Effects of benzyl isothiocyanate and phenethyl isothiocyanate on benzo[a]pyrene metabolism and DNA adduct formation in the A/J mouse
Kristina R.K. Sticha,
Marianne E. Staretz,
Mingyao Wang,
Hong Liang,
Patrick M.J. Kenney and
Stephen S. Hecht1
University of Minnesota Cancer Center, Box 806 Mayo, Minneapolis, MN 55455, USA
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Abstract
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Benzyl isothiocyanate (BITC) inhibits lung tumorigenesis induced in A/J mice by benzo[a]pyrene (B[a]P). In contrast, phenethyl isothiocyanate (PEITC) does not. We tested the hypothesis that BITC inhibits B[a]P tumorigenicity in mouse lung by inhibiting DNA adduct formation, and compared the effects of BITC and PEITC. In mouse liver or lung microsomal incubations, BITC and PEITC inhibited formation of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (B[a]P-7,8-diol) and some other B[a]P metabolites. The metabolism of B[a]P was compared in mouse lung and liver microsomes, 6 or 24h after treatment with BITC or PEITC. In lung, 6 h after treatment, B[a]P-7,8-diol and some other metabolites were inhibited by BITC and PEITC. However, 24 h after treatment, no inhibition of B[a]P-7,8-diol was observed in microsomes from BITC-treated mice, whereas it was substantially increased in mice treated with PEITC. Effects on B[a]P metabolism in liver microsomes were generally modest. Conversion of B[a]P-7,8-diol to mutagens by mouse liver microsomes was more strongly inhibited by BITC than PEITC. Effects on 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA adduct formation were evaluated in DNA from mice treated with isothiocyanates and B[a]P, and killed 2-120h later. The area under the curve (AUC) for BPDE-DNA adducts in lung was 29.5% less (P = 0.001) in the BITC-B[a]P treated mice and 19.0% less (P = 0.02) in the PEITC-B[a]P mice than in the mice treated with B[a]P alone. Similar results were obtained in liver DNA. There were no significant differences between the reduction of BPDE-DNA AUC values by BITC versus PEITC. The results of this study support the hypothesis that BITC inhibits B[a]P-induced lung tumorigenesis in A/J mice by inhibiting the metabolic activation of B[a]P to BPDE-DNA adducts. However, differences in BPDE-DNA adduct formation do not appear to explain fully the contrasting effects of BITC and PEITC on B[a]P-induced lung tumorigenesis.
Abbreviations: 1-OH-B[a]P, 1-hydroxybenzo[a]pyrene; 3-OH-B[a]P, 3-hydroxybenzo[a]pyrene; 9-OH-B[a]P, 9-hydroxybenzo[a]pyrene; AUC, area under the curve; B[a]P, benzo[a]pyrene; B[a]P-4,5-diol, 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene; B[a]P-7,8-diol, 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene; B[a]P-9,10-diol, 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene; B[a]P-quinones, benzo[a]pyrene-1,6-quinone/benzo[a]pyrene-3,6-quinone/benzo[a]pyrene-6,12-quinone; B[a]P-triol, r-7,t-8,c-9-trihydroxy-7,8,9,10-dihydrobenzo[a]pyrene; BITC, benzyl isothiocyanate; BPDE, 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; PAHs, polycyclic aromatic hydrocarbons; PEITC, phenethyl isothiocyanate.
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Introduction
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Lung cancer is the leading cause of cancer death in the USA, with over 150 000 deaths expected in 2000 (1). Approximately 87% of lung cancer is caused by cigarette smoking (1). Worldwide, over 500000 lung cancer deaths in developed countries were caused by smoking in 1995 (2). While it is crucial to prevent addiction to tobacco, and to enhance the efficacy of smoking cessation and reduction programs, these approaches have had little impact in the 1990s, when overall smoking prevalence rates in the USA have remained essentially constant (1). Chemoprevention is one approach to decreasing lung cancer risk in addicted smokers.
