University of Minnesota Cancer Center, Mayo Mail Code 806, 420 Delaware St SE, Minneapolis, MN 55455, USA
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Abstract |
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Abbreviations: B[a]P, benzo[a]pyrene; BITC, benzyl isothiocyanate; BPDE, (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-benzo[a]pyrene; BPDE-N2-dG, N2-[7,8,9-trihydroxy-7,8,9,10-tetrahydro-10-yl]deoxyguanosine; GC-NICI-MS, gas chromatography-negative ion chemical ionization-mass spectrometry; Hb, hemoglobin; HPB, 4-hydroxy-1-(3-pyridyl)-1-butanone; iso-NNAL, 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; P450, cytochrome P450; PEITC, 2-phenethyl isothiocyanate; RBC, red blood cells; trans/anti-B[a]P-tetraol, r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; trans/anti-B[a]P-TME, r-7,t-8,9,c-10-tetramethoxy-7,8,9,10-tetrahydrobenzo[a]pyrene
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Introduction |
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Three recent epidemiologic studies demonstrated that isothiocyanates are protective against lung cancer. London et al. (13) showed that individuals with detectable isothiocyanates in their urine were at decreased risk of lung cancer, and the protective effect was seen particularly in individuals with homozygous deletion of GSTM1 or both GSTM1 and GSTT. Zhao et al. and Spitz et al. obtained similar results (14,15). GSTM1 and GSTT1 are involved in the metabolism of isothiocyanates (16). It has been hypothesized that deletion of these enzymes would result in higher levels of free isothiocyanates in tissues and therefore more effective chemoprevention (17).
Benzo[a]pyrene (B[a]P, Figure 1) and NNK are two of the most important lung carcinogens in tobacco smoke (18). BITC inhibits lung tumor induction by B[a]P and other polycyclic aromatic hydrocarbons in mice while PEITC inhibits lung tumor induction by NNK in mice and rats (1). A mixture of BITC and PEITC, administered in the diet, inhibits lung tumor induction by a mixture of B[a]P and NNK in A/J mice (19). Taken together with the epidemiologic data, these results underline the efficacy and potential suitablility of isothiocyanates for chemoprevention of lung cancer.
Recently, we investigated the mechanism by which a mixture of dietary BITC and PEITC inhibited lung tumorigenesis induced by B[a]P plus NNK in A/J mice (20). The effects of a mixture of BITC and PEITC as well as PEITC alone on DNA adduct formation by B[a]P and NNK were examined (20). A mixture of BITC and PEITC, and PEITC alone, decreased levels of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, Figure 1) releasing DNA adducts of NNK, while there was no effect on levels of O6-methylguanine formed from NNK, or N2-[7,8,9-trihydroxy-7,8,9,10-tetrahydro-10-yl]deoxyguano-sine (BPDE-N2-dG) adducts from B[a]P. These results indicated that one mechanism of chemoprevention of lung tumorigenesis in this A/J mouse model was inhibition of HPB releasing DNA adducts of NNK (20). In the present study, we extended our investigation to an F-344 rat model. Lung tumors can be readily induced in F-344 rats by administration of NNK in the drinking water (2,21). While a mixture of B[a]P plus NNK has not been tested for carcinogenicity in the F-344 rat, dietary administration of B[a]P induces tumors of the forestomach in rats (22). Therefore, a mixture of B[a]P plus NNK would be expected to induce forestomach and lung tumors. An advantage of the rat lung tumorigenesis model is the use of a relatively low daily carcinogen dose (
0.15 mg/kg body wt NNK) (2). The carcinogen dose used in the B[a]P plus NNK A/J mouse lung tumorigenesis model is high: daily doses of 38 mg/kg B[a]P and 31 mg/kg NNK by gavage once weekly for 8 weeks. The chronic daily exposure to carcinogens in the F-344 rat model may be more relevant to human exposure than the dosing regimen used in the A/J mouse model. Therefore, we investigated the effects of dietary BITC, PEITC, and a mixture of BITC plus PEITC on DNA and hemoglobin (Hb) adduct formation, and on urinary metabolites of NNK, in rats treated with B[a]P (2 p.p.m. in the diet) plus NNK (2 p.p.m. in drinking water).
