Comparative effects of phenylenebis(methylene)selenocyanate isomers on xenobiotic metabolizing enzymes in organs of female CD rats

The cancer chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC) inhibits various chemically induced tumors in laboratory animals. We examined the effects of p-XSC and its o- and m-isomers on xenobiotic metabolizing enzymes in vivo. Six-week-old female CD rats were given diets containing o-, m- or p-XSC (5 or 15 p.p.m. as Se), or equimolar amounts (30 or 90 micromol/kg) of 1,4-phenylenebis(methylene)thiocyanate (p-XTC, the sulfur analog of p-XSC) for 1 week. At termination, substrate-specific assays for enzymes of xenobiotic metabolism in various organs were performed. Overall, o-XSC was a more potent enzyme inducer than m- or p-XSC. In hepatic microsomes, o-XSC significantly induced CYP2E1 as detected by increased N-nitrosodimethylamine N-demethylase activity and also by western blot. The activities of CYP1A1 (ethoxyresorufin-O-dealkylase) and CYP1A2 (methoxyresorufin-O-dealkylase) were not affected, but a significant decrease in the activity of CYP2B1 (pentoxyresorufin-O-dealkylase) was observed at the 15 p.p.m. Se level of o-XSC. With the m- and p-XSC isomers or with p-XTC, no significant effect on phase I enzymes was noted. Hepatic UDP-glucuronosyltransferase activities were increased 1.5- to 2-fold by all three XSC isomers at the higher dose level (15 p.p.m. Se), but not by p-XTC; o-XSC again was the most effective. All three XSC isomers were found to increase the alpha, mu and pi isozymes of glutathione S-transferases in the liver, kidney, lung, colon and mammary gland to varying degrees. The XSC isomers also significantly increased glutathione peroxidase in the colon and mammary gland. Although o-XSC was the most powerful in stimulating the enzyme activities, especially in the liver, atomic absorption spectrometry showed that the selenium levels were highest in organs of rats given p-XSC. Thus, the level of tissue distribution of the XSC isomers and/or their metabolite(s) does not correlate with their effects on enzyme activities. The present study demonstrates that individual XSC isomers are capable of modulating specific phase I and/or phase II enzymes involved in the activation and/or detoxification of chemical carcinogens, and provides some mechanistic basis for the cancer chemopreventive efficacy of these organoselenium compounds at the stage of tumor initiation.     

Inhibition of 7,12-dimethylbenz(a)anthracene-induced tumors and DNA adduct formation in the mammary glands of female Sprague-Dawley rats by the synthetic organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate.

We synthesized a novel organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate (XSC), possessing low toxicity by comparison with inorganic Na2SeO3, and several other synthetic organoselenium compounds (K. El-Bayoumy, Cancer Res., 45: 3631-3636, 1985). We tested the effect of XSC treatment during the initiation phase on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma formation. A semipurified high-fat diet containing 80 ppm of XSC (40 ppm as selenium) was fed to 6-wk-old virgin female Sprague-Dawley rats for 2 wk, starting 1 wk before and ending 1 wk after carcinogen treatment. At 7 wk of age, rats were given a single dose of DMBA (5 mg) in 0.2 ml of olive oil by gastric intubation; the experiment was terminated 16 wk later. The development of mammary tumors in those rats that received XSC-supplemented diets was significantly inhibited when compared with the control group (fed the same diet without XSC supplements). This was evident from tumor incidence (percentage of tumor-bearing rats, 88 versus 20) and multiplicity of tumors (mean number of tumors/rats, 3.96 versus 0.28). The finding that XSC acts as a chemopreventive agent in the DMBA mammary tumor model prompted us to examine the effect of dietary XSC on DMBA-DNA binding in both the liver and mammary tissue under conditions identical to those described above for the bioassay. Rats (four/group) were killed 6, 24, 48, and 168 h after [3H]DMBA (5 mg/rat; specific activity, 51.2 mCi/mM) administration. Liver and mammary tissue were obtained and DNA was isolated. Dietary XSC was found to inhibit total DMBA-DNA binding in the mammary tissue, but not in the liver. The most profound effect was observed at early time points, i.e., 24 to 48 h after [3H]DMBA administration. The inhibition in total binding was attributed to a reduction in the formation of the three major adducts derived from bay-region diol-epoxides of DMBA; these were identified as anti-diol-epoxide:deoxyguanosine, syn-diol-epoxide:deoxyadenosine, and anti-diol-epoxide:deoxyadenosine adducts on the basis of their chromatographic characteristics on high-pressure liquid chromatography and on a boronate affinity column. The inhibition of the DMBA-DNA binding in the target tissue provides a plausible explanation for the chemopreventive effect of XSC during the initiation stage of carcinogenesis.     