Our goal is to develop chemopreventive agents that are effective against lung carcinogens in tobacco smoke (3). Polycyclic aromatic hydrocarbons (PAHs), typified by benzo[a]pyrene (B[a]P), and the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are considered to be important carcinogens involved in lung cancer induction in smokers (4). In animal models, isothiocyanates are effective chemopreventive agents against lung tumors induced by B[a]P or NNK (5,6). However, efficacy is highly dependent on the structure of the isothiocyanate. Benzyl isothiocyanate (PhCH2N=C=S; BITC) inhibits lung tumor induction by B[a]P in mice, whereas phenethyl isothiocyanate (PhCH2CH2N=C=S; PEITC) is ineffective (710). In contrast, PEITC is a strong inhibitor of mouse lung tumorigenesis induced by NNK, but BITC is ineffective (6).
Two studies have examined BITC as an inhibitor of B[a]P-induced lung tumorigenesis in A/J mice. Wattenberg administered BITC, at doses of 16.8 or 6.7 µmol, by gavage 15 min prior to gavage of B[a]P (7.9 µmol) (7). This sequence was repeated three times at 2 week intervals. In mice treated with B[a]P only, there were 15.5 lung tumors per mouse. This was decreased to 7.4 and 3.9 lung tumors per mouse in the animals pre-treated with the 6.7 and 16.8 µmol doses of BITC, respectively. We used the same protocol and compared the efficacy of BITC and PEITC, each at doses of 7.9 µmol (8). In the mice treated with B[a]P only, there were 4.8 lung tumors per mouse. This was significantly reduced to 2.6 lung tumors per mouse by BITC, whereas PEITC had no effect. Two other experiments in A/J mice have examined the effect of PEITC on B[a]P-induced lung tumorigenesis (9,10). In one, PEITC was administered by gavage before and after a single i.p. dose of B[a]P while in the other PEITC was given in the diet before and during eight weekly B[a]P administrations by gavage. In neither case was any inhibition of lung tumorigenesis observed.
The results of these studies suggest that BITC could inhibit the lung tumorigenicity of B[a]P by inhibiting its metabolic activation to DNA adducts, or by enhancing its detoxification. An overview of B[a]P metabolism is presented in Figure 1
(11). Initial oxidation catalyzed by a variety of cytochromes P450 produces a series of arene oxides and phenols. The arene oxides can rearrange to phenols or undergo hydration catalyzed by epoxide hydrolase yielding 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene (B[a]P-9,10-diol), 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (B[a]P-7,8-diol) and 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (B[a]P-4,5-diol). B[a]P-7,8-diol is further oxidized to anti- and syn-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Among the four BPDE enantiomers produced in these reactions, the (7R,8S,9S,10R)-enantiomer of anti-BPDE, illustrated in Figure 1
, is generally formed to the greatest extent and shows the highest carcinogenic activity (1114). This diol epoxide reacts with DNA in vitro and in vivo producing a major adduct in which the exocyclic amino group of deoxyguanosine undergoes trans-addition to carbon 10 of BPDE (15,16). Convincing evidence supports the hypothesis that formation of this and related minor DNA adducts is the major metabolic activation pathway of B[a]P (1116). Thus, B[a]P-7,8-diol is a proximate carcinogen of B[a]P whereas BPDE enantiomers are major ultimate carcinogens.
One goal of the present study was to test the hypothesis that BITC inhibits B[a]P tumorigenicity in mouse lung by inhibiting DNA adduct formation. A second goal was to compare the effects of BITC and PEITC on B[a]P metabolism and DNA binding to determine whether their contrasting effects on lung tumorigenicity could be explained.
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Materials and methods
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Chemicals
[G-3H]B[a]P (30 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). It was purified on the day of use by passage through a short column of silica, with hexane as the eluent. Hexane was removed under a stream of N2. HPLC analysis indicated a radiochemical purity of >99.4%. Purified [G-3H]B[a]P was diluted with unlabeled B[a]P obtained from Aldrich Chemical Co. (Milwaukee, WI). All procedures involving B[a]P were performed under subdued light. B[a]P metabolite standards were obtained from the National Cancer Institute Chemical Carcinogen Reference Standard Repository, Midwest Research Institute (Kansas City, MO). BITC and PEITC were procured from Aldrich. All biochemical reagents were purchased from Sigma Chemical Co. (St Louis, MO). OmniSolv H2O for DNA hydrolysis and HPLC analysis was obtained from EM Science (Gibbstown, NJ). Optima grade methanol for HPLC was obtained from Fisher Scientific (Springfield, NJ).
