.0 microM [3H](+)-anti-BPDE, was reduced significantly in cells transfected with mGSTA1-1 compared with HepG2-vector or untransfected HepG2 cells. Consistent with the results with purified protein, overexpression of mGSTA4-4 had no effect on (+)-anti-BPDE-induced DNA damage. The results of the present study indicated that mGSTA1-1 was exceptionally effective in affording protection against (+)-anti-BPDE-induced DNA damage in a cellular system.     

Role of glutathione conjugate efflux in cellular protection against benzo[a]pyrene-7,8-diol-9,10-epoxide-induced DNA damage

Glutathione (GSH) conjugation of (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE], the activated metabolite of benzo[a]pyrene, is believed to be an important mechanism in detoxification of this environmental and dietary carcinogen. Here, we demonstrate that the intracellular accumulation of GSH conjugate of (+)-anti-BPDE (BPD-SG) caused a statistically significant increase in (+)-anti-BPDE-induced DNA adduction. The relationship between intracellular accumulation of BPD-SG and (+)-anti-BPDE-induced DNA adduction was studied using a canine kidney epithelial cell line (MDCKII) and its variants overexpressing multidrug resistance transporter (MDR1) or canalicular multispecific organic anion transporter (cMOAT; also known as multidrug resistance protein 2). MDR1 and cMOAT are implicated in ATP-dependent efflux of anticancer drugs or GSH-xenobiotic conjugates, or both. The GST activity toward (+)-anti-BPDE in parental MDCKII cells did not differ from that in subline overexpressing MDR1 (MDCKII-MDR1) or cMOAT (MDCKII-cMOAT). Intracellular accumulation of BPD-SG, after a 5- or 10-min incubation with 1 microM (+)-anti-BPDE, was significantly higher in parental (41- to 67-fold) and MDCK II-MDR1 cells (31- to 43-fold) than in the MDCKII-cMOAT cells. Interestingly, the levels of DNA adducts of (+)-anti-BPDE, after a 30-min incubation with 0.1 or 0.5 microM [(3)H](+)-anti-BPDE, were significantly higher (about 2.1- and 1.7-fold, respectively) in parental cells than in the MDCKII-cMOAT cells. The results of the present study indicate that in addition to GSH conjugation, the efflux of BPD-SG may be essential for cellular protection against (+)-anti-BPDE-induced DNA damage.     

泛素连接酶RING2对苯并[a]芘染毒人支气管上皮细胞周期和P53蛋白表达的影响

目的:探讨泛素连接酶RING2对苯并[a]芘(BaP)染毒人支气管上皮16HBE细胞周期和P53蛋白表达的影响。方法:以16HBE未处理组为阴性对照组,二甲基亚砜(DMSO)组为溶剂对照组,MOCK组为序列对照组。在使用RNA干扰技术降低16HBE细胞泛素连接酶RING2基因表达前后,分别采用不同浓度BaP(1、2、4、8、16、32 μmol/L)染毒24 h;或16 μmol/L BaP染毒不同时间(1、2、4、8、12、24 h)。用流式细胞术检测干扰前后两组细胞周期分布情况,用Western-blot法检测干扰前后两组细胞P53蛋白表达水平。结果:流式细胞术检测结果显示,与阴性对照组比较,16HBE细胞染毒后各浓度和各时点组S期细胞所占的比例均增加(P<0.05),而16HBE(siRNA-RING2)各浓度和各时点组S期细胞所占的比例均下降(P<0.05)。协方差分析显示分组因素(是否进行RNAi)和染毒浓度都对S期细胞比例有影响(P均<0.01),16HBE(siRNA-RING2)细胞组的修正均数(17.09%)比16HBE细胞组(31.55%)明显降低(P<0.01)。分组因素和染毒时间都对S期细胞比例有影响(P均<0.01),16HBE(siRNA-RING2)细胞组的修正均数(13.07%)比16HBE细胞组(28.04%)明显下降(P<0.01)。Western-blot结果显示,与阴性对照组比较,16HBE 细胞染毒后各浓度和各时点组P53的表达水平均增加(P<0.05),而16HBE(siRNA-RING2)细胞除16 μmol/L染毒8 h组外,其余各组P53的表达水平均降低(P<0.05)。协方差分析显示分组因素和染毒浓度都对P53的表达水平有影响,P值分别为0.026和0.028。16HBE(siRNA-RING2)细胞组的修正均数(0.989)比16HBE细胞组(1.375)明显下降(P<0.05);分组因素 和染毒时间都对P53的表达水平有影响,P值分别为0.007和0.035。16HBE(siRNA-RING2)细胞组的修正均数(0.857)比16HBE细胞组(1.541)明显下降(P<0.05)。结论:RING2参与的组蛋白泛素化修饰可能通过影响 P53表达和细胞周期S期的变化来发挥对DNA损伤修复的调控。     