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Materials and methods |
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Animal experiments
Male F-344 rats were obtained at age 8 weeks from Charles River, Wilmington, MA. They were housed under standard conditions and maintained on NIH-07 diet (Dyets, Bethlehem, PA) (28). After arrival, they were allowed to acclimate to the animal facility for 2 weeks.
The design of the experiment is illustrated in Figure 2. All rats were given B[a]P in the diet (2 mg/kg diet) and NNK in the drinking water (2 µg/ml) ad libitum. The diets containing B[a]P and isothiocyanates were prepared every 3 weeks and stored at -4°C. The drinking water containing NNK was prepared every 34 days. Diets and drinking water were changed in the cages every 34 days. There were 12 rats/group. One week prior to carcinogen treatment, the rats were given diet with additions as follows: group 1, none; group 2, 1 µmol BITC/g diet; group 3, 3 µmol PEITC/g diet; group 4, 1 µmol BITC plus 3 mmol PEITC/g diet. Doses of the isothiocyanates were chosen based on previous studies (2,19,29). Food and water consumption were measured every 34 days during the experiment. Starting 2 weeks after the first carcinogen treatment, every 2 weeks for 16 weeks, four rats from each group were randomly selected, and 0.51.0 ml blood was withdrawn from the orbital sinus. Blood was collected in tubes containing 250 mM EDTA, pH 7.4. The red blood cells (RBC) were pelleted by centrifugation, washed three times with saline (0.9% NaCl) and stored at 80°C. Starting 4 weeks after the first carcinogen treatment, every 4 weeks for 16 weeks, three rats from each group were randomly selected and placed in metabolism cages. Carcinogen treatment was suspended at this time to avoid contamination of urine, but the diets containing the appropriate amounts of isothiocyanates were continued as usual. Twenty-four hour urine samples were collected. Then these rats were returned to the main experiment. Eight weeks after the first carcinogen administration, six rats from each group were randomly selected and killed. Sixteen weeks after the first carcinogen administration, the remaining rats were killed. At the final killing,
5 ml of blood was drawn by cardiac puncture under isofluorane anaesthesia, and lung and liver tissues were harvested and stored at 80°C.
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Precipitation of globin
To precipitate globin, 2 ml of Hb solution was added dropwise to 40 ml ice-cold 1% HClacetone. The supernatant was discarded and the globin was washed twice with 100% acetone. Globin was dissolved in 2 ml H2O and the precipitation was repeated twice. The washed globin was dried at 50°C overnight and stored at -80°C.
DNA isolation
DNA was isolated using the Puregene® DNA isolation kit (Gentra, Minneapolis, MN) according to the manufacturers description. In brief, tissues were homogenized in 3 ml of Cell Lysis Solution using 1050 strokes in a glass homogenizer. To digest the protein, 15 µl Proteinase K Solution (20 mg/ml) was added, and samples were incubated at 55°C for 5 h. Then 15 µl RNase A Solution was added and the samples were incubated at 37°C for 1 h. Proteins were precipitated by adding 3 ml Protein Precipitation Solution followed by centrifugation at 2000 g for 10 min. The pellet was discarded. The DNA was precipitated by slowly adding 3 ml isopropanol, then transferred to 4 ml silanized vials. The DNA was rinsed once with 70% EtOH and twice with 100% EtOH (1 ml each), dried under a gentle stream of N2 and stored at -20°C until analysis.
Gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) analysis of HPB releasing DNA and Hb adducts
The samples were analyzed in sets of 24, including three H2O blanks (negative controls) and one H2O blank spiked with 300 fmol HPB (positive control).
Acid hydrolysis of DNA.
The procedure was performed essentially as described previously (20,31).
Base treatment of Hb.
For the analysis of Hb adducts, 2.0 ml dialyzed Hb solution was used. The Hb concentration was determined by the Drabkin method (Sigma). For the base treatment, 4 N NaOH was added to a final concentration of 0.15 N. Samples were treated for 1 h at room temperature in a sonicator. Hb was precipitated by neutralizing with 4 N HCl. Fifty picograms of 4,4[2H2]HPB (299 fmol) was added as internal standard. The samples were vortexed for 1 min and centrifuged for 15 min at 2000 g. The supernatant was transferred to a new vial. The pH was adjusted to 2.0 ± 0.3 with 4 N HCl.
