anthracene a - American Chemical Society

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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 Heather E. Kleiner, Suryanarayana V. Vulimiri, Melissa J. Reed, Ann Uberecken, and John DiGiovanni* University of Texas MD Anderson Cancer Center, Science Park-Research Division, Department of Carcinogenesis, P.O. Box 389, Smithville, Texas 78957 Received September 14, 2001

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-DMBADEDNA 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 µM bergamottin, imperatorin, isopimpinellin, and 7,8-BF, and with DMBA (2 µM). 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 antiDMBADE, whereas P450 1b1 is primarily responsible for converting DMBA-3,4-diol to synDMBADE. 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.

Introduction The mouse skin model of carcinogenesis is a useful tool to study the mechanisms of chemical carcinogenesis in an epithelial tissue. Polycyclic aromatic hydrocarbons (PAHs)1 such as benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) have been used extensively as initiators or as complete carcinogens in this carcinogenesis model (1). These PAHs are metabolized primarily by the mixed function oxidase system to yield both detoxication products, which are more polar and excretable, and bioactivation products, which are more reactive and therefore toxic. The predominant mutagenic and carcinogenic metabolite of B[a]P is formed by a two* To whom correspondence should be addressed. Phone: (512) 2379414. Fax: (512) 237-3439. E-mail: [email protected].

step oxidation to (+)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide which reacts with DNA, particularly dGuo, which leads to tumor initiation (reviewed in ref 1). In contrast, DMBA bioactivation to mutagenic and carcinogenic 1 Abbreviations: Ah, aryl hydrocarbon; anti-DMBADE, (()anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide; B[a]P, benzo[a]pyrene; 7,8-BF, 7,8-benzoflavone; dAdo, deoxyadenosine; dGuo, deoxyguanosine; DMBA, 7,12-dimethylbenz[a]anthracene; EROD, 7-ethoxyresorufin O-deethylase; DMBA-3,4-diol, 3,4-dihydro-3,4-dihydroxy-7,12dimethylbenz[a]anthracene; DMBA-8,9-diol, 8,9-dihydro-8,9-dihydroxy7,12-dimethylbenz[a]anthracene, Hepa-1 cells, Hepa 1c1c7 cells; 7-OHM12-MBA, 7-hydroxymethyl-12-methylbenz[a]anthracene; 12-OHM-7MBA, 12-hydroxymethyl-7-methylbenz[a]anthracene, R-MEM, R-minimal essential media; PAH, polycyclic aromatic hydrocarbon; PBS, phosphate buffered saline; PLSD, protected least significant difference (PROD); 7-pentoxyresorufin O-dealkylase; PUVA, psoralens with UVA; syn-DMBADE, (()syn-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; 10T1/2 cells, C3H/ 10T1/2 cells.

10.1021/tx010151v CCC: $22.00 © 2002 American Chemical Society Published on Web 01/16/2002

Inhibition of DMBA Metabolic Activation

metabolites results from metabolism to both syn- and anti-DMBA-3,4-diol, 1,2-epoxides (DMBADE) which can bind to both dAdo and dGuo residues in DNA (2, 3). Both the syn- and anti-DMBADE are tumor initiators in mouse epidermis (4). Previous reports have shown that naturally occurring coumarins can modulate P450 activity. 8-Methoxypsoralen, which is used in psoralen phytochemotherapy to treat psoriasis [psoralens with UVA (PUVA) therapy], is bioactivated by P450 to reactive intermediates which bind covalently to the enzyme (5) thereby inhibiting P450-dependent ethoxycoumarin deethylase activity in vitro in liver microsomes of CD-1 mice. Naturally occurring coumarins such as imperatorin, isoimperatorin, and bergapten cause inhibition of drug metabolizing enzymes (6). 8-Acyl-7-hydroxycoumarins inhibit P450-mediated metabolism of B[a]P to dihydrodiols in 3-methylcholanthrene-induced rat liver microsomes (7). Naturally occurring coumarins (e.g., coriandrin, imperatorin) were found to inhibit P450-mediated enzyme activities in vitro (8). Coriandrin and several other naturally occurring coumarins were found to be mechanism-based inactivators of P450s in vitro (9). In addition, bergamottin and coriandrin were found to inhibit metabolism of B[a]P and DNA adduct formation from both B[a]P and DMBA in cultured mouse epidermal keratinocytes (10). Selected naturally occurring coumarins were also found to inhibit skin tumor initiation by B[a]P and DMBA, and imperatorin blocked complete carcinogenesis by DMBA (11). However, differential effects were observed on tumor initiation depending on whether B[a]P or DMBA was used as the initiator. Imperatorin, which preferentially inhibited mouse liver microsomal pentoxyresorufin Odealkylase (PROD) activity, (8) was more effective at blocking skin tumor initiation by DMBA than by B[a]P. Bergamottin, which preferentially inhibited mouse liver microsomal ethoxyresorufin O-deethylase (EROD) activity (8), inhibited skin tumor initiation by B[a]P but was not effective against skin tumor initiation by DMBA at the doses tested (800 nmol) (11). The effect of bergamottin on B[a]P skin tumor initiation was hypothesized to be due to selective inhibition of P450 1a1-mediated metabolism to B[a]P-7,8-dihydrodiol (10, 11). In addition, these studies suggested that B[a]P and DMBA may be bioactivated by different isoforms of P450 in mouse skin and that bergamottin and imperatorin selectively inhibit different isoforms of P450. The current study was designed to determine the mechanistic basis for these differences. In this regard, the effects of bergamottin, imperatorin, and a related compound, isopimpinellin, on DMBA-DNA adduct formation in SENCAR mice were further characterized. In addition, two different cells lines, mouse embryo fibroblast C3H/10T1/2 (10T1/2) cells and mouse hepatoma derived Hepa-1c1c7 (Hepa-1) cells, which preferentially express P450 1b1 and 1a1, respectively (12, 13), were used to investigate the effects of bergamottin, imperatorin, isopimpinellin, and 7,8-benzoflavone (7,8-BF), a known P450 inhibitor, on DMBA metabolism and DNA adduct formation. The results are also discussed in terms of the role of P450 1a1 and 1b1 in metabolic activation of DMBA.