Animal studies
Female A/J mice were obtained at age 7 weeks from Jackson Laboratories (Bar Harbor, ME). They were housed under standard conditions and maintained on AIN-76A modified diet, as described (8). For the microsomal metabolism experiments involving treatment with isothiocyanates, groups of 15 mice each were treated with BITC or PEITC (6.7 µmol) in 0.2 ml cottonseed oil, by gavage, then killed 6 or 24 h later. Liver and lung were isolated and microsomes were prepared as described (17). Five lungs each were used to prepare three pools of lung microsomes. Liver microsomes were prepared from individual tissues.
For the DNA adduct studies, there were 24 groups of four mice each. Isothiocyanates and B[a]P were administered by gavage in 0.2 ml cottonseed oil. At age 9 weeks, mice in groups 18 were treated with 0.2 ml cottonseed oil by gavage, followed 15 min later by treatment with B[a]P (7.9 µmol). Mice were killed 2, 6, 12, 24, 48, 72, 96 and 120h after B[a]P treatment. In groups 916, mice were treated with PEITC (6.7 µmol), and in groups 1724 with BITC (6.7 µmol). Fifteen minutes later, mice in groups 924 were gavaged with B[a]P (7.9 µmol) and killed at the same intervals as above. DNA was isolated as described below.
Microsomal incubations
For the IC50 studies, incubation mixtures (total volume 0.5 ml) contained [G-3H]B[a]P (10 µM, 1.0 µCi) added in 5 µl DMSO, 100 mM potassium phosphate (pH 7.4), 3 mM MgCl2, 1 mM EDTA, an NADPH generating system (5 mM glucose-6-phosphate, 1 mM NADP+ and 1.5 U of glucose-6-phosphate dehydrogenase) and 400 µg liver or lung microsomal protein. Various concentrations of BITC or PEITC were added in 5 µl DMSO. The mixture was incubated at 37°C for 20 min. Reactions were terminated by placing samples on ice and adding an equal volume of acetone. Two volumes of ethyl acetate and the B[a]P metabolite standards were added, and the mixture was vortexed for 2 min. The organic layer was removed and the aqueous layer was extracted two more times with 2 vol ethyl acetate. The combined organic layers were dried in vacuo, redissolved in 70:30 methanol:H2O and analyzed by HPLC with radioflow detection as described previously (18). Each incubation was carried out at least in triplicate. IC50 values were estimated from a plot of percent inhibition versus inhibitor concentration.
Studies with microsomes isolated from mice pre-treated with BITC or PEITC were carried out the same way. Three pools of lung microsomes were used and each incubation was carried out in duplicate. Three liver microsomal samples were each analyzed in duplicate.
Liver microsomes were used for activation of B[a]P-7,8-diol in the umu assay as described previously (19).
Isolation of DNA from tissues
DNA was isolated by modifications of the Kirby method (20). In brief, the tissues were homogenized in 50 mM Tris, 1 mM EDTA pH 7.4, and incubated with 1% sodium dodecyl sulfate and proteinase K (300 µg/ml). The homogenates were extracted twice with phenol/chloroform/isoamyl alcohol (25:24:1) and then once with chloroform/isoamyl alcohol (24:1). DNA was precipitated with ethanol and dissolved in 15 mM sodium citrate, 15 mM sodium chloride, 1 mM EDTA, pH 7.4. The DNA was incubated with RNase A (100 µg/ml) and RNase T1 (2000 U), extracted once with chloroform/isoamyl alcohol and precipitated with ethanol. Absorbance ratios at 260/230 and 260/280 nm of DNA were 2.31 ± 0.17 (n = 382) and 1.74 ± 0.05 (n = 382), respectively. About 100 µg of each DNA sample were used for fluorometric analysis of B[a]P-tetraols and G.
