All-trans retinoic acid suppresses Stat3 signaling during skin carcinogenesis.

Squamous cell carcinoma (SCC) of the skin is the most clinically aggressive form of nonmelanoma skin cancer. We have determined the effects of all-trans retinoic acid (ATRA), a naturally occurring chemopreventive retinoid, on signal transducer and activator of transcription 3 (Stat3) signaling during the development of skin SCC. Stat3 is a transcription factor that plays a critical role in cell proliferation and survival, and it is constitutively active in several malignant cell types. We have previously shown that Stat3 is required for the initiation, promotion, and progression of skin SCC. ATRA is a highly efficient suppressor of tumor formation in the two-stage mouse skin carcinogenesis model and we have shown that this effect correlates with the suppression of the B-Raf/Mek/Erk signaling pathway. In this study, we have determined the pattern of Stat3 phosphorylation throughout the course of the two-stage protocol, both in the presence and absence of ATRA. We have used both SENCAR mice and K5.Stat3C transgenic mice, which express the Stat3C protein, a constitutively active form of Stat3, in the skin. Using Western blotting and immunohistochemical staining with phosphospecific antibodies, we show that coadministration of ATRA suppressed the 12-O-tetradecanoylphorbol-13-acetate-induced phosphorylation of Stat3 in both models, but was only able to suppress tumor formation in the SENCAR mice. Surprisingly, ATRA actually enhanced tumor formation in 12-O-tetradecanoylphorbol-13-acetate-treated K5.Stat3C mice. We hypothesize that ATRA blocks tumor formation, at least in part, by targeting events upstream of Stat3, such as the B-Raf/Mek/Erk pathway, and that in the K5.Stat3C mice, in which Stat3 activity is constitutive, it cannot suppress tumor formation.

Identification of the B-Raf/Mek/Erk MAP kinase pathway as a target for all-trans retinoic acid during skin cancer promotion.

BACKGROUND: Retinoids have been studied extensively for their potential as therapeutic and chemopreventive agents for a variety of cancers, including nonmelanoma skin cancer (NMSC). Despite their use for many years, the mechanism of action of retinoids in the prevention of NMSC is still unclear. In this study we have attempted to understand the chemopreventive mechanism of all-trans retinoic acid (ATRA), a primary biologically active retinoid, in order to more efficiently utilize retinoids in the clinic. RESULTS: We have used the 2-stage dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) mouse skin carcinogenesis model to investigate the chemopreventive effects of ATRA. We have compared the gene expression profiles of control skin to skin subjected to the 2-stage protocol, with or without ATRA, using Affymetrix 430 2.0 DNA microarrays. Approximately 49% of the genes showing altered expression with TPA treatment are conversely affected when ATRA is co-administered. The activity of these genes, which we refer to as 'counter-regulated', may contribute to chemoprevention by ATRA. The counter-regulated genes have been clustered into functional categories and bioinformatic analysis has identified the B-Raf/Mek/Erk branch of the MAP kinase pathway as one containing several genes whose upregulation by TPA is blocked by ATRA. We also show that ATRA blocks signaling through this pathway, as revealed by immunohistochemistry and Western blotting. Finally, we found that blocking the B-Raf/Mek/Erk pathway with a pharmacological inhibitor, Sorafenib (BAY43-9006), induces squamous differentiation of existing skin SCCs formed in the 2-stage model. CONCLUSION: These results indicate that ATRA targets the B-Raf/Mek/Erk signaling pathway in the 2-stage mouse skin carcinogenesis model and this activity coincides with its chemopreventive action. This demonstrates the potential for targeting the B-Raf/Mek/Erk pathway for chemoprevention and therapy of skin SCC in humans. In addition our DNA microarray results provide the first expression signature for the chemopreventive effect of ATRA in a mouse skin cancer model. This is a potential source for novel targets for ATRA and other chemopreventive and therapeutic agents that can eventually be tested in the clinic.

Tissue microarray analysis of eIF4E and its downstream effector proteins in human breast cancer.