Synthesis and excretion profile of 1,4- [14C]phenylenebis(methylene)selenocyanate in the rat

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) inhibits chemically induced tumors in several laboratory animal models. To understand its mode of action, we synthesized p-[14C]XSC, examined its excretion pattern in female CD rats and also the nature of its metabolites. p- [14C]XSC was synthesized from alpha,alpha-dibromo-p-[ring-14C]xylene in 80% yield. The excretion profile of p-[14C]XSC (15.8 mg/kg body wt, 200 microCi/rat, oral administration, in 1 ml corn oil) in vivo was monitored by measuring radioactivity and selenium content. On the basis of radioactivity, approximately 20% of the dose was excreted in the urine and 68% in the feces over 3 days. The cumulative percentages of the dose excreted over 7 days were 24% in urine and 75% in feces, similar to excretion rates of selenium. According to selenium measurement, <1% of the dose was detected in exhaled air; radioactivity was not detected. Only 15% of the dose was extractable from the feces with EtOAc and was identified as tetraselenocyclophane (TSC). Most of the radioactivity remained tightly bound to the feces. Approximately 10% of this bound material converted to TSC on reduction with NaBH4. Organic soluble metabolites in urine did not exceed 2% of the dose; sulfate (9 % of urinary metabolites) and glucuronic acid (19.5% of urinary metabolites) conjugates were observed but their structural identification is still underway. Co-chromatography with a synthetic standard led to the detection of terephthalic acid (1,4- benzenedicarboxylic acid) as a minor metabolite. The major urinary conjugates contained selenium. Despite the low levels of selenium in the exhaled air, the reductive metabolism of p-XSC to H2Se cannot be ruled out. Identification of TSC in vivo indicates that a selenol may be a key intermediate responsible for the chemopreventive action of p- XSC.      

Elevated 8-hydroxy-2'-deoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and inhibition by dietary 1,4-phenylenebis(methylene)selenocyanate

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) is an effective chemopreventive agent against 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK)-induced lung adenoma in female A/J mice. While p-XSC can effectively inhibit NNK-induced DNA methylation in female A/J mice and in male F344 rats, its effect on NNK-induced oxidative DNA damage had not been determined. Thus, the effect of p-XSC on the levels of 8- hydroxy-2'-deoxyguanosine (8-OH-dG) in lung DNA from A/J mice and F344 rats treated with NNK was examined. Mice were given NNK by gavage (0.5 mg/mouse in 0.2 ml corn oil, three times per week for 3 weeks) or by a single i.p. injection (2 mg/mouse in 0.1 ml saline) while maintained on a control diet (AIN-76A) or control diet containing p-XSC at 10 or 15 p.p.m. (as Se) starting 1 week before NNK administration and continuing until termination. Mice were killed 2 h after the last NNK gavage in the multiple administration protocol or 2 h after the single i.p. injection. Treatment with NNK by gavage significantly elevated the levels of 8-OH-dG in lung DNA of A/J mice from 0.7 +/- 0.1 to 1.6 +/- 0.2 adducts/10(5) 2'-deoxyguanosine (dG) (P < 0.001), while dietary p- XSC (at 10 p.p.m. Se) prevented significant elevation of the levels of this lesion caused by NNK, keeping them at 0.9 +/- 0.1 adducts/10(5) dG (P < 0.003). Injection of NNK in saline also significantly increased the levels of 8-OH-dG in lung DNA of A/J mice from 1.2 +/- 0.6 to 3.6 +/- 0.8/10(5) dG adducts (P < 0.01), while dietary p-XSC (at 15 p.p.m. Se) kept these levels at 1.9 +/- 0.5 adducts/10(5) dG (P < 0.03). Rats were given a single i.p. injection of NNK (100 mg/kg body wt) in saline while being maintained on control diet (AIN-76A) or control diet containing p-XSC (15 p.p.m. as Se) starting 1 week before NNK administration and continuing until termination. The rats were killed 2 h after injection. Treatment with NNK using this protocol significantly elevated the levels of 8-OH-dG in lung DNA of F344 rats from 2.6 +/- 0.5 to 3.5 +/- 0.5 adducts/10(5) dG (P < 0.03), while dietary p-XSC (at 15 p.p.m. Se) kept the levels of this lesion at 2.2 +/- 0.6 adducts/10(5) dG (P < 0.01). Our findings suggest that the chemopreventive efficacy of p-XSC against NNK-induced lung tumorigenesis in A/J mice and F344 rats may be due in part to inhibition of oxidative DNA damage.      

