Mark A. Morse

Isothiocyanates and plant polyphenols as inhibitors of lung and esophageal cancer

A group of arylalkyl isothiocyanates were tested for their abilities to inhibit tumorigenicity and DNA methylation induced by both the tobacco-specific nitrosamine, NNK, in A/J mouse lung and the esophageal-specific carcinogen, NMBA, in F344 rat esophagus. In addition, ellagic acid was tested for its ability to inhibit NMBA-induced esophageal tumorigenesis. In the strain A lung tumor model, PEITC effectively inhibited NNK-induced lung tumors at a dose of 5 micromol, but was not inhibitory at lower doses. PPITC, PBITC, PPeITC, and PHITC were all considerably more potent inhibitors of NNK lung tumorigenesis than PEITC, and PHITC was the most potent inhibitor of all. Thus, in the strain A lung tumor model, there was a trend of increased inhibitory efficacy among arylalkyl isothiocyanates with increased alkyl chain length. In the F344 rat esophageal tumor model, PPITC was clearly more potent than PEITC, BITC and PBITC had little inhibitory effect on esophageal tumorigenesis, and in a separate experiment, PHITC actually enhanced esophageal tumorigenesis. Thus, the structure-activity relationships for inhibition of tumorigenesis by arylalkyl isothiocyanates were considerably different in the two animal models. However, the effects of the isothiocyanates on tumorigenesis were well-correlated to their effects on DNA adduct formation in either model. The most likely mechanism of inhibition of tumorigenesis by these isothiocyanates is via inhibition of the cytochrome p450 enzymes responsible for activation of NNK in mouse lung or NMBA in rat esophagus. Ellagic acid was an effective inhibitor of esophageal tumorigenesis, although not as potent as PEITC or PPITC. Like the isothiocyanates, ellagic acid inhibits cytochrome p450-mediated activation of NMBA.

Dose-related inhibition by dietary phenethyl isothiocyanate of esophageal tumorigenesis and DNA methylation induced by N-nitrosomethylbenzylamine in rats

The purpose of this investigation was to establish a dose response for the effects of dietary phenethyl isothiocyanate (PEITC) on N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis and DNA methylation. Groups of 13-27 rats were randomly assigned to AIN-76A diets containing 0, 0.325, 0.75, 1.5 or 3.0 mumol PEITC/g. Two weeks later, rats were administered NMBA subcutaneously at a dose of 0.5 mg/kg once a week for 15 weeks. Animals were maintained on control or experimental diets for an additional 8 weeks and were terminated at week 25 of the experiment. No significant effects on weight gain or food intake were noted for any of the experimental diets when compared with control values. Animals receiving only NMBA developed 9.3 +/- 0.9 tumors/rat, with an incidence of 100%. Dietary PEITC at concentrations of 0.75, 1.5 and 3.0 mumol/g inhibited NMBA-induced esophageal tumor multiplicity by 39%, 90% and 100%, respectively. Esophageal tumor incidence in these groups was reduced by 0%, 40% and 100%, respectively. The 0.325 mumol/g PEITC diet did not significantly affect NMBA-induced esophageal tumorigenesis. These results indicate that the minimum inhibitory dietary concentration of PEITC is between 0.325 and 0.75 mumol/g. Groups of 20 rats were assigned to diets containing 0-3.0 mumol PEITC/g for two weeks as described above, and then sacrificed 24 hours after administration of [3H-methyl]NMBA. The esophageal DNA was isolated, purified, hydrolyzed, and analyzed by HPLC. PEITC inhibited DNA methylation in a dose-dependent manner, as was found in the tumor bioassay. The inhibition of tumor incidence was highly correlated with the percentage inhibition of either 7-methylguanine or O6-methylguanine. These latter results suggest that the inhibitory activity of PEITC in this model is manifested, at least in part, during the functional equivalent of tumor initiation.