Extractions.
The acidic aqueous solutions from DNA or Hb, containing HPB and 4,4[2H2]HPB, were extracted twice with equal volumes of CH2Cl2. The Hb samples were additionally extracted twice with 1 vol hexanes. The aqueous layer was saved and the pH was adjusted to 7.0 with 1 N NaOH. The samples were extracted three times with equal volumes of CH2Cl2. The organic layers were pooled and the solvent was removed at reduced pressure using a Speedvac concentrator (ThermoSavant, Holbrook, NY).
Derivatization and GC-NICI-MS analysis.
These were carried out as described previously (20).
Analyses of NNAL and its glucuronide (NNAL-Gluc) in urine
These analyses were performed essentially as described previously (32). Briefly, 2 ml urine was extracted three times with 1 vol ethyl acetate to obtain NNAL. The aqueous layer containing NNAL-Gluc was incubated overnight with 12 000 U ß-glucuronidase type IX-A from Escherichia coli (Sigma) at 37°C. It was then extracted three times with CH2Cl2. To the ethyl acetate and the CH2Cl2 extracts, 2.7 ng iso-NNAL was added as internal standard and the solvents were removed at reduced pressure.
The samples were purified by HPLC using a 3.9x150 mm Bondclone 10 C18 column (Phenomenex, Torrance, CA) with a gradient from 10 to 45% MeOH in 20 min at a flow rate of 1 ml/min. Samples were dissolved in 500 µl 20 mM KH2PO4 buffer containing 50 µg 3-acetylpyridine and 50 µg 3-pyridylcarbinol acetate as retention time markers. Fractions between the apices of the UV-markers were collected. Solvents were removed and the residues were transferred to 300 µl vials with three portions of 100 µl MeOH. The MeOH was concentrated to dryness, the samples were dissolved in 5 µl bis-trimethylsilyltrifluoroacetamide containing 20 ng N-nitrosopentyl-3-picolylamine as injection standard, and analyzed by GC with nitrosamine selective detection, as described previously (32).
GC-NICI-MS analysis of trans/anti-B[a]P-tetraol releasing DNA and Hb adducts
The procedure was similar to the one described by Melikian et al. (33,34) but using a simplified permethylation method (35).
Release of B[a]P-tetraols from DNA and globin.
B[a]P-tetraols were released from DNA by mild acid hydrolysis. In brief, 100 µg DNA was dissolved in 600 µl H2O. Two picomole trans/anti-[2H8]B[a]P-tetraol was added as internal standard. Samples were hydrolyzed in 0.1 N HCl, 4 h at 80°C. To release B[a]P-tetraols from globin, 50 mg globin was dissolved in 3 ml H2O. The globin solutions were transferred to an 8 ml vacuum hydrolysis vial, 2 pmol trans/anti-[2H8]B[a]P-tetraol was added as internal standard, and B[a]P-tetraols were released by hydrolysis with 0.1 N HCl, 3 h, 80°C under vacuum. After hydrolysis, the globin was precipitated by neutralization with 0.4 N NaOH. The released B[a]P-tetraols were extracted five times with 1 vol ethyl acetate and the combined organic layers were concentrated to dryness on a SpeedVac and stored at -20°C.
HPLC clean up #1.
The released B[a]P-tetraols were further purified by reverse phase HPLC using a 4.6x250 mm 5 µ Ultrasphere ODS C18 column (Beckman, Fullerton, CA). The program was as follows: 20% MeOH in H2O for 10 min, then increase linearly to 55% MeOH in 5 min, and hold for 20 min. The flow rate was 1 ml/min. The samples from globin extracts were dissolved in 70 µl MeOH, while for the DNA samples, the total hydrolysis solution was injected. The retention time of trans/anti-B[a]P-tetraol (2729 min) was determined before each set of samples was injected. Fractions containing trans/anti-B[a]P-tetraol and trans/anti-[2H8]B[a]P-tetraol were collected from 1 min before until 4 min after their elution positions. Solvents were removed with a SpeedVac concentrator and samples were stored at -20°C until derivatization.
Derivatization of trans/anti B[a]P-tetraol to trans/anti-B[a]P-TME.