Hydrolysis of DNA
Prior to hydrolysis, lung or liver DNA samples were analyzed for unbound B[a]P-tetraols. The portion of DNA to be hydrolyzed was rinsed with 100% ethanol, the ethanol was removed and mixed with H2O to make a final concentration of 20% ethanol. The ethanol wash was analyzed for B[a]P-tetraols as described below. The DNA, free of unbound B[a]P-tetraols, was dissolved in H2O and the DNA concentration was determined by UV to ensure sufficient purity and amount of DNA for analysis. The DNA was hydrolyzed as described previously (21,22) by incubation at 80°C for 4 h in a final concentration of 0.1 N HCl. This releases tetraols from BPDEDNA adducts with 90% recovery (21). The structures of the released tetraols are illustrated in Figure 2
. We did not find it necessary to purify the HCl prior to use. The hydrolyzed DNA was stored at 20°C until analyzed by HPLC/fluorescence. Silanized, amber, high recovery autosampler vials were prepared so that 75 µg DNA would be injected in a 900 µl injection. Internal standard, B[a]P-triol, was added to each vial and the total volume in the autosampler vial was brought to 1.0 ml with H2O. The samples in the autosampler vials were first analyzed for B[a]P-tetraols and then for G. Levels of B[a]P-tetraols released from mouse lung and liver DNA were expressed as pmol/µmol G.
Fluorometric analysis of B[a]P-tetraols
The method used in this study is a modification of that described previously (21,23). HPLC analysis was performed with a Hewlett Packard Series 1100 autosampler coupled to a Waters Millipore automated gradient controller and model 510 solvent delivery system. All solvents were sonicated under vacuum prior to use and continuously sparged with high purity He throughout the analyses. The B[a]P-tetraols were concentrated by an initial 10 min isocratic elution of 35% methanol in H2O over a guard column and a 4.6 mmx4.5 cm Beckman Ultrasphere ODS precolumn at 1.0 ml/min. The concentrated B[a]P-tetraols were then eluted from the precolumn by a 30 min, 35100% methanol/H2O linear gradient. The gradient solvents were switched by a Valco Instruments switching valve to flow over a 4.6 mmx25 cm Beckman Ultrasphere ODS analytical column to separate the four B[a]P-tetraol isomers. The B[a]P-tetraols were detected by a Shimadzu RF-10AXL fluorescence detector linked to a Hitachi D-7500 Integrator. The fluorescence excitation wavelength was set at 344 nm and the emission wavelength at 398 nm. The level of each B[a]P-tetraol isomer was determined by comparison with a standard curve generated from the fluorescence areas of authentic B[a]P-tetraol standards analyzed prior to and immediately following the analysis of each set of samples. At 12 h intervals throughout the analysis of a set of samples, the fluorescence area of an authentic B[a]P-tetraol standard was analyzed to validate the standard curve.
Fluorometric analysis of G
This method is similar to that described previously (24). G from about 2 µg DNA per injection was eluted from two Whatman Partisil 10 SCX columns in tandem, isocratically, with 100mM ammonium phosphate, pH2.0, at 1.0ml/min. Buffer was filtered through a 0.2µm nylon membrane and sonicated under vacuum prior to use. G was detected with the fluorescence excitation wavelength set at 286 nm and the emission wavelength at 366 nm. The level of G in each sample was determined by comparison with a standard curve generated from the fluorescence areas of authentic G standards analyzed at 12 h intervals throughout the analysis of a set of samples. Determinations were carried out in duplicate, with variations typically <2%.
Statistical analysis
Comparisons between metabolite levels in microsomes from mice treated with vehicle only, BITC or PEITC were made using Student's t-test. Areas under the curve (AUC) were analyzed by analysis of variance. When the overall F-test was significant, post-hoc t-tests were conducted to determine which of the AUC differed. A P-value of 0.05 or less was considered statistically significant.
