Organ and cell specificity of DNA methylation by N-nitrosomethylamylamine in rats

N-Nitrosomethylamylamine (NMAA) is a potent carcinogen in rodents with the esophagus as the principal target organ. The present study aims at an assessment of DNA methylation by NMAA in various rat tissues and an identification of cell populations actively involved in its bioactivation. Adult male F344 rats received a single i.p. dose of N-nitroso[methyl-14C]amylamine (0.1 mmol/kg). After 6 h organs were removed and the DNA was extracted, hydrolyzed in 0.1 M HCl, and subjected to radiochromatography on Sephasorb-HP. Highest levels of DNA alkylation were found in esophagus (798 mumol 7-methylguanine/mol mol guanine), followed by nasal epithelium (672 mumol) and liver (624 mumol). Trachea, lung, forestomach, and kidney had considerably lower levels of alkylation and in glandular stomach, spleen, and duodenum, values were close to the limit of detection. Specific target cell populations were identified autoradiographically and by immunohistochemistry using a rabbit antiserum to O6-methyldeoxyguanosine. In the esophagus, NMAA was selectively metabolized by the basal cells of the mucosa. In the respiratory tract, O6-methyldeoxyguanosine was almost exclusively present in the tracheal and bronchiolar epithelia. In the nasal cavity, labeled nuclei were found in both the olfactory and the respiratory epithelium and in the serous glands. Our studies indicate that NMAA and related asymmetrical nitrosamines are, in addition to liver, preferentially metabolized in tissues derived from the ventral entoderm, including the upper respiratory and gastrointestinal tract.     

异硫氰酸苯己酯诱导Molt-4细胞p15基因去甲基化的实验研究

 目的　研究异硫氰酸苯己酯（PHI）对急性T淋巴细胞性白血病Molt-4细胞p15基因的去甲基化作用及转录激活作用。方法　采用甲基特异性聚合酶链反应（MSP）检测PHI作用前后Molt－4细胞株p15基因甲基化状态的变化情况;RT-PCR检测p15基因的mRNA的表达变化;Western blotting检测p15蛋白的表达变化。结果　PHI作用于Molt-4细胞5 d后,p15基因的异常甲基化现象被逆转,基因的甲基化程度减弱;基因转录激活,p15 mRNA、p15蛋白表达呈浓度依赖性增加。各组p15 mRNA条带灰度值与β-actin比值为：空白对照组（0.17±0.12）,PHI 10 μmol／L组（0.29±0.14）,PHI 20 μmol／L组（0.55±0.07）,PHI 40 μmol／L组（0.93±0.13）,各加药组与空白对照组相比,差异均有统计学意义（P＜0.05）。结论　PHI有DNA去甲基化的作用,能诱导沉默的p15基因重新表达。     

Enhancement of esophageal carcinogenesis in male F344 rats by dietary phenylhexyl isothiocyanate

The purpose of this study was to evaluate the potential effectsof dietary 6-phenylhexyl isothiocyanate (PHITC) on N-nitrosomethylbenzylamine(NMBA)-induced esophageal carcinogenesis in rats. Groups of15 male F344 rats received weekly s.c. injections of NMBA in20% dimethylsulfoxide or the vehicle alone for 15 consecutiveweeks. Two weeks prior to initiation of carcinogen or vehicleinjections rats were provided with modified AIN-76A diet ormodified AIN-76A diet containing PHITC at levels of 0.4, 1.0or 2.5 µmol/g diet. Experimental controls consisted ofgroups that received only the vehicle (vehicle controls), NMBA(carcinogen controls) or PHITC at the high dose level of 2.5µmol/g diet. No esophageal tumors or preneoplastic lesionswere detected in rats that received the vehicle or PHITC alone.In contrast, all rats treated with NMBA alone or PHITC + NMBAexhibited esophageal tumors and preneoplastic esophageal lesions.In groups that received PHITC + NMBA tumor multiplicity wasincreased by 21–69% when compared with rats treated withNMBA alone, indicating that PHITC enhanced esophageal tumorigenesisin this model system. These results, in conjunction with ourprevious work, demonstrate that arylalkyl isothiocyanates mayinhibit or enhance esophageal tumorigenesis in the NMBA-treatedrat. The ability of isothiocyanates to inhibit or enhance experimentaltumorigenesis may depend on alkyl chain length of the isothiocyanate,the animal species examined and the specific carcinogen employed.     