The derivatization was carried out at room temperature. Samples were dissolved in 100 µl DMSO. A magnetic miniature stirrer and 2 mg of dry NaH were added and the mixture was stirred for 2 min. Then 50 µl CH3I was added and the reaction was allowed to proceed for 15 min with stirring at room temperature. The reaction was quenched by adding 500 µl H2O. Trans/anti-B[a]P-TME was extracted with benzene (3x1 ml). The benzene was removed and the samples were stored at -20°C.
HPLC clean up #2.
Trans/anti-B[a]P-TME was purified by reverse phase HPLC using the ODS C18 column operated with a gradient from 70 to 100% MeOH in H2O in 30 min at a flow rate of 1 ml/min. Samples were dissolved in 50 µl of MeOH containing 50 ng of hexanophenone and octanophenone as retention time markers. The entire sample was injected. Fractions between apices of the UV markers were collected. Solvents were removed with a SpeedVac and the residues were transferred to 300 µl insert vials with 3x250 µl MeOH and stored at -20°C until analysis.
GC-NICI-MS.
Samples were dissolved in 10 µl benzene containing 150 fmol trans/anti-[13CH3]4 trans/anti-B[a]P-TME as injection standard. Five microliters were injected in splitless mode.
The analysis was performed on a Finnigan TSQ 7000 instrument (FinniganMAT/Thermoquest, San Jose, CA) interfaced with a CTC A200SE autosampler (Leap Technologies, Carrboro, NC) and a HP5890 series II gas chromatograph (Agilent, Wilmington, DE). A DB-17 MS (30 m, ID 0.25 mm, film thickness, 0.15 µm) capillary GC column (J&W Scientific, Folsom, CA), connected to a 2 mx0.530 µm fused silica uncoated deactivated retention gap, was interfaced to the CI source operated in negative ion mode. The oven temperature was held at 60°C for 1 min, then increased to 300°C at 20°C/min. The temperature was held at 300°C for 15 min. The MS parameters were as follows: ion-source temperature, 150°C; emission current, 700 mA; electron energy, 150 eV. Ultra high-purity methane was the reagent gas. The molecular ions m/z 376 (trans/anti-B[a]P-TME, analyte), m/z 380 (trans/anti-[13CH3]4B[a]P-TME, injection standard) and m/z 384 (trans/anti-[2H8]B[a]P-TME, internal standard) were monitored.
Statistical analyses
Adduct and urinary metabolite levels were compared using analysis of variance. When the overall F-test was significant, subsequent pairwise comparisons were tested using Students t-test. A P value of 0.05 or less was considered statistically significant.
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Results |
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Table II and Figure 4A
summarize adduct levels for each group after 8 and 16 weeks of treatment. HPB releasing DNA adducts were significantly higher in lung than liver. PEITC and BITC plus PEITC significantly reduced HPB releasing DNA adduct levels in lung after 8 and 16 weeks of treatment by 50%. PEITC and BITC plus PEITC reduced levels of HPB releasing DNA adducts in lung to levels similar to those seen in the liver DNA (Figure 4A
). There were no effects of the isothiocyanates on levels of HPB releasing DNA adducts in liver.
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There were no effects of the isothiocyanates on levels of trans/anti-B[a]P-tetraol releasing DNA adducts in lung or liver (Table II, Figure 4B
) or on levels of trans/anti-B[a]P-tetraol releasing Hb adducts (Table II
, Figure 5B
).
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Discussion |
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In A/J mice treated weekly by gavage with a mixture of B[a]P plus NNK, addition of PEITC or BITC plus PEITC to the diet, at the doses used here, reduced lung tumor multiplicity by 40% (19). No inhibition was observed with dietary BITC. In experiments with single carcinogens administered by the same protocol, PEITC inhibited lung tumorigenesis induced by NNK but not by B[a]P (19). Therefore, we concluded that the inhibitory effect of a mixture of dietary BITC and PEITC on lung tumorigenesis by B[a]P plus NNK in A/J mice was due mainly to the effect of PEITC on NNK. The results of our previous DNA adduct experiments in mice, taken together with the present results, strongly support this conclusion.