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Results
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IC50 values were determined for inhibition of B[a]P metabolism by BITC or PEITC in mouse lung or liver microsomes. These data are summarized in Table I
. In lung, BITC strongly inhibited formation of B[a]P-7,8-diol, B[a]P-4,5-diol and 1-hydroxybenzo[a]pyrene (1-OH-B[a]P), all with IC50 < 15 µM. Moderate inhibitory effects (IC50 = 1530 µM) were observed for B[a]P-9,10-diol, 3-hydroxybenzo[a]pyrene (3-OH-B[a]P) and B[a]P-quinones. PEITC strongly inhibited (IC50 < 15 µM) the formation of all B[a]P metabolites in lung, except B[a]P-quinones and 9-hydroxybenzo[a]pyrene (9-OH-B[a]P). The inhibitory effects of PEITC were stronger than those of BITC for B[a]P-9,10-diol, B[a]P-4,5-diol, 1-OH-B[a]P and 3-OH-B[a]P. The inhibitory effects of BITC and PEITC on B[a]P metabolism in liver were less pronounced than in lung except for 9-OH-B[a]P. Whereas the effects of PEITC were consistently greater than those of BITC, inhibition was relatively strong only in the case of B[a]P-4,5-diol.
Lung and liver microsomes were isolated from mice killed 6 or 24 h after treatment with 6.7 µmol BITC or PEITC. These microsomes were used for metabolism of B[a]P. The results for lung at 6 h are illustrated in Figure 3A
. B[a]P-4,5-diol, B[a]P-7,8-diol, B[a]P-quinones, 1-OH-B[a]P and 3-OH-B[a]P were all significantly inhibited by both BITC and PEITC. BITC inhibited B[a]P-4,5-diol by 29%, B[a]P-7,8-diol by 44%, B[a]P-quinones by 50%, 1-OH-B[a]P by 42% and 3-OH-B[a]P by 43%. PEITC inhibited B[a]P-4,5-diol by 46%, B[a]P-7,8-diol by 28%, B[a]P-quinones by 54%, 1-OH-B[a]P by 53% and 3-OH-B[a]P by 52%. Small but significant opposing effects were seen on B[a]P-9,10-diol, which was enhanced by BITC but inhibited by PEITC. There were no effects on 9-OH-B[a]P.

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Fig. 3. Formation of B[a]P metabolites in mouse lung (A and B) and liver (C and D) microsomes, 6 (A and C) or 24 h (B and D) after treatment by gavage with vehicle (open bars), BITC (hatched bars), or PEITC (cross-hatched bars). *, P < 0.05; **, P < 0.005, compared with vehicle control. The data represent means ± SD for six values.
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In lung, 24 h after treatment, the picture was quite different (Figure 3B
). The significant inhibitory effects of BITC on B[a]P-4,5-diol, B[a]P-quinones, 1-OH-B[a]P and 3-OH-B[a]P persisted. However, no inhibition of B[a]P-7,8-diol formation was observed. PEITC significantly enhanced levels of B[a]P-9,10-diol (by 83%) and B[a]P-7,8-diol (by 139%), whereas its inhibitory effects on other metabolites persisted.
In liver, 6 h after treatment, the effects of both BITC and PEITC were generally modest (Figure 3C
). BITC significantly enhanced the formation of B[a]P-7,8-diol (46%), B[a]P-quinones (36%), 9-OH-B[a]P (17%), 1-OH-B[a]P (23%) and 3-OH-B[a]P (32%). PEITC significantly inhibited B[a]P-9,10-diol (22%), B[a]P-4,5-diol (24%), 1-OH-B[a]P (14%) and 3-OH-B[a]P (36%). In liver, 24 h after treatment, there were no significant effects other than slight enhancement of B[a]P-9,10-diol and 9-OH-B[a]P by PEITC (Figure 3D
).
The effects of various concentrations of isothiocyanates on mouse liver microsomal metabolism of B[a]P-7,8-diol to mutagenic products was examined using the umu gene response in the chimeric plasmid pSK1002, carried in Salmonella typhimurium TA 1535, as an endpoint. The results, which are summarized in Figure 4
, demonstrate that BITC was more effective than PEITC as an inhibitor of mutagenic response in this system, presumably resulting from inhibition of BPDE formation.

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Fig. 4. Effects of BITC ( ) or PEITC () on A/J mouse liver microsomal metabolism of B[a]P-7,8-diol to mutagenic products, as determined by umu gene response.