Benzo[a]pyrene promotes proliferation of human lung cancer cells by accelerating the epidermal growth factor receptor signaling pathway

Smoking is an independent prognostic factor of lung adenocarcinoma. Benzo[a]pyrene (B[a]P) is one of the strongest carcinogens and it is present in both the environment and cigarette smoke. In this study, the effect of B[a]P on the proliferative activity of lung adenocarcinoma cells was investigated. A lung adenocarcinoma cell line, A549, was cultured with B[a]P for various periods, and its proliferative activity was examined by an MTS assay. To investigate the intracellular events related to the proliferative activity, the gene expression profile was investigated by a microarray analysis and a quantitative RT-PCR, and the protein expression and activation status of Akt, ERK 1/2 and the epidermal growth factor receptor (EGFR) were examined by a western blot analysis. Following the culture with B[a]P for 24 weeks, the serum-independent proliferative activity was increased. A microarray analysis revealed that a reversible upregulation of the EGFR and epiregulin genes was recognized in the B[a]P treated cells, in which the overexpression of the phosphorylated EGFR protein was also recognized. The EGFR tyrosine kinase inhibitor reduced the cellular proliferation and the level of phosphorylation of ERK1/2, which is a downstream signal of the EGFR, in the B[a]P-treated A549 cells. Moreover, the B[a]P treatment increased the mRNA expressions of the ligands for EGFR such as amphiregulin and epiregulin. B[a]P increases the proliferative potential of lung adenocarcinoma cells through the EGFR signaling pathway.     

Effects of benzo[a]pyrene and ({+/-})-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene on mitosis in Chinese hamster V79 cells with stable expression of rat cytochrome P4501A1 or 1A2

The effect of bioactivation of benzo[a]pyrene (B[a]P) and (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(B[a]P-7,8-diol) on spindle disturbances and toxicity has beeninvestigated in V79 Chinese hamster cells genetically engineeredto express cytochrome P4501A1 (CYP1A1) and cytochrome P4501A2(CYP1A2). B[a]P induces spindle disturbances in native V79 Chinesehamster cells. This effect was enhanced by the expression ofCYP1A1 but not CYP1A2. The increased effect seen in the CYP1A1-expressingcell line could be brought back to the level seen in the nativecell line by     

Effects of chronic alcohol consumption on DNA damage and immune regulation induced by the environmental pollutant dibenzo[a,l]pyrene in oral tissues of mice

Previously, we showed that oral application of the environmental pollutant dibenzo[a,l]pyrene (DB[a,l]P) induces oral tumors in mice. Thus, in the present investigation we examined the effect of alcohol on DB[a,l]P-induced DNA damage and immune regulation; we showed that alcohol (6.4% v/v in the diet, 35% of Calories) significantly enhanced the levels of (?)-anti-trans-DB[a,l]P-dA while decreased the levels of GSH in the mouse oral tissues. Analysis of RNA expression revealed that DB[a,l]P alone upregulates inflammatory genes while alcohol suppresses several markers of immune surveillance. Collectively, these results suggest that alcohol may enhance oral carcinogenesis induced by DB[a,l]P.     

The time-dependent increase in the binding of benzo[a]pyrene to DNA through (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide in primary rat hepatocyte cultures results from induction of cytochrome P450IA1 by benzo[a]pyrene treatment.