Inhibition of the Tobacco‐Specific Nitrosamine‐Induced Lung Tumorigenesis by Compounds Derived from Cruciferous Vegetables and Green Tea

We have shown that PEITC and I3C, both of cruciferous origin, inhibited lung tumor formation induced by the tobacco-specific nitrosamine NNK. The inhibition by PEITC is due largely to its inhibitory effect on the enzymes of NNK metabolism, whereas; the inhibition by I3C may be attributed to its ability to induce hepatic enzyme activity of NNK metabolism, which resulted in decreased availability of NNK to the lung. On a molar basis, PEITC is considerably more effective than I3C. PEITC was released upon consumption of watercress. The N-acetylcysteine conjugate of PEITC is a promising urinary marker for quantitating uptake of this dietary anticarcinogen in humans. These studies also showed that green tea polyphenol EGCG inhibited the NNK-induced lung tumorigenesis, probably due to its antioxidant property. These studies provide for the first time evidence for the involvement of free radicals in nitrosamine tumorigenesis. The mechanism by which free radicals are generated by NNK treatment is not yet known. The reduced levels of oxidative lesions in lung as a result of EGCG treatment may be related to its ability to reduce reactive oxygen species and/or to chelate iron ion resulting in a decreased production of hydroxyl radicals. Overall, these studies have identified ingredients in cruciferous vegetables and green tea that are inhibitory against lung tumorigenesis induced by NNK in rodents.

Effects of theaflavins on N-nitrosomethylbenzylamine-induced esophageal tumorigenesis

The purpose of this experiment was to compare the inhibitory effects of the polyphenol fraction of black tea, theaflavins (TF), the polyphenol fraction of green tea, and (-)-epigallocatechin-3-gallate (EGCG) in the rat esophageal tumor model. The tea fractions were administered in the drinking water at concentrations of 360 and 1,200 ppm for two weeks before administration of the esophageal carcinogen N-nitrosomethylbenzylamine (NMBA). NMBA was administered subcutaneously in 10% dimethyl sulfoxide three times weekly for five weeks. Additional groups of rats received only vehicle and plain drinking water or vehicle and drinking water containing 1,200 ppm of each tea fraction. Twenty-five weeks after NMBA administration began, the experiment was terminated and esophagi were excised and scored for tumors. Rats that were not dosed with NMBA had no tumors. Rats treated with NMBA only had an esophageal tumor incidence of 100% and a multiplicity of 3.3 +/- 0.4 tumors/rat. The proportion of rats developing tumors was not significantly reduced by any of the four tea fractions at the concentrations tested. However, the 1,200 ppm concentrations of each tea fraction in the drinking water produced some reduction in esophageal tumor multiplicity, although only TF significantly reduced tumor multiplicity compared with rats treated with NMBA only. The rates of esophageal tumor formation were significantly reduced at 360 and 1,200 ppm by TF and EGCG.

Effect of dietary aromatic isothiocyanates fed subsequent to the administration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone on lung tumorigenicity in mice

Naturally-occurring aromatic isothiocyanates, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), were tested for their post-treatment effects on lung tumorigenicity by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. Mice at 7 weeks of age were administered a single i.p. dose of NNK (10 mumol/mouse). One week after NNK dosing, mice were placed on AIN-76A diet containing 1 or 3 mumol/g diet of BITC or PEITC. The control group was maintained on AIN-76A diet after NNK administration. Mice were killed 16 weeks after NNK treatment and lung adenomas were counted. The results showed mice fed control diet developed 7.8 tumors/mouse. Mice fed PEITC at concentrations of 1 or 3 mumol/g diet had 8.2 or 6.1 tumors/mouse, respectively. Feeding BITC at 1 mumol/g diet resulted in a tumor yield of 8.0 tumors/mouse, whereas BITC diet at 3 mumol/g diet gave 5.2 tumors/mouse, a small but significant inhibition. However, in the high BITC dose group, a loss in weight gain due to reduced food intake was noted. The results of this study showed that post-treatment of aromatic isothiocyanates had little, if any, effect on NNK lung tumorigenicity in A/J mice. This is in contrast to our previous findings in which pretreatment with PEITC greatly inhibited lung tumor induction by NNK in A/J mice and suggests that tumor inhibition by PEITC is due to inhibition of NNK metabolic activation.