Experimental Procedures Caution. 7,12-Dimethylbenz[a]anthracene and DMBA-3,4diol are carcinogenic and mutagenic and should be handled with

Chem. Res. Toxicol., Vol. 15, No. 2, 2002 227 extreme caution using the guidelines for carcinogenic materials developed by the National Cancer Institute. In addition, several of the linear furanocoumarins are phototoxic and should be handled with extreme care. Materials. DMBA was purchased from Eastman Kodak Co. (Rochester, NY). [3H]DMBA (sp. act. 62.0 Ci/mmol) was obtained from Amersham Co. (Arlington Heights, IL) and diluted with unlabeled DMBA to specific activity of 1 or 10 Ci/mmol. Bergamottin and isopimpinellin were purchased from Indofine Chemical Co. (Somerville, NJ). Imperatorin was synthesized by Wayne Ivie (U.S. Department of Agriculture, College Station, TX) as previously described (14). DNase I (bovine pancreas, EC 3.1.4.1), 7,8-BF, snake venom phosphodiesterase (Crotalus atrox, EC 3.1.4.1), and alkaline phosphatase (Escherichia coli, type III, EC 3.1.3.1) were supplied by Sigma Chemical Co. (St. Louis, MO). Sephadex LH-20 was obtained from Pharmacia, Inc. (Piscataway, NJ). Sep-pak (C-18) cartridges were supplied by Waters Corp. (Milford, MA). Basal Medium Eagle, R-MEM, and antibiotic/antimycotic, were purchased from Life Technologies (Frederick, MD). Fetal bovine serum was obtained from Atlanta Biologicals (Norcross, GA). HPLC-grade acetone and methanol were purchased from EM Science (Gibbstown, NJ), and HPLCgrade ethyl acetate was from Fisher Chemical Co. (Fair Lawn, NJ). DMBA-3,4-diol and DMBA-8,9-diol were generous gifts from Dr. Ronald Harvey (U. Chicago). In addition, 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA), 12-hydroxymethyl-7-methylbenz[a]anthracene (12-OHM-7-MBA), 7,12dihydroxymethylbenz[a]anthracene, and phenols were obtained as previously described (15). [3H]DMBA-DNA adduct markers were constructed as described below. Preparation of Deoxyribonucleoside Hydrocarbon Adduct Markers. PAH and furanocoumarins are light sensitive. All studies were performed under subdued lighting. DMBAderived DNA adduct markers were generated by a procedure described previously (16). Briefly, calf thymus DNA was labeled with [3H]dAdo or [3H]dGuo, using a nick translation procedure (Gibco BRL, Gaitherburg, MD). The (()-syn- and (()-anti-diol epoxides of DMBA (obtained from Ronald Harvey, U. Chicago) were individually reacted with either unlabeled or radiolabeled calf thymus DNA at 37 °C for 16 h in 0.05 M Tris buffer, pH 7.0. DNA was extracted with 2 vol of n-butanol, followed by two extractions with water-saturated ethyl acetate. DNA was precipitated from the aqueous phase using 1/10 volume 4 M sodium acetate buffer, pH 4.5, and 2 vol of ice-cold absolute ethanol. DNA was sequentially hydrolyzed to deoxyribonucleosides using DNase I, snake venom phosphodiesterase, and alkaline phosphatase as described previously (17). Sephadex LH-20 columns or C18 Sep-pak cartridges were used to isolate the modified deoxyribonucleosides from the hydrolysate (18). HPLC analysis of DMBA-DNA adducts was conducted by a method previously described (11, 18). Treatment of Mice for DNA Adduct Studies. The backs of female SENCAR mice (7-9 weeks of age) (obtained from the National Cancer Institute, Frederick, MD) were shaved 2 days prior to treatment, and only those mice in the resting phase of the hair cycle were used. Increasing doses of the furanocoumarins (100-3200 nmol) or 7,8-BF (5-40 nmol) (each dissolved in 0.2 mL acetone) were applied to the dorsal skin of mice (4-6 mice/group) 5 min prior to treatment with [3H]DMBA (10 nmol, 10 Ci/mmol, also dissolved in 0.2 mL acetone). Control mice were pretreated with acetone vehicle only. Mice were sacrificed by cervical dislocation 24 h after DMBA treatment, and the epidermis was scraped and pooled for DNA isolation and DNA adduct analysis. Epidermal scrapings were homogenized using a Polytron PT10 in 6% (w/v) 4-amino salicylate, 1% (w/v) sodium chloride, 1% (w/v) triiso-propylnaphthalenesulfonic acid sodium salt, and 6% (v/v) sec-butanol as described previously (19). Culture and Treatment of Cells. Mouse embryo fibroblast cells [C3H/0T1/2 cells (10T1/2 cells), clone 8] and mouse hepatoma derived cells (Hepa 1c1c7, Hepa-1) were purchased from ATCC (Manassas, VA). 10T1/2 cells were cultured in Eagle’s Basal medium containing Earle’s salts, L-glutamine, 100