BACKGROUND: Eukaryotic initiation factor 4E (eIF4E) is elevated in many cancers and is a prognostic indicator in breast cancer. Many pro-tumorigenic proteins are selectively translated via eIF4E, including c-Myc, cyclin D1, ornithine decarboxylase (ODC), vascular endothelial growth factor (VEGF) and Tousled-like kinase 1B (TLK1B). However, western blot analysis of these factors in human breast cancer has been limited by the availability of fresh frozen tissue and the labor-intensive nature of the multiple assays required. Our goal was to validate whether formalin-fixed, paraffin-embedded tissues arranged in a tissue microarray (TMA) format would be more efficient than the use of fresh-frozen tissue and western blot to test multiple downstream gene products. RESULTS: Breast tumor TMAs were stained immunohistochemically and quantitated using the ARIOL imaging system. In the TMAs, eIF4E levels correlated strongly with c-Myc, cyclin D1, TLK1B, VEGF, and ODC. Western blot comparisons of eIF4E vs. TLK1B were consistent with the immunohistochemical results. Consistent with our previous western blot results, eIF4E did not correlate with node status, ER, PR, or HER-2/neu. CONCLUSION: We conclude that the TMA technique yields similar results as the western blot technique and can be more efficient and thorough in the evaluation of several products downstream of eIF4E.

Comparison of citrus coumarins on carcinogen-detoxifying enzymes in Nrf2 knockout mice.

Naturally occurring coumarins possess anti-carcinogenic activities in part by inducing carcinogen-detoxifying enzymes glutathione S-transferase (GST) and/or NAD(P)H quinone oxidoreductase (NQO1). Our goal was to determine whether citrus coumarins induce hepatic GST and/or NQO1 via activation of Nrf2 and the antioxidant response element (ARE). First, HepG2 cells stably transfected with the ARE and a green fluorescent protein (GFP) reporter were treated with increasing concentrations of coumarins and compared to positive controls. tert-Butylhydroquinone (TBHQ) and oltipraz increased GFP fluorescence, as did coumarin, limettin, auraptene, imperatorin, and 7,8-benzoflavone, suggesting that they activate the ARE, whereas isopimpinellin did not increase GFP fluorescence. Next, the effects of orally administered coumarins and oltipraz on hepatic GST and NQO1 activities were compared in Nrf2 knockout mice or Nrf2 heterozygous mice exhibiting the wild-type phenotype. Oltipraz, auraptene, imperatorin, isopimpinellin, and auraptene all significantly increased liver cytosolic GST activities in Nrf2 heterozygous mice. This effect was abrogated in Nrf2(-/-) mice dosed with oltipraz, attenuated in mice Nrf2(-/-) mice treated with auraptene and imperatorin, and still significant in Nrf2(-/-) mice treated with isopimpinellin. Of these compounds, only isopimpinellin significantly increased liver cytosolic NQO1 activities, and this effect was not attenuated in Nrf2(-/-) mice. These results strongly suggest that imperatorin and auraptene induce murine liver cytosolic GST activities via the Nrf2/ARE mechanism. Although structurally similar, isopimpinellin did not appear to activate HepG2-ARE-GFP and the Nrf2 knockout mouse study suggests that isopimpinellin may induce GST and NQO1 via additional mechanisms.

Effects of naturally occurring coumarins on hepatic drug-metabolizing enzymes in mice.

Cytochromes P450 (P450s) and glutathione S-transferases (GSTs) constitute two important enzyme families involved in carcinogen metabolism. Generally, P450s play activation or detoxifying roles while GSTs act primarily as detoxifying enzymes. We previously demonstrated that oral administration of the linear furanocoumarins, isopimpinellin and imperatorin, modulated P450 and GST activities in various tissues of mice. The purpose of the present study was to compare a broader range of naturally occurring coumarins (simple coumarins, and furanocoumarins of the linear and angular type) for their abilities to modulate hepatic drug-metabolizing enzymes when administered orally to mice. We now report that all of the different coumarins tested (coumarin, limettin, auraptene, angelicin, bergamottin, imperatorin and isopimpinellin) induced hepatic GST activities, whereas the linear furanocoumarins possessed the greatest abilities to induce hepatic P450 activities, in particular P450 2B and 3A. In both cases, this corresponded to an increase in protein expression of the enzymes. Induction of P4502B10, 3A11, and 2C9 by xenobiotics often is a result of activation of the pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR). Using a pregnane X receptor reporter system, our results demonstrated that isopimpinellin activated both PXR and its human ortholog SXR by recruiting coactivator SRC-1 in transfected cells. In CAR transfection assays, isopimpinellin counteracted the inhibitory effect of androstanol on full-length mCAR, a Gal4-mCAR ligand-binding domain fusion, and restored coactivator binding. Orally administered isopimpinellin induced hepatic mRNA expression of Cyp2b10, Cyp3a11, and GSTain CAR(+/+) wild-type mice. In contrast, the induction of Cyp2b10 mRNA by isopimpinellin was attenuated in the CAR(-/-) mice, suggesting that isopimpinellin induces Cyp2b10 via the CAR receptor. Overall, the current data indicate that naturally occurring coumarins have diverse activities in terms of inducing various xenobiotic metabolizing enzymes based on their chemical structure.