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The effects of BITC and PEITC on BPDEDNA adduct formation in mouse lung and liver were examined by a fluorometric method (21,23). B[a]P-tetraols were released from DNA by acid hydrolysis (Figure 2
). The hydrolysis was carried out at 80°C, rather than 90°C, to reduce decomposition of cis-syn-B[a]P-tetraol (22). The hydrolysates were analyzed essentially as described (21,23) with two major modifications: continuous sparging of solvents with high purity He and elution of the B[a]P-tetraols by a gradient of methanol in H2O.
Sparging was necessary to prevent quenching of the fluorescence of the B[a]P-tetraol standards over time, presumably by introduction of O2 into the solvents exposed to air. With He sparging, there was <4% variation in the B[a]P-tetraol standard run at 12 h intervals throughout the analysis. Sparging the solvents with He also resulted in decreasing the limit of detection from 5 fmol (1.3 pg) to 2 fmol (0.6 pg), with a signal to noise ratio of 2.0.
A typical chromatogram is shown in Figure 5
. Occasionally, peaks other than those corresponding to authentic B[a]P-tetraols were observed, including a shoulder on the trans-anti-B[a]P-tetraol. Identification of the source of these fluorescent peaks was not pursued; however, they were not observed in DNA from untreated mice. Analysis of ethanol washes of DNA prior to hydrolysis did not reveal free B[a]P-tetraols; thus, all B[a]P-tetraols analyzed here were released from adducted DNA by acid hydrolysis.

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Fig. 5. Typical reverse-phase HPLC fluorometric profile of lung or liver DNA after acid hydrolysis. The lung DNA (75 µg) analyzed in this chromatogram is from a mouse killed 72 h after treatment with 7.9 µmol B[a]P. Details of the animal treatments, and the procedures for the separation and detection of B[a]P-tetraols by fluorescence are given in Materials and methods, I.S., internal standard.
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Continual automated HPLC analysis was used in this study, as each chromatographic run required 80 min. Internal standard, B[a]P-triol, was added to each sample to detect and quantify variations in fluorometric analysis. B[a]P-triol was chosen because of its similar structure to the B[a]P-tetraols, its convenient retention time, and the similar sensitivity of its fluorescence to oxygen quenching. Variations in the area of the internal standard were <5%. With the addition of the internal standard to each sample, and analysis of B[a]P-tetraol standards every 12 h, we could detect any variations in the analysis conditions using the automated method.
For determination of the effects of isothiocyanates on B[a]P-DNA adduct formation, mice were treated with BITC or PEITC (6.7 µmol) by gavage, then 15 min later with B[a]P (7.9 µmol) by gavage. They were killed at various intervals after B[a]P treatment and DNA was isolated and analyzed. The results of the analyses in lung are summarized in Figure 6A
J. In mice treated with BITC and B[a]P, adduct formation was consistently inhibited 12120 h after B[a]P treatment, compared with the mice treated with B[a]P alone (Figure 6A
). The AUC in the BITCB[a]P group was 29.5% less (P = 0.001) than in the B[a]P group (Table II
). In mice treated with PEITC and B[a]P, adduct formation was reduced by 19.0% compared with the B[a]P group (P = 0.02). The inhibition was not as strong and adduct levels from 12120 h were not consistently lower than in the B[a]P group (Figure 6B
). Similar analyses were carried out for each B[a]P-tetraol isomer (Figure 6CJ
; Table II
). The results were similar to those obtained for total B[a]P-tetraols, with the exception of trans-syn-B[a]P-tetraol (Figure 6G and H
), which is the least abundant isomer. In this case, the scatter was greater and no significant differences were observed among the groups. There were no significant differences between the AUCs in the BITCB[a]P and PEITCB[a]P groups for any of the tetraols.
The results of the adduct analyses in mouse liver are illustrated in Figure 7A and B
. The results were remarkably similar to those obtained in lung. Adduct formation in the BITCB[a]P group was consistently inhibited 12120 h after B[a]P treatment compared with the group treated with B[a]P alone; overall reduction was 35.6% (P = 0.0007; Table II
). In the mice treated with PEITC and B[a]P, adduct formation was inhibited only at the 12 and 24 h time points, but the overall AUC was reduced by 25.1%, which was significantly lower than in the B[a]P treated mice (P = 0.006). As in lung, data for the individual tetraols generally resembled that for the total, except in the case of trans-syn-B[a]P-tetraol (Table II
). There were no significant differences between the BITCB[a]P and PEITCB[a]P groups.