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units/mL penicillin G sodium, 100 µg/mL streptomycin sulfate, 0.25 µg/mL amphotericin B, supplemented with 10% heatinactivated fetal bovine serum in a humidified atmosphere at 5% CO2 at 37 °C. Hepa-1 cells were cultured in R-MEM medium without nucleosides, containing L-glutamine, 100 units/mL penicillin G sodium, 100 µg/mL streptomycin sulfate, 0.25 µg/ mL amphotericin B, supplemented with 5% heat-inactivated fetal bovine serum in a humidified atmosphere at 5% CO2 at 37 °C. Both lines of cells were passaged using 5% trypsin/0.03% EDTA in PBS at 80% confluence. For experiments, 10T1/2 cells (passage 12-18) were seeded in polystyrene tissue culture plates at a density of 1 × 106 cells/35 mm dish or 5 × 106 cells/100 mm dish. Hepa-1 cells were seeded in polystyrene tissue culture plates at a density of 0.5 × 106 cells/35 mm dish or 2.5 × 106 cells/100 mm dish. At ∼80% confluence, cells were treated with [3H]DMBA (1 Ci/mmol for metabolism studies or 10-20 Ci/mmol for DNA adduct studies) at a final media concentration of 2 µM. Cells were co-treated with acetone or the test compounds dissolved in acetone. Total acetone concentration in media was 0.75% (v/v). For DNA adduct analysis, at 24 h after treatment, media was removed, and cells were washed twice with PBS. Cells were lysed with 0.75 M guanidine isothiocyanate, and the DNA was subsequently isolated as described (18). DMBA-DNA adducts were analyzed as described below. For metabolism studies, media was removed and extracted as described below. Analysis of DMBA Metabolites in Media from Cell Cultures. Media samples were extracted twice with 2 vol of ethyl acetate:acetone (2:1, v/v), as previously described (10). Organic solvent soluble metabolites were analyzed by HPLC as previously described (10) with radiomatic detection (Flo-one beta, Packard Instrument Co., Meriden, CT). The HPLC effluent was mixed with Ultima-Flo AP at a ratio of 1:3 and detected through a 0.5 mL cell. Retention times of experimental metabolites were identified by comparison of retention times with authentic standards. Under these conditions, retention times of peaks were as follows: DMBA-8,9-diol, 19.7 min; DMBA-3,4diol, 34.3 min; 7-OHM-12-MBA, 42.9 min; 12-OHM-7-MBA, 45.2 min; phenols, 50.7 min; and DMBA, 64.4 min. Data were represented as percentage of total disintegrations per minute (dpm) eluted. Analysis of DMBA-DNA Adducts. DNA was isolated by phenol extraction and RNase A digestion as described previously (19). Purity of DNA was determined spectrophotometrically and DNA content was estimated using calf thymus DNA as a standard (20). Due to the limited quantities of DNA from 10T1/2 or Hepa-1 cells, DNA was quantitated spectrophotometrically using absorbance at 260 nm. To quantitate total DNA adducts, DNA was hydrolyzed with DNase I, and aliquots were analyzed by liquid scintillation spectroscopy. Specific activity of DNA binding was represented as picomoles of DMBA bound per milligram of DNA (pmol DMBA bound/mg of DNA). To analyze individual DNA adducts, the DNase I DNA hydrolysates were sequentially hydrolyzed with snake venom phosphodiesterase and alkaline phosphatase as described previously (17) and purified through Sephadex LH-20 columns or Sep-pak (C-18) cartridges (18). HPLC analysis of DMBA-DNA adducts was conducted by a method previously described (11, 18), using either a fraction collector and liquid scintillation counting of fractions, or using radiomatic detection as described above. By comparing retention times of individual peaks with the synthetic standards (described above) and with previous identification of adducts (10), the three major adduct peaks were identified as anti-DMBADE-dGuo (peak I), syn-DMBADE-dAdo (peak II), anti-DMBADE-dAdo (peak III). Minor peaks included peak a, syn-DMBADE-dGuo, peak b, anti-DMBADE-dGuo, peak f, synDMBADE-dAdo, and peak g, syn-DMBADE-dAdo. These adducts have been well-characterized in the past (10, 21). Statistical Analysis. Regression analysis was performed using SPSS software, version 10 (SPSS, Inc., Chicago, IL). Analysis of covariance was performed as previously described (22) and as implemented by D. A. Johnston ([email protected]). All other data were analyzed by

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Figure 1. Structures of bergamottin, imperatorin, isopimpinellin, and 7,8-benzoflavone.

Figure 2. Effects of naturally occurring furanocoumarins on the formation of DMBA-DNA adducts in SENCAR mice. Furanocoumarins (100-3200 nmol) were applied five min prior to the addition of [3H]DMBA (10 nmol, 10 Ci/mmol). Control mice received acetone five min prior to [3H]DMBA. The data are expressed as specific activity (S. A., pmol of DMBA equivalents bound per mg DNA) (mean ( SE of at least three experiments). (*) P < 0.05 (Fischer’s PLSD test). analysis of variance followed by Fisher protected least significant difference (PLSD) test. Fisher PLSD tests were performed on a Macintosh computer with Statview 5.0 software (Altura Software, Inc.).