Effects of a combination of docosahexaenoic acid and 1,4-phenylene bis(methylene) selenocyanate on cyclooxygenase 2, inducible nitric oxide synthase and beta-catenin pathways in colon cancer cells

Epidemiological and preclinical studies suggest that diets that are rich in n-3 polyunsaturated fatty acids (PUFAs) and selenium (Se) reduce the risk of colon cancer. Studies conducted in our laboratory have indicated that synthetic organoselenium 1,4-phenylene bis(methylene) selenocyanate (p-XSC) is less toxic and more effective than inorganic Se and selenomethionine, the major Se compound in natural selenium yeast. Through cDNA microarray analysis, we have demonstrated earlier that the n-3 PUFA docosahexaenoic acid (DHA), modulated more than one signaling pathway by altering several genes involved in colon cancer growth. There is increasing interest in the use of combinations of low doses of chemopreventive agents that differ in their specific modes of action as this approach can minimize toxicity and increase efficacy in model assays. In the present study we assessed the efficacy of DHA and p-XSC individually and in combination at low doses in CaCo-2 colon cancer cells, using cell growth inhibition and apoptosis as measures of chemopreventive efficacy. On the basis of western blot and RT-PCR analysis, we also determined the effects of DHA and p-XSC on the levels of expression of cyclooxygenase-2, inducible nitric oxide synthase, cyclin D1, beta-catenin and nuclear factor kappaB, all of which presumably participate in colon carcinogenesis. A 48 h incubation of CaCo-2 cells with 5 microM each DHA or p-XSC induced cell growth inhibition and apoptosis and altered the expression of the above molecular parameters. Interestingly, the modulation of these cellular and molecular parameters was more pronounced in cells treated with low doses of DHA and p-XSC (2.5 microM each) in combination than in cells treated with these agents individually at higher concentrations (5.0 microM each). These findings are viewed as highly significant since they will provide the basis for the development of combinations of low dose regimens of DHA and p-XSC in preclinical models against colon carcinogenesis and, ultimately, in human clinical trials.     

Chemoprevention of lung tumorigenesis induced by a mixture of benzo(a)pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by the organoselenium compound 1,4-phenylenebis(methylene)selenocyanate