Pro-apoptotic effects of 1'-acetoxychavicol acetate in human breast carcinoma cells.

The tropical ginger compound, 1'-acetoxychavicol acetate (ACA) possesses cancer chemopreventive properties in several models but its effects on breast cancer have not been fully evaluated. In this study, the effects of ACA on human breast carcinoma-derived MCF-7 and MDA-MB-231 cell viability were assessed using trypan blue exclusion analysis. ACA significantly decreased cell viability in a time- and dose-dependent manner, with effective concentrations 10-50 microM. Apoptosis was confirmed by morphological examination of cells through light microscopy, 4,6-diamidino-2-phenylindole dihydrochloride staining, and annexin V/Alexa Fluor 488 staining visualized using flow cytometry. ACA also increased protein expression of the activated form of caspase-3 in MDA-MB-231 cells. Addition of antioxidants N-acetylcysteine, ascorbic acid, or trolox prevented the loss of viability caused by ACA using trypan blue uptake as a marker. These results suggest ACA may have potential anticancer effects against breast carcinoma cells by inducing apoptosis.

Naturally occurring coumarins inhibit 7,12-dimethylbenz[a]anthracene DNA adduct formation in mouse mammary gland.

Naturally occurring coumarins (NOCs) are anti-carcinogenic in the mouse skin model. To characterize the chemopreventive potential of NOCs against breast cancer, we first examined their effects on 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adduct formation in mouse mammary gland. We hypothesized that those NOCs that both inhibited cytochrome P450 1A1/1B1 and induced hepatic glutathione S-transferases (GSTs) would be the most effective in blocking DMBA-DNA adduct formation in mouse mammary gland. To address this hypothesis, simple coumarins (e.g. coumarin and limettin, which induced mouse hepatic GSTs but had little effect on P4501A1/1B1) and linear furanocoumarins (e.g. imperatorin and isopimpinellin, which induced hepatic GSTs and were potent inhibitors of P4501A1/1B1) were compared. Mice were pretreated with NOCs (150 mg/kg body wt, by gavage) prior to either a single dose of DMBA (50 microg) or multiple doses of DMBA (20 microg daily for 3 and 6 weeks). Mammary DMBA-DNA adduct formation was quantitated by the nuclease P1-enhanced 32P-postlabeling assay. With the single dose of DMBA, coumarin, limettin, imperatorin and isopimpinellin inhibited DMBA-DNA adduct formation by 50, 41, 79 and 88%, respectively. Coumarin, limettin and imperatorin blocked DMBA-DNA adduct formation by 36, 60, and 66% at 3 weeks, and by 0, 49 and 55% at 6 weeks of DMBA dosing, respectively. In a 6 week dose-response study of select NOCs and 7,8-benzoflavone (a potent P4501 inhibitor that had little effect on GSTs), DMBA-DNA adduct formation was inhibited by 0, 43 and 24% in the limettin groups; by 26, 26 and 69% in the isopimpinellin groups; and by 80, 96 and 97% in the 7,8- benzoflavone groups at 35, 70 and 150 mg/kg, respectively. Taken together, these results suggest that linear furanocoumarins had a greater inhibitory effect on DMBA-DNA adduct formation in mouse mammary glands compared with simple coumarins, and that the predominant effect may be P4501 inhibition.

Coumarins are competitive inhibitors of cytochrome P450 1B1, with equal potency for allelic variants.

OBJECTIVES: Coumarins are naturally occurring chemicals with potential as chemopreventive agents, several with known action on the cytochrome P450 1A family. We examined whether cytochrome P450 1B1 (CYP1B1) was inhibited by coumarins, whether such inhibition was competitive, and whether inhibition varied between common polymorphic variants of this enzyme. METHODS: We tested the inhibition properties of four coumarins, bergamottin, isopimpinellin, isoimperatorin, and imperatorin in an assay for oxidation of (-)benzo[a]pyrene-7R-trans-7,8-dihyrodiol (B[a]P-7,8-diol) by CYP1B1 using yeast-microsome expressed enzymes. These assays were performed with wild-type enzyme and five single-amino acid polymorphic variants. RESULTS: All four coumarins are competitive inhibitors of CYP1B1, with Ki values equal to 587, 11, 6 and 1 muM respectively. Inhibition parameters were consistent between five haplotypes of CYP1B1, three representing common haplotypes in Asians, African-Americans and European-Americans, and two with baseline kinetic parameters previously shown to be potentially different from wild-type. CONCLUSIONS: Coumarins are capable of inhibiting carcinogen activation by CYP1B1 with varying potencies, and their efficacy as chemopreventive agents is not likely to be affected by polymorphism in this enzyme.