S-Adenosylmethionine metabolism and DNA methylation in hydrazine-treated rats

The treatment of rats with hepatotoxic doses of hydrazine (NH₂-NH₂)induces the rapid formation of 7-methylguanine and O⁶-methylguaninein liver DNA. The methyl moiety in these reactions might bederived from the cellular S-adenosyl-methionine pool becauseradioactivity administered to these rats as methionine rapidlyappears in the DNA as methylated guanine. An increased incorporationof radioactivity into 5-methylcytosine was previously reportedfollowed by subsequent suppression. This increased radiolabelingof 5-methylcytosine coincided with time of maximal DNA guaninemethylation. To determine the nature of S-adenosyl-methioninemetabolism during the period of DNA methylation induced by hydrazinetreatment, and to determine if the increased radiolabeling of5-methylcytosine at this time reflected an actual increase in5-methylcytosine synthesis, liver DNA synthesis and S-adenosylmethioninelevels and turnover were assayed. Liver S-adenosylmethionineconcentrations varied slightly between control rats and hydrazine-treatedrats during the first five hours after hydrazine ad ministration,and no difference was detectable in the incorporation of administered[³H]methionine into S-adenosylmethionine. Because S-adenosylmethioninespecific radio activity in hydrazine-treated rats was not differentfrom control rats, the previously observed increased radiolabelingof 5-methylcytosine appeared to represent an actual increasein synthesis. This conclusion was supported by finding thatin corporation of radioactive thymidine into DNA was also acceleratedimmediately following hydrazine administration, again followedby a decrease. 5-Methylcytosine synthesis, therefore, appearsto follow DNA synthesis during hydrazine toxicity, and formationof 7-methylguanine and O⁶-methylguanine in liver DNA of hydrazine-treatedrats occurs during a short period of increased DNA synthesisand 5-methylcytosine formation very early in hydrazine toxicity.     

Strong promoting activity of phenylethyl isothiocyanate and benzyl isothiocyanate on urinary bladder carcinogenesis in F344 male rats

Post-initiation effects of phenylethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC) on hepatocarcinogenesis and urinary bladder carcinogenesis were examined in rats pretreated with diethylnitrosamine (DEN) and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Groups of 21 rats received a single intraperitoneal injection of 200 mg/kg body weight of DEN. Starting 2 days thereafter, they were administered 0.05% BBN in the drinking water for 4 weeks. Three days after completion of the carcinogen treatment, they were placed on a diet containing PEITC or BITC at a dose of 0.1%, or a basal diet alone for 32 weeks and then killed for autopsy. Further groups of 6 rats each were similarly treated with PEITC, BITC or basal diet alone for 32 weeks without prior DEN and BBN exposure. In the liver, although the incidences of liver tumors were not significantly affected, the multiplicity of foci larger than 0.5 cm in diameter was slightly increased by PEITC. In the urinary bladder, the incidences of papillary or nodular (PN) hyperplasias and carcinomas were significantly elevated by PEITC or BITC after DEN and BBN initiation. In the groups without initiation, PN hyperplasia was found in all rats of both PEITC and BITC groups, along with papillomas and carcinomas in some animals. Tumors and PN hyperplasias in the groups treated with PEITC and BITC are characterized by downward growth. Our results thus showed PEITC and BITC to be strong promoters of urinary bladder carcinogenesis with some complete carcinogenic potential. Int. J. Cancer 77:773–777, 1998. © 1998 Wiley-Liss, Inc.     