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Discussion
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BITC is known to inhibit tumor induction by B[a]P and 7,12-dimethylbenz[a]anthracene (7,8,25,26), but there have been no previous reports on the effects of BITC on the metabolism of these PAH carcinogens. The results of this study support the hypothesis that BITC inhibits the lung tumorigenicity of B[a]P by inhibiting its metabolic activation. In vitro, BITC inhibited the metabolism of B[a]P in mouse lung with particularly strong effects on the formation of B[a]P-7,8-diol, the proximate carcinogen of B[a]P (Table I
). In lung microsomes obtained from mice treated with BITC, inhibition of B[a]P-7,8-diol formation was observed 6 h after treatment, although this effect disappeared 24 h after (Figure 3A and B
). In addition, BITC inhibited the formation of mutagens from B[a]P-7,8-diol (Figure 4
). The results of the BPDEDNA adduct studies are consistent with these findings. In mouse lung, BITC inhibited adduct formation 12120 h after treatment (Figure 6A
). An exception to this consistent pattern was the in vitro results in liver, where we observed an enhancement of several B[a]P metabolites 6 h after treatment with BITC (Figure 3C
). The increased formation of B[a]P-7,8-diol may have been compensated for by the inhibition of conversion of B[a]P-7,8-diol to BPDE, since BPDEDNA adduct formation was inhibited by BITC in liver, as observed in lung (Figures 4 and 7A
). The increased metabolism of B[a]P in liver could decrease the dose of B[a]P to the lung.
The most likely mechanism by which BITC inhibits the metabolic activation of B[a]P is by inhibition of cytochrome P450 enzymes. Isothiocyanates are known to be effective inhibitors of cytochromes P450 (5,6,27). Treatment of A/J mice with BITC inhibits lung microsomal ethoxyresorufin-O-dealkylase activity and pentoxyresorufin-O-dealkylase activity, indicating inhibition of cytochromes P450 1A and 2B1, respectively (28). BITC is also a mechanism-based inactivator of cytochrome P450 2E1 (29). As B[a]P metabolism is known to be catalyzed in part by cytochromes P450 1A1 and 1A2 (1114), and there is evidence for the presence of these enzymes in mouse liver and lung (30,31), it seems probable that inhibition of cytochrome P450 1A enzymes by BITC partially explains the observed inhibition of B[a]P metabolism. Inhibition of other cytochromes P450, such as P450 1B1, may also be involved (32). BITC is also known to induce phase II enzymes (33). Pre-treatment of mice with BITC results in induction of glutathione S-transferases in liver, lung and other tissues (3335). Enhancing effects on the activity of NAD(P)H:(quinone acceptor)oxidoreductase have also been observed (33). Since glutathione S-transferases are involved in the detoxification of BPDE as well as other epoxide metabolites of B[a]P (1114), this could have played a role in the decreased levels of BPDEDNA adducts observed here.