The proportion and amount of benzo[a]pyrene (B[a]P) that binds to DNA through the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] increases with time of exposure to B[a]P in cell cultures derived from a number of species. Pretreatment of primary rat hepatocyte cultures for 12 h with 1 microgram B[a]P/ml medium increased the subsequent metabolism of [3H]B[a]P by 47% and [3H]B[a]P-DNA binding by 53% compared with acetone-pretreated hepatocytes. The amount of (+)-anti-BPDE bound to DNA in the B[a]P-pretreated hepatocytes increased 175%. B[a]P pretreatment also increased DNA-binding 2-fold in hepatocytes treated with [3H]7,8-dihydroxy-7,8-dihydro-B[a]P but had no effect on DNA binding in cells treated with anti-B[a]P-7,8-diol-9,10-epoxide. Western blotting showed that cytochrome P450IA1, which was not detectable prior to B[a]P treatment, was selectively increased by B[a]P treatment. A monoclonal antibody that specifically inhibits cytochrome P450IA1 reduced the binding of B[a]P to DNA by greater than 90% in microsomal preparations from B[a]P-pretreated hepatocytes. These results indicate that the time-dependent increase in the formation of (+)-anti-BPDE-DNA adducts results from an increase in the amount and proportion of B[a]P metabolized to this ultimate carcinogen by P450IA1 that is induced by the B[a]P treatment. The importance of P450IA1 induction by the B[a]P for its activation to this ultimate carcinogenic metabolite suggests that long-term exposure of cells to B[a]P could result in activation of a higher proportion of the B[a]P to the carcinogenic (+)-anti-BPDE.     

Oral benzo[a]pyrene in Cyp1a1/1b1(–/–) double‐knockout mice: Microarray analysis during squamous cell carcinoma formation in preputial gland duct

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) found in combustion processes. Cytochrome P450 1A1 and 1B1 enzymes (CYP1A1, CYP1B1) and other enzymes can activate PAHs to reactive oxygenated intermediates involved in mutagenesis and tumor initiation; also, CYP1 enzymes can detoxify PAHs. Cyp1(+/+) wild‐type (WT) and Cyp1b1(–/–) knockout mice receiving oral BaP (12.5 mg/kg/day) remain healthy for >12 months. In contrast, we found that global knockout of the Cyp1a1 gene (1a1KO) results in proximal small intestine (PSI) adenocarcinoma within 8–12 weeks on this BaP regimen; striking compensatory increases in PSI CYP1B1 likely participate in initiation of adenocarcinoma in 1a1KO mice. Cyp1a1/1b1(–/–) double‐knockout (DKO) mice on this BaP regimen show no PSI adenocarcinoma, but instead preputial gland duct (PGD) squamous cell carcinoma (SCC) occurs by 12 weeks. Herein, we compare microarray expression of PGD genes in WT, 1a1KO and DKO mice at 0, 4, 8, 12 and 16 weeks of oral BaP; about four dozen genes up‐ or down‐regulated during most critical time‐points were further verified by qRT‐PCR. In DKO mice, CYP3A59 was unequivocally identified as the BaP‐inducible and BaP‐metabolizing best candidate responsible for initiation of BaP‐induced SCC. Striking increases or decreases were found in 26 cancer‐related genes plus eight Serpin genes in DKO, but not in 1a1KO or WT, mice on this BaP regimen; of the 26, 8 were RAS‐related oncogenes. The mechanism by which cancer‐related genes are responsible for SCC tumor progression in the PGD remains to be elucidated.     

Oral benzo[a]pyrene‐induced cancer: Two distinct types in different target organs depend on the mouse Cyp1 genotype

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) found in combustion processes. Cytochrome P450 1A1 and 1B1 enzymes (CYP1A1 and CYP1B1) can both detoxify PAHs and activate them to cancer‐causing reactive intermediates. Following high dosage of oral BaP (125 mg/kg/day), ablation of the mouse Cyp1a1 gene causes immunosuppression and death within ～28 days, whereas Cyp1(+/+) wild‐type mice remain healthy for >12 months on this regimen. In this study, male Cyp1(+/+) wild‐type, Cyp1a1(?/?) and Cyp1b1(?/?) single‐knockout and Cyp1a1/1b1(?/?) double‐knockout mice received a lower dose (12.5 mg/kg/day) of oral BaP. Tissues from 16 different organs—including proximal small intestine (PSI), liver and preputial gland duct (PGD)—were evaluated; microarray cDNA expression and >30 mRNA levels were measured. Cyp1a1(?/?) mice revealed markedly increased CYP1B1 mRNA levels in the PSI, and between 8 and 12 weeks developed unique PSI adenomas and adenocarcinomas. Cyp1a1/1b1(?/?) mice showed no PSI tumors but instead developed squamous cell carcinoma of the PGD. Cyp1(+/+) and Cyp1b1(?/?) mice remained healthy with no remarkable abnormalities in any tissue examined. PSI adenocarcinomas exhibited striking upregulation of the Xist gene, suggesting epigenetic silencing of specific genes on the Y‐chromosome; the Rab30 oncogene was upregulated; the Nr0b2 tumor suppressor gene was downregulated; paradoxical overexpression of numerous immunoglobulin kappa‐ and heavy‐chain variable genes was found—although the adenocarcinoma showed no immunohistochemical evidence of being lymphatic in origin. This oral BaP mouse paradigm represents an example of “gene‐environment interactions” in which the same exposure of carcinogen results in altered target organ and tumor type, as a function of just 1 or 2 globally absent genes.     