异硫氰酸苯己酯调控肝癌细胞株SMMC-7721组蛋白乙酰化及诱导细胞凋亡

目的 研究异硫氰酸苯己酯(PHI)对肝癌SMMC-7721细胞组蛋白乙酰化调控及凋亡的影响.方法 采用台盼蓝拒染直接计数法观察PHI对SMMC-7721细胞增殖的影响,采用原位末端标记(TUNEL)法检测PHI对SMMC-7721细胞凋亡的影响,采用Western blot法检测PHI对SMMC-7721细胞组蛋白乙酰化及凋亡相关蛋白表达的影响.结果 PHI可抑制SMMC-7721细胞的增殖,与0 μmol/L作用组比较,5、10、20、40和80 μmol/L的PHI对SMMC-7721细胞均有不同程度的增殖抑制作用.PHI可诱导SMMC-7721细胞产生凋亡,PHI作用于SMMC-7721细胞7 h后,10、20和40 μmol/LPHI组的细胞凋亡率分别为6.9%±2.4%、17.5%±4.2%和54.5%±5.4%,明显高于0 μmol/L PHI组(4.5%±2.3%,P＜0.05).PHI作用于SMMC-7721细胞3 h时,与0 μmol/L PHI组比较,10、20和40 μmol/L PHI组中Bcl-2、Procaspse-9和Procaspse-3的表达下降,caspase-9和caspase-3的表达上升,而Procaspase-8的表达未见明显变化;作用7 h时,这种变化趋势更加明显.PHI作用于SMMC-7721细胞3 h时,与0 μmol/L PHI组比较,10、20和40 μmol/L PHI组中组蛋白H3的乙酰化分别增加了1.87倍、2.43倍和3.67倍,组蛋白H4的乙酰化分别增加了1.29倍、1.45倍和2.25倍;作用7 h时,这种变化趋势更加明显.结论 PHI是一种组蛋白去乙酰化酶抑制剂,可调控组蛋白的乙酰化水平,影响其表观遗传学,并通过线粒体凋亡途径诱导细胞凋亡.     

The phenylhexyl isothiocyanate induces apoptosis and inhibits leukemia cell growth in vivo

Isothiocyanates are potent chemopreventive agents for carcinogen-induced cancers in rodents. The major mode of action for chemoprevention is cytoprotection i.e. inducing detoxifying enzymes to remove the carcinogens, thus blocking the initiation of carcinogenesis. Analysis has indicated that isothiocyanates also act at the post-initiation levels of carcinogenesis. We have also reported that the phenylhexyl isothiocyanate (PHI) induced growth arrest and apoptosis in human leukemia HL-60 cells in culture. Since then we have investigated the in vivo efficacy of PHI. The effects of PHI were evaluated in immunodeficient mice, with xenografts of human leukemia HL-60 cells. The maximum tolerated dose (MTD) was determined. The experimental mice received 80% of the MTD. Oral feedings of PHI significantly reduced tumor incidence (p<0.05) without overt toxicity. PHI inhibited cell cycle progression through the down-regulation of cyclin expression, Rb phosphorylation and the up-regulation of the cdk-inhibitors. Apoptosis was significantly increased in the treated tumors but not in the normal mouse tissues. In conclusion, PHI induced apoptosis and inhibited the growth of xenografts by targeting the cell cycle regulators. PHI induced selective apoptosis effects in the rapidly growing tumor cells but not in the normal tissues.     

Inhibition of DNA methylation and reactivation of silenced genes by zebularine

BACKGROUND: Gene silencing by abnormal methylation of promoter regions of regulatory genes is commonly associated with cancer. Silenced tumor suppressor genes are obvious targets for reactivation by methylation inhibitors such as 5-azacytidine (5-Aza-CR) and 5-aza-2'-deoxycytidine (5-Aza-CdR). However, both compounds are chemically unstable and toxic and neither can be given orally. We characterized a new demethylating agent, zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one], which is a chemically stable cytidine analog. METHODS: We tested the ability of zebularine to reactivate a silenced Neurospora crassa gene using a hygromycin gene reactivation assay. We then analyzed the ability of zebularine to inhibit DNA methylation in C3H 10T1/2 Cl8 (10T1/2) mouse embryo cells as assayed by induction of a myogenic phenotype and in T24 human bladder carcinoma cells, using the methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay. We also evaluated the effects of zebularine (administered orally or intraperitoneally) on growth of EJ6 human bladder carcinoma cells grown in BALB/c nu/nu mice (five mice per group) and the in vivo reactivation of a methylated p16 gene in these cells. All statistical tests were two-sided. RESULTS: In N. crassa, zebularine inhibited DNA methylation and reactivated a gene previously silenced by methylation. Zebularine induced the myogenic phenotype in 10T1/2 cells, which is a phenomenon unique to DNA methylation inhibitors. Zebularine reactivated a silenced p16 gene and demethylated its promoter region in T24 bladder carcinoma cells in vitro and in tumors grown in mice. Zebularine was only slightly cytotoxic to T24 cells in vitro (1 mM zebularine for 48 hours decreased plating efficiency by 17% [95% confidence interval (CI) = 12.8% to 21.2%]) and to tumor-bearing mice (average maximal weight change in mice treated with 1000 mg/kg zebularine = 11% [95% CI = 4% to 19%]). Compared with those in control mice, tumor volumes were statistically significantly reduced in mice treated with high-dose zebularine administered by intraperitoneal injection (P<.001) or by oral gavage (P<.001). CONCLUSIONS: Zebularine is a stable DNA demethylating agent and the first drug in its class able to reactivate an epigenetically silenced gene by oral administration.     