Consistent with the DNA adduct data, we observed significant decreases in levels of HPB releasing Hb adducts of NNK in rats treated with PEITC or BITC plus PEITC, but not with BITC. There were no effects of the isothiocyanates on levels of Hb adducts of B[a]P, which is also consistent with the DNA adduct data. Hb and DNA are traps for electrophiles formed in the metabolic activation of B[a]P and NNK. These results indicate that PEITC inhibits the metabolic activation of NNK to pyridyloxobutylating agents, resulting in a decrease in both DNA and Hb adducts. The observed decrease in DNA adduct levels is therefore unlikely to be due to effects on DNA repair. Neither PEITC nor BITC plus PEITC appears to have any significant effect on the metabolic activation of B[a]P to BPDE.
Levels of HPB releasing Hb adducts of NNK decreased between 12 and 16 weeks in the rats treated with B[a]P plus NNK or with B[a]P plus NNK and BITC. In a previous study, we also observed a decrease in Hb adduct levels in rats during chronic treatment with NNK, 2 p.p.m. in the drinking water (2). The decrease occurred during months 59 of treatment. The origin of this effect is not known at present. Previous studies demonstrated that chronic NNK treatment of rats resulted in a decrease in lung microsomal -methylene hydroxylation of NNK as well as levels of pulmonary O6-methylguanine, a product of
-methylene hydroxylation (39,40). Further studies demonstrated that this was due to NNK treatment and not to the age of the rats (40). However, no such effects were observed on
-methyl hydroxylation, the pathway which leads to the formation of the HPB releasing Hb and DNA adducts studied here (39,40). Therefore, effects of NNK on its own metabolism are unlikely to explain this observation, and further studies are required.
Dietary PEITC, or BITC plus PEITC, significantly inhibited levels of HPB releasing DNA adducts of NNK in lung but not in liver. These results are consistent with a previous study in which we showed that chronic treatment of rats with PEITC inhibited pulmonary but not hepatic metabolic activation of NNK, by both -hydroxylation pathways (40). In tandem with the inhibitory effects of PEITC on pulmonary HPB releasing DNA adducts of NNK observed in the present study, we saw a substantial increase in levels of urinary NNAL and NNAL-Gluc. These results indicate a link between inhibition of pulmonary NNK
-hydroxylation and increased urinary NNAL and NNAL-Gluc. We have recently shown that NNAL accumulates in rat lung, possibly due to binding of (S)-NNAL to a receptor site (41). PEITC is known to inhibit pulmonary metabolism of NNAL to NNK, as well as
-hydroxylation of NNAL and NNK (40). Inhibition of these metabolic steps in the lung could lead to further accumulation of NNAL and ultimately to increased urinary excretion of NNAL. There was no effect of BITC on HPB releasing DNA adducts of NNK in lung or liver, nor was there any consistent effect of BITC on urinary NNAL or NNAL-Gluc levels. Collectively, these results indicate that pulmonary NNK and NNAL metabolism is a factor in urinary NNAL levels. These conclusions are consistent with a previous study, which compared NNK metabolism in vivo in the rat with its metabolism by primary lung and liver cells (42). The authors concluded that, at low doses of NNK, the profile of urinary metabolites is determined mainly by pulmonary metabolism. These results suggest that levels of NNAL and NNAL-Gluc in human urine could be a biomarker of NNK activation in the lung, and therefore could be used to determine the effects of chemopreventive agents such as PEITC.
HPB releasing DNA adducts of NNK were significantly higher in lung than liver after 8 and 16 weeks of treatment. These results are consistent with the known organoselectivity of NNK for induction of lung tumors in rats and with the importance of the lung in NNK metabolism, as discussed above. Previous studies indicated that HPB releasing DNA adducts and O6-methylguanine are important in lung tumor induction by NNK in rats (36,39,43). However, there have been few comparisons of HPB releasing adduct levels in rat liver and lung. A study by Murphy et al. (44) compared levels of HPB releasing DNA adducts of NNK in rat lung and liver after 4 daily i.p. injections of radiolabeled NNK. At low doses (150 µg/kg/day or less) the amount of HPB released from lung DNA was greater than from liver DNA, whereas at higher doses HPB releasing DNA adduct levels in liver exceeded those in lung. The daily dose in the present experiment was 150 µg/kg/day. Therefore, our results are consistent with those of Murphy et al., although the routes of administration were different. Morse et al. (38) found higher levels of HPB releasing DNA adducts of NNK in liver than in lung after treatment of rats with four consecutive daily s.c. doses of 600 µg/kg body wt NNK, consistent with Murphys study. Similar results were obtained by Peterson et al. (45). The present study is the only one to compare levels of pulmonary and hepatic HPB releasing DNA adducts of NNK using a protocol similar to that which induces lung tumors in rats, e.g. chronic treatment with 2 p.p.m. NNK in the drinking water.