Role of cytochrome p4501 family members in the metabolic activation of polycyclic aromatic hydrocarbons in mouse epidermis.

Polycyclic aromatic hydrocarbons (PAHs) are known to be activated by the cytochrome P450 (P450) 1 family. However, the precise role of individual P4501 family members in PAH bioactivation remains to be fully elucidated. We therefore investigated the formation of PAH-DNA adducts in the epidermis of Cyp1a2(-/-), Cyp1b1(-/-), and Ahr(-/-) knockout mice. A panel of different PAHs was used, ranging in carcinogenic potency. Mice were treated topically on the dorsal skin with the following tritium-labeled PAHs: dibenzo[a,l]pyre-ne (DB[a,l]P), 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B[a]P), dibenzo[a,h]anthracene (DB[a,h]A), benzo[g]chrysene (B[g]C), and benzo[c]phenanthrene (B[c]P). At 24 h after treatment, mice (two male and two female mice per group) were sacrificed, and epidermal DNA was isolated and hydrolyzed with DNase I; subsequently, DNA adducts were quantitated by liquid scintillation counting. In the DB[a,l]P-treated mice, levels of DNA adducts were significantly lower in Cyp1a2(-/-) and Cyp1b1(-/-) mice by 57 and 46%, respectively, as compared to wild-type (WT) mice (C57BL/6 background). The levels of DB[a,l]P DNA adducts formed in Ahr(-/-) mice were 26% lower, but this was not statistically significant. The levels of DMBA-DNA adducts in Cyp1a2(-/-) mice were not different than the WT mice but were significantly lower in Cyp1b1(-/-) and Ahr(-/-) mice by 64 and 52%, respectively. DMBA-DNA adduct samples were further analyzed by HPLC following further digestion to deoxyribonucleosides. HPLC analysis of individual DMBA-DNA adducts revealed differences in the ratio of syn-DMBA-diol epoxide- to anti-DMBA-diol epoxide-derived adducts in the Ahr(-/-) and Cyp1b1(-/-) mice. The ratio of syn-/anti-derived adducts in WT mice was 0.49. This ratio was 0.23 in the Cyp1b1(-/-) mice and 0.87 in the Ahr(-/-) mice. In contrast to the results with DB[a,l]P and DMBA, the levels of B[a]P-, DB[a,h]A-, B[g]C-, and B[c]P-DNA adducts were significantly lower in Ahr(-/-) mice by 73, 75, 50, and 81%, respectively, as compared to WT mice but were not significantly lower in the Cyp1a2(-/-) or Cyp1b1(-/-) mice. Collectively, these and other results support a role for both P4501A1 and P4501B1 in the bioactivation of DMBA; P4501A2, P4501B1, and possibly P4501A1 in the bioactivation of DB[a,l]P; and P4501A1 in the bioactivation of B[a]P, DB[a,h]A, B[g]C, and B[c]P in mouse epidermis. Furthermore, in the metabolic activation of DMBA in mouse epidermis, P4501B1 shows a preference for the formation of syn-DMBA-diol epoxide adducts, whereas P4501A1 shows a preference for the formation of anti-DMBA-diol epoxide adducts.

High levels of oxidative DNA damage in lymphocyte DNA of premenopausal breast cancer patients from Egypt.