Results Effects of Naturally Occurring Furanocoumarins and 7,8-BF on DMBA-DNA Adduct Formation in SENCAR Mice. The effects of pretreatment with naturally occurring furanocoumarins and 7,8-BF (structures shown in Figure 1) on the formation of DMBA-DNA adducts in SENCAR mice were assessed (Figure 2). Imperatorin significantly (P e 0.05) inhibited total DNA adduct formation at all doses tested, by 43% to 81% (100 to 1600 nmol). Isopimpinellin also blocked DMBA-DNA


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Figure 4. Effects of coumarin pretreatment on the ratio of synto anti-DMBADE-DNA adducts in SENCAR mouse epidermis. Specific DMBA-DNA adducts were analyzed by HPLC as described in Experimental Procedures. Furanocoumarins (100800 nmol) were applied 5 min prior to the addition of [3H]DMBA (10 nmol, 10 Ci/mmol). Control mice received acetone five min prior to [3H]DMBA. The data are expressed as the ratio of the major syn-DMBADE-derived DNA adduct divided by the major anti-DMBADE-derived DNA adducts (mean ( SE of at least three experiments).

Figure 3. Representative radiochromatograms of DMBA-DNA adducts in SENCAR mouse epidermis. Furanocoumarins (400 nmol) or 7,8-BF (20 nmol) were applied 5 min prior to the addition of [3H]DMBA (10 nmol, 10 Ci/mmol). Control mice received acetone 5 min prior to [3H]DMBA. DNA hydrolysates were analyzed by HPLC and liquid scintillation spectroscopy as described in Experimental Procedures. Radioactive peaks were identified by comparison of retention times with authentic standards generated by nick translation (as described in Experimental Procedures). Peak a, syn-DMBADE-dGuo; peak b, anti-DMBADE-dGuo; peak I, anti-DMBADE-dGuo; peak II, synDMBADE-dAdo; peak III, anti-DMBADE-dAdo; peak f, synDMBADE-dAdo; and peak g, syn-DMBADE-dAdo. All chromatograms were normalized to the retention time of the internal standard, B[a]P-9,10-diol, which eluted at 53 min. Arrows (f) in panel C represent the positions of the radioactive standards, anti-DMBADE-dGuo, syn-DMBADE-dAdo, and anti-DMBADEdAdo.

adduct formation by up to 69%, at doses of g400 nmol. In contrast, lower doses (100-400 nmol) of bergamottin appeared to elevate DMBA-DNA adduct formation by up to 47% compared to the acetone-pretreated control, although this difference was not statistically significant. At the higher doses of bergamottin (800 to 1600 nmol), adduct levels were slightly lower than control, but these values were also not significantly different than the control value. Pretreatment with 7,8-BF blocked DMBADNA adduct formation by 13-46% (5-40 nmol). It was previously reported that a dose of 92 nmol of 7,8-BF blocked DMBA-DNA binding by 78% in the skin of female NIH Swiss mice (23). Effects of Naturally Occurring Furanocoumarins and 7,8-BF on Individual DMBA-DNA Adducts in SENCAR Mice. To further understand the effects of naturally occurring furanocoumarins on DMBA-DNA adduct formation, DNA hydrolysates were analyzed by HPLC after cleanup on Sephadex LH20 or Sep-pak cartridges. Figure 3 shows representative HPLC radiochromatograms of either the control group (mice pretreated with acetone vehicle and treated with DMBA) or groups pretreated with the different furanocoumarins or 7,8-BF. Bergamottin selectively reduced formation of anti-DMBADE-DNA adducts and increased levels of synDMBADE-DNA adducts, resulting in no overall inhibition of total DMBA-DNA adducts. In contrast, imperatorin and isopimpinellin inhibited the formation of DMBADNA adducts derived from both anti- and syn-DMBADE. Pretreatment with 7,8-BF reduced DNA adduct levels from both anti- and syn-DMBADE, but there was some selectivity for reduction of syn-DMBADE-DNA adducts (Figure 3). The dose-dependent effects of the furanocoumarins and 7,8-BF on the ratio of syn-DMBADE derived DNA adducts (peak II) compared to anti-DMBADE derived DNA adducts (peaks I and III) are further illustrated in Figure 4. The ratio of peak II/(peaks I + III) in the control group was 0.41. Bergamottin selectively inhibited peaks I and III and elevated peak II, resulting in a shift in this ratio to nearly 1.0, at the highest doses examined. Imperatorin and isopimpinellin inhibited formation of all three major

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Figure 5. Effects of naturally occurring furanocoumarins on DMBA metabolism to DMBA-3,4-diol (A) and DMBA-DNA adduct formation (B) in 10T1/2 cells. Cells were cultured as described in Experimental procedures. (A) Increasing concentrations of test compounds (0.4-10 µM) were co-incubated with DMBA (2 µM) for 24 h. Symbols are as follows: (O) 7,8-BF; (b), bergamottin; (9) isopimpinellin; (4) imperatorin. Figures represent the percentage of total radioactive peaks eluted. Data represent the average of two or three experiments (at 2 µM test compounds). Error bars represent means ( SE at the acetone control and the 2 µM dose of test compounds (n g 3). (B) Cells were co-incubated with DMBA (2 µM) and test compounds (2 µM each) for 24 h. Figures represent specific activities (S. A.) of total DMBA-DNA adduct levels (pmol/mg DNA) (means ( SE, n ) 4-7). (*) Significantly different from acetone control (P < 0.05) as determined by ANOVA followed by Fischer’s PLSD test.