PEITC also inhibited B[a]P metabolism in vitro, with IC50 values similar to, or lower than, those of BITC (Table I
). In lung microsomes isolated from mice treated with PEITC, inhibition of metabolism was similar to that observed with BITC, 6 h after treatment (Figure 3A
). However, a major difference between PEITC and BITC was seen 24 h after treatment, when levels of B[a]P-7,8-diol and B[a]P-9,10-diol were substantially increased by PEITC, whereas they remained unaffected by BITC (Figure 3B
). A second potentially important difference between PEITC and BITC was in their effects on conversion of B[a]P-7,8-diol to mutagens; PEITC was a relatively ineffective inhibitor (Figure 4
). These observations are consistent with the smaller inhibition of BPDEDNA adduct formation in lung and liver by PEITC compared with BITC. Whereas BITC inhibited BPDEDNA adduct formation by 29.5 and 35.6% in lung and liver, respectively, the corresponding figures for PEITC were 19.0 and 25.1%. While the AUC data for PEITC and BITC were not significantly different, they do indicate a less pronounced inhibitory effect of PEITC on BPDEDNA adduct formation. Nevertheless, based on these data, we might have expected inhibition of B[a]P-induced lung tumorigenesis by PEITC, but this was not observed (8,9). It is possible that there are larger differences between inhibition of BPDEDNA adduct formation by BITC and PEITC in particular lung cell types, which were not examined here. It is also possible that DNA adducts other than those formed from BPDE are important in A/J mouse lung tumorigenesis. As we did not use radiolabelled B[a]P in this study, we would not have detected other DNA adducts. In previous studies, we and others have observed early eluting radioactivity in enzymatic hydrolysates of DNA from the lungs and livers of mice treated with [3H]B[a]P (36,37). This radioactivity has not been characterized. Moreover, `depurinating-DNA adducts' formed by one electron oxidation of B[a]P have also been proposed to play a role in B[a]P tumorigenesis, but they were not measured here (3840). Consideration of the extent of inhibition of BPDEDNA adducts versus inhibition of tumorigenesis suggests that other DNA adducts, or events other than adduct formation, are involved. Inhibition of mouse lung BPDEDNA adduct formation by BITC was 29.5%, but inhibition of tumor multiplicity in two studies was consistently ~50% (7,8).
BITC and PEITC both induce sustained activation of c-Jun N-terminal kinase in a dose-dependent manner (41). This activation is associated with induction of apoptosis in various cell types (41). Treatment with apoptosis-inducing concentrations of isothiocyanates also causes rapid and transient induction of caspase-3/CPP32-like activity and stimulates proteolytic cleavage of poly(ADP-ribose) polymerase (42). PEITC blocks tumor promoter-induced cell transformation in mouse epidermal JB6 cells, and this inhibitory activity is correlated with induction of apoptosis (43). Apoptosis induction in this system occurs through a p53-dependent pathway. These results indicate that isothiocyanates have potentially important effects downstream from DNA adduct formation. It would be of interest to examine the comparative efficacy of BITC and PEITC with respect to these parameters in the A/J mouse lung tumorigenesis model.
The protocol used in this study is based on a previous tumorigenicity experiment in which the inhibitory activities of BITC and PEITC were directly compared (8). A more realistic protocol with respect to human exposure might have treated the mice with isothiocyanates prior to, or concurrent with, multiple doses of B[a]P. This would allow long term assessment of isothiocyanate effects on B[a]P metabolism, which would be important for developing chemoprevention strategies. These aspects should be investigated in future studies.
Levels of BPDEDNA adducts in liver and lung were similar (Figures 6A and 7A
). Our data are consistent with previous studies of BPDEDNA adduct formation in A/HeJ mice, which also showed little difference in adduct levels among various tissues, independent of their susceptibility to tumor formation (44). The A/J mouse lung is highly susceptible to tumor induction by DNA damaging agents (45). Clearly, factors other than adduct formation are involved in this response, although there is a good correlation between pulmonary PAHDNA adduct levels and lung tumorigenicity in this mouse strain (46).
In summary, the results of this study support the hypothesis that BITC inhibits B[a]P-induced lung tumorigenesis by inhibiting its metabolic activation to BPDEDNA adducts in mouse lung. However, differences in BPDEDNA adduct formation do not appear to explain fully the contrasting inhibitory properties of BITC and PEITC on B[a]P-induced lung tumorigenesis suggesting that other events are also involved.
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Notes
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1 To whom correspondence should be addressed Email: hecht002{at}tc.umn.edu 
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Acknowledgments
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This study was supported by National Cancer Institute grant CA-46535. We thank Robin Bliss, University of Minnesota Cancer Center Biostatistics Core, for help with the statistical analyses. The Biostatistics Core is partially supported by Cancer Center Support grant CA-77598. We thank Gunnar Boysen and Steven Carmella for help in developing the HPLC-fluorescence assay. S.S.H. is an American Cancer Society Research Professor, supported by ACS Grant RP-00-138.
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Received February 24, 2000;
revised May 18, 2000;
accepted May 24, 2000.