Egypt shows a parallel increase in premenopausal breast cancer and environmental pollution. The purpose of this study is to explore a possible relationship between oxidative DNA damage, urinary estrogen metabolites and breast cancer in Egyptian premenopausal women. We conducted a pilot study of Egyptian breast cancer involving 29 cases and 32 controls and analysed lymphocyte DNA levels of 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-dG), a measure of oxidative DNA damage using high performance liquid chromatography with electro-chemical detection (HPLC-ECD) method. We analysed levels of urinary estrogen metabolites, 2-hydroxyestrone (2-OHE) and 16alpha-hydroxyestrone (16alpha-OHE) by an enzyme immuno assay. We also collected residential, occupational, and reproductive histories of all study subjects. We detected, in all subjects, exceptionally high levels of 8-oxo-dG and thus oxidative DNA damage, the levels (mean 8-oxo-dG/10(5) dG+/-SD) were significantly (P<0.01) higher in breast cancer cases (139.4+/-78.4) than in controls (60.9+/-51.5). Urinary 2-OHE and 16alpha-OHE or their ratio was not significantly different between cases and controls. However, 8-oxo-dG levels were positively correlated (P<0.05) with 2-OHE and 16alpha-OHE from cases while controls showed a negative correlation (P<0.05). Urban residence (Odds Ratio [OR] 3.1; Confidence interval [CI], 1.1-9.3), infertility (OR [9.8]; CI [1.1-89.7]), age (OR [2.6]; CI [1.4-4.6]) and 8-oxo-dG (OR 5.8; CI 1.9-17.5) levels were found to be significant predictors of breast cancer. Our finding of exceptionally high levels of 8-oxo-dG, a common result of oxidative DNA damage, warrant future studies on a larger population of premenopausal women in Egypt with consideration of other susceptibility markers and dietary characteristics.

Naturally occurring coumarins inhibit human cytochromes P450 and block benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene DNA adduct formation in MCF-7 cells.

Naturally occurring coumarins (NOCs) inhibit polycyclic aromatic hydrocarbon-induced skin tumor initiation in mice by blocking cytochrome P450 (P450)-mediated bioactivation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA). Bergamottin selectively inhibits tumor initiation by B[a]P, whereas imperatorin and isopimpinellin inhibit tumor initiation with both carcinogens. The goals of the current study were to examine the ability of NOCs to inhibit human P450s in vitro and to establish whether NOCs, which are anticarcinogenic in mice, can block carcinogen bioactivation in cultured human cells. For the initial experiments, incubations containing 5 microM P450, P450 substrate, an NADPH generating system, and NOCs were used to determine the concentrations of each inhibitor that blocked 50% of P450 activity (IC(50)). These results confirmed that NOCs are capable of inhibiting multiple human P450s and that they exhibit selectivity for certain isoforms of human P450s. In subsequent experiments, we examined the effects of bergamottin, imperatorin, and isopimpinellin on DMBA and B[a]P DNA adduct formation in the human breast MCF-7 adenocarcinoma cell line. Coincubation of cells with the three different NOCs significantly inhibited DMBA DNA adduct formation by 29-82% at doses ranging from 2 to 10 microM and significantly inhibited B[a]P DNA adduct formation by 37-80% at doses ranging from 20 to 80 microM. HPLC analysis of the DNA hydrolysates demonstrated that inhibition of DNA adducts corresponded to inhibition of the major B[a]P and DMBA diol-epoxide-derived adducts. Although bergamottin was not effective at blocking DMBA bioactivation in the mouse skin model, it was similar in effectiveness to imperatorin and isopimpinellin in MCF-7 cells. These results demonstrate that NOCs, which are present in citrus fruits and other components of the human diet, are capable of inhibiting carcinogen metabolizing enzymes and blocking bioactivation of both B[a]P and DMBA in MCF-7 cells.

Oral administration of the citrus coumarin, isopimpinellin, blocks DNA adduct formation and skin tumor initiation by 7,12-dimethylbenz[a]anthracene in SENCAR mice.