Effects of phenylethyl isothiocyanate on early molecular events in N-nitrosomethylbenzylamine-induced cytotoxicity in rat esophagus

There is little information on early molecular events in the development of N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis and of the effects of chemopreventive agents on these events. In this study, we identified genes in rat esophagus that were differentially expressed in response to short-term NMBA treatment and modulated by cotreatment with phenylethyl isothiocyanate (PEITC). Rats were fed AIN-76A diet or AIN-76A diet containing PEITC for 3 weeks. During the 3rd week of dietary treatment, they were administered three s.c. doses of NMBA (0.5 mg/kg body weight). Rats were sacrificed 24 h after the last treatment; esophagi were excised and processed for histologic grading, microarray and real-time PCR analysis. Histopathologic analysis showed that treatment of rats with PEITC had a protective effect on NMBA-induced preneoplastic lesions in the rat esophagus. We identified 2,261 genes that were differentially expressed in the NMBA-treated versus control esophagi and 1,936 genes in the PEITC + NMBA versus NMBA-treated esophagi. The intersection of these two sets resulted in the identification of 1,323 genes in NMBA-treated esophagus, the vast majority of which were modulated by PEITC to near-normal levels of expression. Measured changes in the expression levels of eight selected genes were validated using real-time PCR. Results from 12 microarrays indicated that PEITC treatment had a genome-wide modulating effect on NMBA-induced gene expression. Samples obtained from animals treated with PEITC alone or cotreated with PEITC + NMBA were more similar to controls than to samples treated with NMBA alone.     

Inhibition of DNA methylation by antisense oligonucleotide MG98 as cancer therapy

Results of preclinical studies and clinical phase I/II trials suggest that the antisense oligodeoxynucleotide and DNA methyltransferase inhibitor MG98 can safely and effectively lead to reactivation of methylation silence tumor suppressor genes. It is possible that daily or continuous dosing of MG98 might be more active and less toxic than intermittent dosing. Combination of MG98 with other agents having completely different mechanisms of action seems reasonable. One clinical trial now under way is evaluating the use of MG98 in combination with interferon-alpha in patients with advanced renal cell carcinoma (RCC). Because of the current preclinical and clinical evidence, further trials of MG98 as therapy for RCC would be of interest.     

Inhibition of N'-nitrosonornicotine-induced esophageal tumorigenesis by 3-phenylpropyl isothiocyanate

The ability of dietary isothiocyanates to inhibit the esophageal metabolism of N'-nitrosonornicotine (NNN) was examined in F344 rats. Following feeding of benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), 4- phenylbutyl isothiocyanate (PBITC) or 6-phenylhexyl isothiocyanate for 2 weeks, rats were killed and the esophagi were incubated in vitro with [5-3H]NNN. While dietary BITC, PEITC and PBITC all decreased NNN metabolism, dietary PPITC had the greatest effect, yielding inhibition ranging from 55 to 91% of the control production of various NNN metabolites. To determine the chemopreventive efficacy of PPITC on NNN- induced esophageal tumorigenesis, rats were fed AIN-76A diets containing 0, 1.0 or 2.5 micromol/g PPITC and were given untreated drinking water or drinking water containing 5 p.p.m. NNN. After 87 weeks, the experiment was terminated and the esophageal tumors were counted. Rats that were given untreated drinking water developed no tumors. Rats that were given 5 p.p.m. NNN and unadulterated AIN-76A diet had an esophageal tumor incidence of 71% and a multiplicity of 1.57 tumors/animal. The two dietary concentrations of PPITC reduced the incidence and multiplicity of NNN-induced esophageal tumors by >95%. These results demonstrate the remarkable chemopreventive efficacy of PPITC in the NNN-induced esophageal tumor model.      