Effect of frequency of isothiocyanate administration on inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced pulmonary adenoma formation in A/J mice

The abilities of phenethyl isothiocyanate (PEITC) and 6-phenylhexyl isothiocyanate (PHITC) to inhibit 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenicity, when administered by a standard four-dose protocol or by a single-dose protocol, were determined. Corn oil or isothiocyanates were administered once or for four consecutive days by gavage, with the final (or single) administration of corn oil or inhibitor occurring 2 h prior to a single i.p. injection of NNK (10 mumol/mouse). Sixteen weeks following NNK administration, the experiment was terminated and pulmonary adenomas were quantitated. Pretreatment with PEITC at a dose of 5 mumol or PHITC at a dose of 0.2 mumol resulted in significant reductions of tumor multiplicity compared to control, regardless of whether each isothiocyanate was administered once or four times. For both isothiocyanates, there were no statistically significant differences between dosing frequencies in inhibitory effects on tumor multiplicities and tumor incidences. Furthermore, the results achieved following a single pretreatment with either isothiocyanate were in good agreement with previous results obtained utilizing the four-dose protocol. Thus, it appears that most or all of the inhibitory potential of the four-dose protocol is due to the final dose of isothiocyanate.

Inhibitory effects of 2-mercaptoethane sulfonate and 6-phenylhexyl isothiocyanate on urinary bladder tumorigenesis in rats induced by N-butyl-N-(4-hydroxybutyl)nitrosamine

The effects of 2-mercaptoethane sulfonate (MESNA) and phenethyl and 6-phenylhexyl isothiocyanates (PEITC and PHITC) on urinary bladder tumorigenesis were investigated in Wistar rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BHBN). A total of 190 male rats were divided into eight groups. Animals in groups 1, 3 and 4 were administered BHBN in drinking water (0.025%) and group 2 rats were given 0.025% BHBN plus 0.05% MESNA solution for 15 weeks. Rats in groups 3 and 4 were fed a diet supplemented with PEITC or PHITC at a dose level of 0.5 micromol/g for 16 weeks starting 1 week prior to the BHBN treatment. Rats in groups 5-7 served as MESNA, PEITC or PHITC alone controls. Group 8 served as a non-treatment control. The chromatographic profile of drinking water for group 2 animals indicated no chemical interaction between BHBN and MESNA. The incidences of papillomas in the bladder and total bladder tumors including transitional cell carcinomas were significantly (P<0.01) reduced in group 2 as compared to that in group 1. The multiplicities of papillomas or total bladder tumors were significantly (P<0.01) lower in group 2 and 4 rats treated with MESNA and PHITC, respectively, than in group 1 rats. The PEITC treatment, however, did not affect the BHBN-induced bladder tumorigenesis. No bladder tumors were found in animals of groups 5-8. The results in the present study clearly indicate that both MESNA and PHITC are potent chemopreventive agents against bladder tumor development in rats induced by BHBN.

Mechanism of enhancement of esophageal tumorigenesis by 6-phenylhexyl isothiocyanate

6-Phenylhexyl isothiocyanate (PHITC) enhances esophageal tumorigenesis induced by the carcinogen N-nitrosomethylbenzylamine (NMBA) in rats while its shorter chain analog, phenethyl isothiocyanate (PEITC), inhibits NMBA-induced esophageal tumorigenesis. A significant increase in O6-methylguanine levels in esophageal DNA at 72 h after NMBA administration to rats pretreated with PHITC suggested that PHITC might enhance NMBA metabolic activation or inhibit DNA repair. To test this hypothesis, groups of 20 rats were administered PEITC or PHITC at concentrations of 0, 1.0, or 2.5 mmol/kg in modified AIN-76A diet for 2 weeks. The esophagi were removed from rats, stripped, split, and maintained in HEPES buffered saline (HBS) for assays of NMBA metabolism (n = 5 per group) or were snap frozen for DNA repair assays (n = 15 per group). The principal metabolites of NMBA produced by esophageal explants were: two unidentified peaks, benzyl alcohol (at 4 h only), and benzoic acid. Esophageal explants from PEITC-treated animals showed a significantly decreased ability to metabolize NMBA as expected. PHITC-treated animals showed a slight inhibition in the formation of most NMBA-related metabolites, rather than an overall increase in NMBA activation. This inhibition was less than that observed with PEITC. No inhibitory effects were observed on O6-alkylguanine transferase (AGT) activity in the esophagi of rats treated with 1.0 micromol/g or 2.5 micromol/g PHITC. Thus, effects of PHITC on esophageal metabolism and DNA repair do not account for the enhancement of NMBA tumorigenicity by PHITC.