PEITC and BITC plus PEITC, reduced levels of HPB releasing DNA adducts in lung by 50%, from 1.22.0 to 0.40.9 fmol/µg DNA. These residual adduct levels in lung, which were unaffected by the isothiocyanate treatments, were approximately the same as the adduct levels observed in liver (0.40.5 fmol/µg DNA), independent of isothiocyanate treatment (Figure 4A
). This suggests that the HPB releasing DNA adducts were either PEITC-sensitive or PEITC-insensitive. The PEITC-sensitive adducts are found only in lung and are reduced by PEITC treatment. It is unlikely that there are structural differences between HPB releasing DNA adducts in lung and liver. It is more likely that this observation is related to effects of PEITC on cytochrome P450 (P450) enzymes in rat lung and liver. PEITC is known to specifically inhibit
-methyl hydroxylation of NNK in rat lung (40,46). The effects of PEITC on P450s are complex and dependent on route of administration, dose, and timing (47). PEITC is known to inhibit P450 2B-mediated activity in rat lung, but it can induce P450 2B1 in rat liver. PEITC can also inhibit P450 1A2 and 3A related activities in rat liver (47). These P450s, as well as P450 2A3, which is present in rat lung but not liver, are involved in NNK
-methyl hydroxylation (36,48,49). There may be differential effects of PEITC on these P450s, which produce the PEITC-sensitive and PEITC-insensitive DNA adducts.
The results of this study are fully consistent with our previous investigation in which we determined levels of HPB releasing Hb adducts and urinary NNAL and NNAL-Gluc in rats treated with NNK (2 p.p.m. in drinking water) with or without dietary PEITC (3 µmol/g diet) (2). This was a carcinogenicity study in which PEITC completely inhibited lung tumor induction by NNK. Adduct measurements and urinary metabolite determinations were carried out at intervals during the experiment. Levels of HPB releasing Hb adducts were 1.8 times lower in the PEITC-treated animals than in the controls, throughout the 2 year experiment. Although, as mentioned above, levels of HPB releasing Hb adducts decreased in the control animals during the course of the experiment, a corresponding decrease was also seen in the PEITC-treated rats, which is somewhat different from the present results. Levels of NNAL and NNAL-Gluc in urine also increased markedly (four to six times) as in the present study. Our results indicate that PEITC or BITC plus PEITC would inhibit lung tumor formation in rats treated with NNK in the drinking water and B[a]P in the diet.
There were no effects of BITC on biomarkers of B[a]P metabolic activation in this study. Evidently, the dose of BITC, which is limited to 1 µmol/g diet (9 mg/kg body wt/day) by considerations of palatability, is insufficient. In A/J mice, 1 µmol/g diet BITC (15 mg/kg body wt/day) had no effect on lung tumor induction by B[a]P plus NNK (19). On the other hand, gavaged BITC is a very effective inhibitor of B[a]P-induced lung tumorigenesis in A/J mice (50,51). In a recent study, we observed 64 and 91% inhibition of lung tumor multiplicity by gavage of 6.7 or 13.4 µmol of BITC (50 or 100 mg/kg body wt), respectively, 15 min before each of 8 weekly treatments with 3 µmol B[a]P (52). We observed modest inhibition of B[a]PDNA adduct formation in mice treated with BITC by gavage prior to B[a]P administration (53). The single high dose of BITC may have other effects, such as induction of apoptosis, which are also important in lung tumor inhibition.
In summary, our results clearly show that PEITC or a mixture of BITC plus PEITC inhibits the formation of HPB releasing DNA adducts in the lungs of rats treated with B[a]P plus NNK. BITC had no effect on adducts derived from B[a]P or NNK. These results are consistent with previous studies, which assessed the inhibitory effects of these isothiocyanates on lung tumor formation, demonstrating the central role of inhibition of NNK metabolic activation by PEITC as a mechanism of chemoprevention.
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Notes |
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Acknowledgments |
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References |
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