DMBA-DNA adducts, and there was a modest increase in the ratio of peak II/(peaks I + III) with imperatorin pretreatment, but not with isopimpinellin. In contrast, 7,8-BF caused a decrease in the ratio of syn- to antiderived DMBADE-DNA adducts to 0.28, again supporting a preference for blocking the formation of synDMBADE-DNA adducts by this compound. Effects of Naturally Occurring Furanocoumarins on DMBA Metabolism and DNA Adduct Formation in 10T1/2 Cells. To further examine the effects of naturally occurring furanocoumarins on DMBA metabolism, 10T1/2 cells that predominantly express P450 1b1 (12, 13) were used. Equimolar concentrations (2 µM) of furanocoumarins or 7,8-BF were co-incubated with DMBA for 3, 6, or 24 h. Of the time-points tested, maximum DMBA metabolism was observed at 24 h (data not shown), so this time-point was selected for further studies. In cells treated with acetone and DMBA, the major organic solvent soluble peaks detected in media at 24 h were DMBA-8,9-diol, DMBA-3,4-diol, phenols, and DMBA which represented 23%, 13%, 12%, and 48% of the total peaks eluted (data not shown). Figure 5A

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illustrates the effects of the inhibitors on metabolism of DMBA to its corresponding 3,4-diol. At equimolar concentrations (2 µM), metabolism of DMBA to 8,9-diol, 3,4diol, and phenols was significantly (P < 0.05, Fisher’s PLSD test) inhibited by 45%, 44%, and 45% (imperatorin), 43%, 48%, and 48%, (isopimpinellin), and 83%, 100%, and 91% (7,8-BF), respectively (Figure 5A and data not shown). In contrast, co-incubation with 2 µM bergamottin was not effective at blocking DMBA metabolism (Figure 5A and data not shown). Inhibition of DMBA metabolism to DMBA-3,4-diol in 10T1/2 cells was dose-dependent (Figure 5A). Data were processed on a log scale using regression analysis followed by analysis of covariance. When examining the entire dose-response curve, all treatment groups were significantly different (P < 0.05) from the zero concentration (acetone control). In addition, the 7,8-BF group was significantly different (P < 0.05) from imperatorin, isopimpinellin, and bergamottin. The slopes of the lines were much steeper in the 7,8-BF, imperatorin, and isopimpinellin groups compared to bergamottin, with the 7,8-BF slope being the steepest. Notably, the most potent inhibitor tested, 7,8-BF, blocked DMBA metabolism by 92% at 0.4 µM (Figure 5A). With increasing doses of imperatorin and isopimpinellin (0.4-10 µM), DMBA-3,4diol formation was inhibited by up to 75% (Figure 5A). In contrast, a concentration of greater than 10 µM bergamottin was required to inhibit metabolism of DMBA to DMBA-3,4-diol by 50% (Figure 5A). At equimolar concentrations (2 µM), imperatorin and isopimpinellin significantly inhibited formation of DMBADNA adducts in 10T1/2 cells by 59% and 56%, respectively (Figure 5B). Bergamottin did not significantly block DNA adduct formation by DMBA (Figure 5B). However, 7,8-BF (2 µM) blocked DNA adduct formation by 98% (Figure 5B). Thus, effects of the test compounds on DMBA metabolism to the 3,4-diol in 10T1/2 cells correlated with their effects on DNA adduct formation. HPLC analysis of DNA hydrolysates revealed one major adduct (peak II) and several minor adducts (peaks a, b, I, III, f, and g) (Figure 6). The syn-DMBADE DNA adducts were more prominent than the anti-DMBADE DNA adducts (Figure 6). The ratio of peak II/(peaks I + III) in the acetone co-treated 10T1/2 cells was 1.2, compared to 0.4 observed in mouse skin (Figure 4). This ratio increased somewhat with imperatorin and isopimpinellin co-treatment. Imperatorin inhibited formation of peaks I, II, and III by 69%, 41%, and 56%, respectively; isopimpinellin inhibited formation of peaks I, II, and III by 56%, 32%, and 51%, respectively. In contrast, at the dose tested (2 µM), bergamottin did not significantly block formation of any of the major DMBA-DNA adducts (Figures 5B and 6). There were no DMBA-DNA adducts detected by HPLC in cells co-treated with DMBA and 7,8BF (Figure 6). Thus, the effects of furanocoumarins and 7,8-BF on individual DMBA-DNA adducts corresponded to the overall effects on total adducts, shown in Figure 5B. Effects on Naturally Occurring Furanocoumarins and 7,8-BF on DMBA Metabolism and DNA Adduct Formation in Hepa-1 Cells. In further experiments, the effects of naturally occurring furanocoumarins on DMBA metabolism were examined in Hepa-1 cells, which predominantly express P450 1a1 (12, 13). Equimolar concentrations (2 µM) of furanocoumarins or 7,8-BF were co-incubated with DMBA for 1.5, 3, 6, or 24 h. Metabolism