METABOLISM OF N-NITROSOBENZYLMETHYLAMINE BY HUMAN CYTOCHROME P-450 ENZYMES

N-Nitrosobenzylmethylamine (NBzMA) is a potent esophageal carcinogen in rodents, and has been found as a dietary contaminant in certain areas of China where esophageal cancer is endemic. To determine which cytochrome P-450 enzymes in humans are primarily responsible for NBzMA metabolism, microsomes from lymphoblastoid cell lines expressing a panel of human cytochrome P-450s (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP2C9, CYP2C19, and CYP3A4) and a panel of 10 different human liver microsomal preparations were examined for their abilities to metabolize [3H]NBzMA. In addition, the ability of human liver microsomes to form various NBzMA metabolites was correlated with the abilities of these preparations to metabolize coumarin, ethoxyresorufin, chlorzoxazone, 7-ethoxy-4-trifluoromethylcoumarin, S-mephenytoin, and nifedipine. NBzMA metabolites were quantitated by reversed-phase high-performance liquid chromatography (HPLC) coupled with flow-through radioactivity detection. Major metabolites included benzaldehyde, benzyl alcohol, benzoic acid, and several uncharacterized radioactive peaks. Of the representative P-450 activities, only CYP2E1 and CYP2A6 catalyzed substantial metabolism of NBzMA. Compared to CYP2E1, CYP2A6 metabolized NBzMA more readily. NBzMA acted as a potent inhibitor of coumarin 7-hydroxylation in CYP2A6 microsomes. Human liver microsomes metabolized NBzMA readily. NBzMA metabolite formation was most highly correlated with coumarin 7-hydroxylase activity, a marker of CYP2A6 activity. 8-Methoxypsoralen substantially inhibited NBzMA metabolism in human hepatic microsomes. When the effects of the potent isothiocyanates PEITC and PHITC were analyzed on microsomes from cell lines expressing CYP2E1 and CYP2A6, it was found that PEITC inhibited both enzymes, PHITC was the more effective inhibitor of CYP2E1, and PHITC was an ineffective inhibitor of CYP2A6. Collectively, these data indicate that CYP2A6 and, to a lesser degree, CYP2E1 are important P-450 enzymes in the activation of NBzMA in human systems.

Inhibition of metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by limonene

Previous work by others shows that d-limonene (LIM) inhibits carcinogen-induced lung tumorigenesis in mice and strongly suggests that LIM can inhibit the metabolic activation of nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Thus, in the current study, the ability of LIM and other monoterpenes to inhibit the activation of the tobacco-specific NNK was examined in murine pulmonary and hepatic microsomes after addition in vitro or administration in vivo. LIM inhibited the metabolic activation of NNK in both pulmonary and hepatic microsomes. Perillyl alcohol was a more potent inhibitor than LIM, while p-menth-1-ene was equipotent with LIM. After administration of LIM, limonene 1,2-oxide, or perillyl alcohol in vivo, significant inhibition of cytochrome P450-mediated metabolites (NNK N-oxide and HPB) was found at 1 and 4 h after administration of monoterpene. These results indicate that LIM and other monoterpenes are effective inhibitors of NNK metabolic activation, and that other monoterpenes such as perillyl alcohol may be effective chemopreventive agents against NNK-induced lung tumorigenesis.