CYP1A1 and CYP1B1 gene expression and DNA adduct formation in normal human mammary epithelial cells exposed to benzo[a]pyrene in the absence or presence of chlorophyllin

Benzo[a]pyrene (BP) is a potent pro-carcinogen and ubiquitous environmental pollutant. Here, we examined the induction and modulation of CYP1A1 and CYP1B1 and 10-(deoxyguanosin-N²-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG) adduct formation in DNA from 20 primary normal human mammary epithelial cell (NHMEC) strains exposed to BP (4 μM) in the absence or presence of chlorophyllin (5 μM). Real-time polymerase chain reaction (RT-PCR) analysis revealed strong induction of both CYP1A1 and CYP1B1 by BP, with high levels of inter-individual variability. Variable BPdG formation was found in all strains by r7, t8-dihydroxy-t-9, 10 epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence assay (CIA). Chlorophyllin mitigated BP-induced CYP1A1 and CYP1B1 gene expression in all 20 strains when administered with BP. Chlorophyllin, administered prior to BP-exposure, mitigated CYP1A1 expression in 18/20 NHMEC strains (p < 0.005) and CYP1B1 expression in 17/20 NHMEC strains (p < 0.005). Maximum percent reductions of CYP1A1 and CYP1B1 gene expression and BPdG adduct formation were observed when cells were pre-dosed with chlorophyllin followed by administration of the carcinogen with chlorophyllin (p < 0.005 for CYP1A1 and CYP1B1 expression and p < 0.0005 for BPdG adducts). Therefore, chlorophyllin is likely to be a good chemoprotective agent for a large proportion of the human population.     

The K-region trans-8,9-diol does not significantly contribute as an intermediate in the metabolic activation of dibenzo[a,l]pyrene to DNA-binding metabolites by human cytochrome P450 1A1 or 1B1.

Metabolic activation of the K-region trans-8,9-diol of the highly carcinogenic hexacyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) by human cytochrome P-450 (P450) 1A1 and 1B1 was investigated in Chinese hamster V79 cell lines expressing human P450 1A1 or 1B1. P450 1A1 and 1B1 are the major P450s involved in metabolic activation of polycyclic aromatic hydrocarbons in human cells. The major DNA adducts formed by metabolism of DB[a,l]P in cultures expressing P450 1A1 or 1B1 resulted mainly from the fjord region (-)-anti-DB[a,l]P-11,12-diol 13,14-epoxide [(-)-anti-DB[a,l]PDE] and, to a lesser extent, (+)-syn-DB[a,l]PDE. In V79 cells expressing human P450 1A1, high amounts of as yet unidentified highly polar DNA adducts are formed in addition to the DNA adducts derived from DB[a,l]PDEs. Human P450 1A1 has been found to metabolize DB[a,l]P on its K-region to the trans-8,9-diol, and it has been proposed that the DNA binding of the parent compound in P450 1A1-expressing tissues may be partially mediated by activation of the K-region trans-8,9-diol to form bis-diol epoxides. V79 cells expressing human P450 1A1 or 1B1 formed only low amounts of DNA adducts after treatment with high doses of the K-region trans-8,9-diol. None of the adducts formed were identical to the main adducts formed in the same cell lines by metabolic activation of DB[a,l]P or (-)-DB[a,l]P-trans-11,12-diol. These results demonstrate that the K-region trans-8,9-diol does not significantly contribute to the genotoxicity of the very potent carcinogen DB[a,l]P in human cells or tissues expressing P450 1A1 or 1B1.