Figure 7. Effects of naturally occurring furanocoumarins on DMBA metabolism to DMBA-3,4-diol (A) and DMBA-DNA adduct formation (B) in Hepa-1 cells. Cells were cultured as described in Experimental Procedures. (A) Increasing concentrations of test compounds (0.4-10 µM) were co-incubated with DMBA (2 µM) for 6 h. Symbols are as follows: (O) 7,8-BF; (b) bergamottin; (0) isopimpinellin; (4) imperatorin. Figures represent the percent of total radioactive peaks eluted. Data represent the average of two experiments, or three experiments (at 2 µM test compounds). Error bars represent means ( SE at the acetone control and the 2 µM dose of test compounds (n g 3). (B) Cells were co-incubated with DMBA (2 µM) and test compounds (2 µM each) for 24 h. Figures represent specific activities (S.A.) of total DMBA-DNA adduct levels (pmol/mg DNA) (means ( SE, n ) 4-7). (*) Significantly different from acetone control (P < 0.05) as determined by ANOVA followed by Fischer’s PLSD test.

Figure 6. Representative radio-chromatogram of DMBA-DNA adducts in 10T1/2 cells. Peak a, syn-DMBADE-dGuo; peak b, anti-DMBADE-dGuo; peak I, anti-DMBADE-dGuo; peak II, synDMBADE-dAdo; peak III, anti-DMBADE-dAdo; peak f, synDMBADE-dAdo; and peak g, syn-DMBADE-dAdo. All chromatograms were normalized to the retention time of the internal standard, B[a]P-9,10-diol, which eluted at 53 min.

of DMBA into organic solvent soluble metabolites increased with each time point tested, reaching a maximum at 6 h (data not shown). At 24 h, the DMBA peak was absent, and DMBA-3,4-diol was not detected (data not shown). Therefore, the 6 h time point was selected for further studies. In cells treated with acetone and DMBA, the organic solvent soluble peaks identified in media at 6 h were DMBA-8,9-diol, DMBA-3,4-diol, phenols, and DMBA which represented 34%, 0.9%, 1.1%, and 46% of the total peaks eluted (data not shown). Figure 7A illustrates the effects of the test compounds on metabolism of DMBA to its corresponding 3,4-diol. At equimolar concentrations, metabolism of DMBA to 8,9-diol, 3,4-diol, and phenols was significantly (P < 0.05, Fisher’s PLSD

test) inhibited by 92%, 87%, and 77% (bergamottin) and 42%, 25%, and 63%, (imperatorin), respectively (Figure 7A and data not shown). In contrast, co-incubation with either isopimpinellin or 7,8-BF failed to block DMBA metabolism (Figure 7A and data not shown). Data on the effects of increasing doses of test compounds on DMBA metabolism to DMBA-3,4-diol in Hepa-1 cells (Figure 7A) were also processed on a log scale using regression analysis followed by analysis of covariance. When examining the entire dose-response curve, only the bergamottin and imperatorin groups were significantly different (P < 0.05) from the zero concentration (acetone control). In addition, the bergamottin group, which had the steepest curve, was significantly different (P < 0.05) from isopimpinellin. Imperatorin was also significantly different (P < 0.05) from isopimpinellin. With increasing doses of bergamottin and imperatorin, up to 90% and 80%, respectively, of DMBA-3,4-diol formation was inhibited in Hepa-1 cells (0.4-10 µM) (Figure 7A). In contrast, even at concentrations of 10 µM, isopimpinellin, and 7,8-BF did not significantly block metabolism of DMBA to DMBA-3,4-diol.

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II/(I + III) of ∼0.15. This ratio did not change appreciably with inhibitor co-treatment. Inhibition of individual DNA adducts corresponded to overall inhibition of total DNA adducts. Individual peaks (I-III) were inhibited by 100% (bergamottin), 65-73% (imperatorin), and 41-44% (isopimpinellin). Consistent with the lack of effect on total DMBA-DNA adduct formation, 7,8-BF did not significantly block formation of any of the individual DMBADNA adducts.

Discussion

Figure 8. Representative radio-chromatogram of DMBA-DNA adducts in Hepa-1 cells. Peak a, syn-DMBADE-dGuo; peak b, anti-DMBADE-dGuo; peak I, anti-DMBADE-dGuo; peak II, synDMBADE-dAdo; peak III, anti-DMBADE-dAdo. All chromatograms were normalized to the retention time of the internal standard, B[a]P-9,10-diol, which eluted at 53 min.

At equimolar concentrations, bergamottin, imperatorin, and isopimpinellin significantly inhibited formation of DMBA-DNA adducts in Hepa-1 cells by 91%, 50%, and 45%, respectively (Figure 7B). In contrast, 7,8-BF did not significantly reduce total DNA adduct formation by DMBA (Figure 7B). Thus, with the exception of isopimpinellin, the effects of the test compounds on DMBA metabolism in Hepa-1 cells correlated with their effects on DMBA-DNA adduct formation. HPLC analysis of DNA hydrolysates revealed two major DNA adducts, both of which were anti-DMBADE-derived (peaks I and III) (Figure 8). In contrast to the 10T1/2 cells (Figure 6) and in vivo in epidermis of SENCAR mice (Figure 3), peak II represented a smaller proportion of total adducts formed in Hepa-1 cells (Figure 8), resulting in a ratio of peak