Inhibition of endothelial cell differentiation and proinflammatory cytokine production during angiogenesis by allyl isothiocyanate and phenyl isothiocyanate

Angiogenesis is a crucial step in the growth and metastasis of cancers. The activation of endothelial cells and their further behavior are very critical during angiogenesis. The authors analyze the effect of allyl isothiocyanate (AITC) and phenyl isothiocyanate (PITC) on angiogenesis in an in vitro model using human umbilical vein endothelial cells (HUVECs). AITC and PITC significantly inhibited endothelial cell migration, invasion, and tube formation. (3)H-thymidine proliferation assay showed that AITC and PITC significantly inhibited the proliferation of HUVECs in vitro. The authors also studied the effect of AITC and PITC on the serum cytokine profiles of angiogenesis-induced animals and found that these compounds are highly potent in the downregulation of vascular endothelial growth factor (VEGF) and proinflammatory cytokines such as interleukin (IL)-1beta , IL-6, granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-alpha). Treatment with these compounds showed an elevation in the levels of IL-2 and tissue inhibitor of metalloproteinases (TIMP)-1, which are antiangiogenic factors. Moreover, studies using B16F-10 melanoma cells showed that both AITC and PITC significantly reduced VEGF mRNA expression. These findings suggest that AITC and PITC act as angiogenesis inhibitors through the downregulation of VEGF and proinflammatory cytokines such as IL-1beta, IL-6, GM-CSF, and TNF-alpha and upregulation of IL-2 and TIMP.     

Reversibility of proliferative lesions and induction of non-papillary tumors in rat urinary bladder treated with phenylethyl isothiocyanate

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Limited tumor-initiating activity of phenylethyl isothiocyanate by promotion with sodium L-ascorbate in a rat two-stage urinary bladder carcinogenesis model

Initiation activity of phenylethyl isothiocyanate (PEITC) was examined in a two-stage urinary bladder carcinogenesis model. Male 6-week-old Fischer 344 rats were fed diet containing 0.1% PEITC for 12 or 24 weeks, with or without subsequent administration of 5% sodium l-ascorbate (Na-AsA) in diet until week 48, or for the entire experimental period. After 12 weeks of PEITC-treatment, both simple hyperplasia and papillary or nodular (PN) hyperplasia had developed in all animals, but the majority of these lesions had disappeared at week 48, irrespective of the Na-AsA-treatment. The same lesions after 24 weeks of PEITC-treatment had progressed to dysplasia and carcinoma, in a small number of cases by week 48 (6% in incidence for each lesion), but enhancement by the Na-AsA-treatment was evident only with simple hyperplasias (from 56 to 100% in incidence) and PN hyperplasias (from 19 to 56%). The results suggest a limited initiation activity of PEITC with induction of irreversible lesions by 24 weeks of exposure.     

Reversibility of proliferative lesions and induction of non-papillary tumors in rat urinary bladder treated with phenylethyl isothiocyanate