The current study was designed to evaluate the effects of oral administration of the citrus coumarin, isopimpinellin, on skin tumor initiation by topically applied benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA). To evaluate the effects of orally administered isopimpinellin on skin tumor initiation by B[a]P and DMBA, its effects on DNA adduct formation were first evaluated. Female SENCAR mice were pre-treated twice with corn oil, or isopimpinellin (70 mg/kg body wt per os) at 24 h and 2 h prior to topical treatment with B[a]P or DMBA. Another citrus coumarin, imperatorin, was also included in these experiments for comparison. Orally administered isopimpinellin and imperatorin significantly inhibited B[a]P-DNA adduct formation by 37 and 26%, respectively. Imperatorin also blocked DMBA-DNA adduct formation by 43%. In a second dose-response study, orally administered isopimpinellin (35, 70 and 150 mg/kg) blocked DMBA-DNA adduct formation by 23, 56 and 69%, respectively. For the tumor study, mice were pretreated orally with corn oil or isopimpinellin at 24 and 2 h prior to initiation with DMBA, and 2 weeks later promotion began with 12-O-tetradecanoylphorbol-13-acetate (TPA). Isopimpinellin significantly reduced the mean number of papillomas per mouse by 49, 73 and 78% compared to corn oil controls at 30, 70 and 150 mg/kg body wt, respectively. Orally administered isopimpinellin also significantly reduced the percentage of mice with papillomas at the highest dose tested (150 mg/kg). The effectiveness of isopimpinellin given topically over a broad dose range against DMBA tumor initiation was also evaluated for comparison. As part of this study, several parameters of systemic toxicity were evaluated following oral dosing with isopimpinellin and imperatorin. Mice were treated orally with corn oil, isopimpinellin or imperatorin (35, 70 and 150 mg/kg body wt per os) once daily for four consecutive days, killed at 24 h after the last dose, and livers, lungs, and kidneys evaluated histologically. In addition, urinary parameters of nephrotoxicity, blood parameters of liver and kidney function, and thrombin clotting time were assayed. No significant changes in blood clotting, or renal or hepatic function were observed. There was, however, a significant increase in liver wt accompanied by cytoplasmic vacuolation of hepatocytes. There were no histopathological changes in lungs or kidneys. Overall, these data indicate that isopimpinellin (and imperatorin) have chemopreventive effects when administered orally on skin tumor initiation by DMBA.

Role of cytochrome P450 1a1 and 1b1 in the metabolic activation of 7,12-dimethylbenz[a]anthracene and the effects of naturally occurring furanocoumarins on skin tumor initiation.

The current study was designed to determine the mechanistic basis for differences in the effects of naturally occurring furanocoumarins on skin tumor initiation by 7,12-dimethylbenz[a]anthracene (DMBA). Female SENCAR mice were pretreated topically with bergamottin, imperatorin, or isopimpinellin (100-3200 nmol), 7,8-benzoflavone (7,8-BF, 5-40 nmol, a known inhibitor of DMBA skin carcinogenesis in mice), or acetone (vehicle control) 5 min prior to topical treatment with DMBA (10 nmol). Imperatorin, isopimpinellin, and 7,8-BF, but not bergamottin, significantly blocked total DMBA-DNA adduct formation. HPLC analysis of DNA adducts revealed that bergamottin preferentially inhibited formation of anti-DMBA diol-epoxide (DMBADE) derived DNA adducts, imperatorin, and isopimpinellin inhibited both anti- and syn- derived adducts, whereas 7,8-BF showed some selectivity for reduction of syn-DMBADE-DNA adducts. Mouse embryo fibroblast C3H/10T1/2 (10T1/2) cells, and mouse hepatoma-derived 1c1c7 (Hepa-1) cells, which preferentially express P450 1b1 and P450 1a1, respectively, were co-incubated with 2 microM bergamottin, imperatorin, isopimpinellin, and 7,8-BF, and with DMBA (2 microM). Hepa-1 cells (P450 1a1) formed mainly anti-DMBADE-DNA adducts. In contrast, 10T1/2 cells (P450 1b1) formed mainly syn-DMBADE-DNA adducts. Bergamottin inhibited DMBA metabolism to DMBA-3,4-diol and blocked DNA adduct formation in Hepa-1 cells, but had little effect in 10T1/2 cells. In contrast, 7,8-BF completely blocked DMBA metabolism and DNA adduct formation in 10T1/2 cells, but had little effect in Hepa-1 cells. Imperatorin and isopimpinellin inhibited DMBA bioactivation in both cell lines. These results indicate that bergamottin is a more selective inhibitor of P450 1a1 and overall a less effective inhibitor of the metabolic activation of DMBA in mouse epidermis. In contrast, imperatorin, isopimpinellin, and especially 7,8-BF, which block metabolic activation of DMBA in mouse epidermis, appear more selective for P450 1b1. On the basis of our studies using 10T1/2 cells and Hepa-1 cells, it appears that P450 1a1 is primarily responsible for converting DMBA-3,4-diol to anti-DMBADE, whereas P450 1b1 is primarily responsible for converting DMBA-3,4-diol to syn-DMBADE. These data demonstrate the role of P450 1a1 and 1b1 in the metabolic activation of DMBA in mouse epidermis and provide a mechanistic explanation for the differential effects of naturally occurring furanocoumarins (and 7,8-BF) on polycyclic aromatic hydrocarbon skin carcinogenesis.