The current study was designed to further characterize the effects of bergamottin, imperatorin, and isopimpinellin on DMBA metabolism and DNA adduct formation and to understand the mechanistic basis for their differential effects on PAH skin tumor initiation in SENCAR mice. In previous work from our laboratory, imperatorin and isopimpinellin were found to inhibit skin tumor initiation by both B[a]P and DMBA, whereas bergamottin inhibited tumor initiation by B[a]P, but not DMBA, and actually enhanced DMBA tumor initiation at the lower doses tested (11). In addition, 7,8-BF was used for comparison since previous work from several laboratories had shown this compound to be an effective inhibitor of DMBA skin tumor initiation but not B[a]P skin tumor initiation (24, 25). The results of this study indicate that (i) bergamottin is a selective inhibitor of mouse P450 1a1 whereas imperatorin and isopimpinellin are effective inhibitors of both P450 1a1 and 1b1 with some preference for the latter P450 (especially isopimpinellin); (ii) formation of DMBA-3,4-diol is catalyzed mainly by P450 1b1 in mouse epidermis although P450 1a1 can also catalyze this reaction to a lesser extent; (iii) formation of antiDMBADE-DNA adducts following DMBA-3,4-diol production is primarily a result of P450 1a1 metabolism whereas formation of syn-DMBADE-DNA adducts is primarily a result of P450 1b1 metabolism; (iv) 7,8-BF is a highly selective inhibitor of mouse P450 1b1; and (v) the differential effects of bergamottin, imperatorin, isopimpinellin, and 7,8-BF on skin tumor initiation by B[a]P and DMBA can be explained by their differential inhibitory effects on mouse P450 1a1 and 1b1. The data from the current study also provide evidence for an important role of both P450 1a1 and 1b1 in the metabolic activation of DMBA to DNA binding intermediates in mouse skin. In SENCAR mouse epidermis, the effects of pretreatment with furanocoumarins on total DMBA-DNA adducts corresponded with their effects on skin tumor initiation in SENCAR mice (11, and unpublished data). In this regard, pretreatment with imperatorin and isopimpinellin inhibited, whereas bergamottin had no statistically significant effect on DMBA-DNA adduct formation in epidermis of SENCAR mice. Analysis of DMBA-DNA adduct profiles revealed some interesting findings. Bergamottin appeared to selectively block formation of anti-DMBADE-DNA adducts and increased formation of syn-DMBADE-DNA adducts. For comparison, we also pretreated mice with 7,8-BF. A dose of 7,8BF was selected which would inhibit total DMBA-DNA adducts by less than 50% because previous reports had shown no differential effect of 7,8-BF on individual DMBA-DNA adducts (23). However, the doses used (92367 nmol) in these earlier experiments were high enough that almost complete inhibition of total DMBA-DNA


adducts was achieved (23). At the lower doses (5-40 nmol) of 7,8-BF used in the current study, we observed some selectivity for inhibition of syn-DMBADE-DNA adducts, causing a decrease in the ratio of syn-/antiderived DNA adducts from 0.41 in the acetone control to 0.28 in the 7,8-BF group. On the basis of these observations, we hypothesized that differential effects of the furanocoumarins and 7,8-BF on skin tumor initiation with DMBA and/or B[a]P were the result of compound selective effects on specific mouse epidermal P450s. To further explore the effects of the furanocoumarins on mouse P450s involved in DMBA metabolic activation and DNA binding, we employed mouse 10T1/2 cells and Hepa-1 cells. The 10T1/2 cells predominantly express P450 1b1 and have negligible P450 1a1 activity (12, 13, 26-29). In contrast, Hepa-1 cells predominantly express P450 1a1, with negligible levels of P450 1b1 (30). The regioselectivity of DMBA metabolism in 10T1/2 cells differs from that in Hepa-1 cells (12, 13, 26-28). Our findings were consistent with these earlier reports, in that DMBA-3,4-diol was a major metabolite of 10T1/2 cells incubated with DMBA, but only a relatively minor metabolite of Hepa-1 cells (Figures 5 and 7). When the various compounds were tested in these cell culture systems, imperatorin and isopimpinellin were generally found to block formation of DMBA-3,4-diol, supporting the hypothesis that these compounds inhibit both P450 1a1 and 1b1 (although isopimpinellin was less effective than imperatorin at blocking DMBA-3,4-diol formation in Hepa-1 cells). In contrast, bergamottin was less effective at blocking DMBA-3,4-diol formation in 10T1/2 cells, whereas it was a potent inhibitor of DMBA-3,4-diol formation in Hepa-1 cells. These data support the hypothesis that bergamottin is a relatively specific inhibitor of mouse P450 1a1 in Hepa-1 cells. In contrast to bergamottin, 7,8-BF had no effect on DMBA-3,4-diol formation in Hepa-1 cells but was a very potent inhibitor of DMBA-3,4-diol formation in 10T1/2 cells, suggesting that 7,8-BF is a relatively selective inhibitor of mouse P450 1b1 in 10T1/2 cells. The effects of all these compounds on formation of total DMBA-DNA adducts correlated with their effects on DMBA-3,4-diol formation in both cell types, with the exception of the effects of isopimpinellin in Hepa-1 cells. In this regard, isopimpinellin may block the conversion of DMBA-3,4-diol to its corresponding diol-epoxide in Hepa-1 cells, thereby blocking the formation of DMBA-DNA adducts. Further analyses of specific DMBA-DNA adducts formed in 10T1/2 and Hepa-1 cells yielded some very interesting results. Whereas both syn- and anti-DMBADE derived DNA adducts were formed in mouse epidermis, there appeared to be preferential formation of syn-DMBADEDNA adducts after treatment of DMBA in 10T1/2 cells, which predominantly express P450 1b1. Similar to our results with 10T1/2 cells, syn-DMBADE-dAdo was also the major DNA adduct formed in bone marrow cells of wild-type mice treated with DMBA (31), in which the stromal cells exclusively express P450 1b1 (29). In contrast, DMBA-DNA adducts were negligible in bone marrow cells of Cyp1b1(-/-) knockout mice (31). Taken together, these observations suggest that murine P450 1b1 is capable of activating DMBA-3,4-diol into predominantly syn-DMBADE. In contrast, Hepa-1 cells, which predominantly express P450 1a1, formed mainly antiDMBADE-DNA adducts. Previous evidence has suggested that formation of anti-DMBADE-DNA adducts