Phenylethyl isothiocyanate (PEITC), generally thought to be a chemopreventive agent for various kinds of genotoxic carcinogens, has been found to induce rat urinary bladder carcinomas in our laboratory. To cast light on the underlying mechanisms, the reversibility of urothelial proliferative lesions and the frequencies of H-ras and p53 mutations in the induced rat urinary bladder tumors were investigated. F344 male rats were given diet containing 0.1% PEITC for 48 weeks and then killed, or for 32 weeks and then returned to normal diet without supplement for 1, 3, 7 days or 16 weeks before death. At 7 days after withdrawal of PEITC treatment, carcinomas were observed in only two of 24 rats but a high incidence of dysplasias was evident. Furthermore, 16 weeks after withdrawal, seven of 12 (58.3%) rats had carcinomas. In addition, carcinomas were induced in 11 of 12 (91.7%) rats continuously receiving PEITC for 48 weeks. Most of the carcinomas were characterized as of non-papillary transitional cell type with occasional squamous cell differentiation and/or glandular components. Bromodeoxyuridine labeling indices (LIs) were increased by PEITC administration even in normal-looking epithelium. After withdrawal of treatment, LIs in simple and papillary or nodular (PN) hyperplasias were markedly decreased and these lesions gradually disappeared, while values for dysplasias and carcinomas, which persisted, were only slightly decreased. A silent point mutation was found in H-ras in one of 12 tumor samples (8.3%), whereas seven (58.3%) had mutations in p53. These results indicate that PEITC itself is a carcinogen for the rat urinary bladder, and that while the simple and PN hyperplasia induced by PEITC are reversible, dysplasia is irreversible with the potential to give rise to non-papillary carcinomas with frequent p53 mutations.     

DNA methylation and hepatocarcinogenesis in rats fed a choline-devoid diet

Groups of male Fischer-344 rats were fed either a choline-supplementedor a choline-devoid (CD) diet, for up to 14 months. In ratsfed the CD diet, hepatic lesions developed and progressed throughtwo distinct stages, the first characterized by severe steatosisand an increase in cell turnover and the second by gradual clearanceof the deposited fat, fibrosis and parenchymal nodularity. Largehepatocellular carcinomas were found in rats killed at 14 months.DNA was purified from the livers of all groups of rats and fromthe tumors, and its level of methylations was analyzed usingthe restriction endonucleases Hpall and Mspl. DNA under-methylationwas detected only in the livers of rats fed the CD diet for14 months, whether bearing tumors or not, and in three of fourhepatocellular carcinomas. Undermethylation of liver total DNAis therefore a late effect of dietary choline deficiency inthe rat.     

Inhibition of DNA cytosine methyltransferase by chemopreventive selenium compounds, determined by an improved assay for DNA cytosine methyltransferase and DNA cytosine methylation

The organoselenium compounds benzyl selenocyanate (BSC) and 1,4- phenylenebis(methylene)selenocyanate (p-XSC), as well as sodium selenite, are effective chemopreventive agents for various chemically induced tumors in animal models at both the initiation and postinitiation stages. The mechanisms involved at the postinitiation stage are not clear. Because several lines of evidence indicate that inhibition of excess DNA (cytosine-5)-methyltransferase (Mtase) may be a sufficient factor for the suppression or reversion of carcinogenesis, we examined the effects of sodium selenite, BSC, p-XSC and benzyl thiocyanate (BTC), the sulfur analog of BSC, on Mtase activity in nuclear extracts of human colon carcinomas, and of p-XSC on the Mtase activity of HCT116 human colon carcinoma cells in culture. For this purpose, we developed an improved Mtase assay, in which the incorporation of the methyl-[3H] group from S-adenosyl[methyl- 3H]methionine into deoxycytidine of poly(dI-dC)-poly(dI-dC), is specifically determined by HPLC with radioflow detection after enzymatic hydrolysis, enhancing specificity and reliability. In a variation, using SssI methyltransferase and labeled S- adenosylmethionine, the overall methylation status of DNA in various tissues can also be compared. Selenite, BSC and p-XSC inhibited Mtase extracted from a human colon carcinoma with IC50s of 3.8, 8.1 and 5.2 microM, respectively; BTC had no effect. p-XSC also inhibited the Mtase activity and growth of human colon carcinoma HCT116 cells, with an IC50 of approximately 20 microM. The improved Mtase assay should prove to be a reliable method for screening potential Mtase inhibitors, especially using cells in culture. We suggest that inhibition of Mtase may be a major mechanism of chemoprevention by selenium compounds at the postinitiation stage of carcinogenesis.      

Effects of benzyl isothiocyanate and phenethyl isothiocyanate on benzo[a]pyrene metabolism and DNA adduct formation in the A/J mouse

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.