Figure 9. Proposed mechanisms of selective anti-carcinogenesis by bergamottin versus imperatorin, isopimpinellin, and 7,8BF in DMBA treated mouse skin.

required induction of P450(s) such as 1a1 (32, 33). Formation of these adducts was inhibited by bergamottin, which from our studies, appeared to be a fairly specific P450 1a1 inhibitor. In contrast, 7,8-BF, which appeared to be a potent and selective inhibitor of mouse P450 1b1 had no effect on formation of anti-DMBADE-DNA adducts in Hepa-1 cells. Taken together these observations suggest that P450 1a1 is primarily responsible for converting DMBA-3,4-diol to anti-DMBADE. On the basis of the data in our current and previous studies, and other data in the literature, we offer the following model for involvement of mouse P4501 family members in metabolic activation of DMBA and in the action of naturally occurring furanocoumarins toward DMBA metabolic activation in mouse epidermis. As shown in Figure 9, DMBA is metabolized to DMBA-3,4diol primarily by P450 1b1, although P450 1a1 can also catalyze this reaction but to a much lesser extent. 7,8BF, isopimpinellin, and imperatorin can effectively block this reaction whereas bergamottin does not. Consequently, 7,8-BF, isopimpinellin, and imperatorin block formation of both anti- and syn-DMBADE-DNA adducts and block DMBA skin tumor initiation. Several isoforms of P450 have been detected either constitutively or are inducible in mouse skin, including P450 1a1/1a2 and 1b1 (34-36). 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 antiDMBADE whereas P450 1b1 is primarily responsible for converting DMBA-3,4-diol to syn-DMBADE. Bergamottin, which is highly selective for inhibiting P450 1a1 showed preferential inhibition of anti-DMBADE-DNA adducts while actually enhancing syn-DMBADE-DNA adducts in mouse epidermis, supporting this conclusion. Our results indicate that 7,8-BF is a potent and selective inhibitor of P450 1b1 compared to P450 1a1 in mouse cells. The preference of 7,8-BF for P450 1b1 may explain the older data showing the lack of effect of this compound on B[a]P tumor initiation (24, 25). 7,8-BF is reportedly 10 times more potent at inhibiting EROD activity using recombinant human P450 1B1 compared to P450 1A1 or 1A2 (37). When we examined the effects of 7,8-BF at low doses, we found some preference for inhibition of synDMBADE-DNA adducts as expected based on the model proposed in Figure 9. However, the effects of 7,8-BF are complicated by several factors. First, if P450 1b1 also mediates the first oxidation step of DMBA, then 7,8-BF blocks conversion of DMBA to DMBA-3,4-diol, thereby reducing all further metabolism of DMBA to both diolepoxides. Furthermore, 7,8-BF can also act as a partial agonist of the Ah receptor (38). Studies in progress with

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Cyp1a1(-/-) and Cyp1b1(-/-) knockout mice will help to further delineate differences in the role of specific P4501 family members in metabolism and metabolic activation of DMBA. In conclusion, our current results have increased our understanding of the mechanism(s) for differences in the abilities of bergamottin, imperatorin, and isopimpinellin to block skin tumor initiation in mice. These results also suggest that P450 1a1 and P450 1b1 may mediate the formation of anti- and syn-DMBADE, respectively (Figure 9). On the basis of our current results, it appears that compounds that can inhibit both P450 1a1 and 1b1 are the most effective inhibitors of PAH-DNA adduct formation and tumor initiation in mouse epidermis. Imperatorin and isopimpinellin possess these properties and appear to be good candidates for further anti-carcinogenesis studies. P450 1A1 is expressed in extra-hepatic tissues and is induced in humans in many tissues, including the lung and placenta following exposure to inducers such as cigarette smoke (39, 40). P450 1A1 is important in the metabolism of carcinogens, particularly B[a]P and other PAH. Human P450 1B1 catalyzes the activation of a number of diverse pro-carcinogens (41), and is expressed in a variety of extra-hepatic sites, including steroid responsive and steroidogenic tissues (13, 26, 42). Future studies will focus on the effects of naturally occurring furanocoumarins on inhibition of carcinogen metabolism by human P450.

Acknowledgment. WeacknowledgeDennisA.Johnston and Facility Core 6 (Biostatistics and Data Processing) of the Center for Research on Environmental Disease. H.K. was a recipient of the Research Training in Carcinogenesis and Mutagenesis fellowship (CA09480). This research was supported by NCI Grant CA 79442 (J.D.), UTMDACC Core Grant CA 16672, and UTMDACC Science Park-Research Division NIEHS Center Grant ES07783.

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