We evaluated the chemopreventive efficacy of the organoselenium compound 1,4-phenylenebis(methylene)selenocyanate (p-XSC) against the development of tumors of the lung and forestomach induced by a mixture of benzo(a)pyrene (B(a)P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), two of the major lung carcinogens present in tobacco smoke. A/J mice (20 mice/group) were given intragastric doses of a mixture of B(a)P (3 micromol/mouse) and NNK (3 micromol/mouse) in cottonseed oil (0.1 ml) once a week for eight consecutive weeks. Mice were fed either AIN-76A control diet or control diet containing p-XSC (10 ppm selenium), either during or after carcinogen administration. Dietary p-XSC significantly reduced lung tumor multiplicity, regardless of whether it was given during or after carcinogen administration. p-XSC was also an effective inhibitor of tumor development in the forestomach. To provide some biochemical insights into the protective role of p-XSC, its effect on selected phase I and II enzyme activities involved in the metabolism of NNK and B(a)P was also examined in vivo in this animal model. Dietary p-XSC significantly inhibited the activities of the phase I enzymes, methoxyresorufin O-dealkylase (MROD) and N-nitrosodimethylamine N-demethylase (NDMAD), in mouse liver, but it had no effect on ethoxyresorufin O-dealkylase (EROD), pentoxyresorufin O-dealkylase (PROD), and erythromycin N-demethylase (ERYTD). Total glutathione S-transferase (GST) enzyme activity, as well as GST-pi and GST-mu enzyme activities, were significantly induced by dietary p-XSC in both the lung and liver. Glutathione peroxidase (GPX) activity was also induced by p-XSC in mouse lung, but not in the liver. Dietary p-XSC had no effect on selenium-dependent glutathione peroxidase (GPX(Se)), GST-alpha, and UDP-glucuronosyl transferase (UDPGT) enzyme activities in either the lung or the liver. These studies suggest that the chemopreventive efficacy of p-XSC, when fed during carcinogen administration, may be, in part, due to the inhibition of certain phase I enzymes involved in the metabolic activation of these carcinogens, and the induction of specific phase II enzymes involved in their detoxification. The mechanisms that account for the effect of p-XSC when fed after carcinogen administration remain to be determined.     

In vitro and in vivo effects of sodium selenite on 7,12-dimethylbenz[a]anthracene--DNA adduct formation in isolated rat mammary epithelial cells

Supplementation with increasing quantities of selenium (Se), as sodium selenite, to cultures of rat mammary epithelial cells resulted in a proportional depression in 7,12-dimethylbenz[a]anthracene (DMBA) binding to DNA. A depression in the two major anti bay-region dihydrodiol epoxide-deoxyribonucleoside adducts largely accounted for the reduced binding. DMBA-DNA binding in freshly isolated mammary cells from rats fed a diet containing 2.0 p.p.m. Se and incubated in culture with DMBA for 24 h was decreased 32% compared to binding in cells obtained from rats fed 0.1 p.p.m. Se. DMBA-DNA binding in mammary cells obtained from rats fed supplemental Se for 2 weeks or more was depressed compared to that occurring in cells from unsupplemented rats. The reduced ability of cells obtained from rats previously fed a 2.0 p.p.m. Se diet to activate DMBA to intermediates capable of binding to DNA became increasingly apparent with the duration of exposure to the carcinogen. Consistent with the in vitro supplementation study, cells isolated from rats previously fed a diet containing 2.0 p.p.m. Se had a reduced occurrence of the two major anti dihydrodiol epoxide adducts. The depression in DMBA binding following selenite supplementation, both in vitro and in vivo, supports the ability of Se to inhibit the initiation phase of carcinogenesis.     

Chemoprevention of colon carcinogenesis by the synthetic organoselenium compound 1,4-phenylenebis(methylene)selenocyanate.

The chemopreventive effect of 40% and 80% maximum tolerated dose (MTD) levels of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) administered in the diet during the initiation phase (2 weeks before, during, and up to 3 days after carcinogen administration) and the post-initiation phase (3 days after carcinogen treatment until termination) of azoxymethane (AOM)-induced colon carcinogenesis was studied in male F344 rats. The MTD of p-XSC was determined in male F344 rats and found to be 50 ppm. Beginning at 5 weeks of age, all animals were divided into various experimental groups (42 rats/group) and fed the high-fat semipurified diet or diets containing 20 (40% MTD) and 40 (80% MTD) ppm p-XSC. At 7 weeks of age, all animals (30 rats/group) except the vehicle-treated groups (12 rats/group) were administered s.c. injections of AOM (15 mg/kg body weight/week for 2 weeks). Three days after the second injection of AOM or vehicle (normal saline), groups of animals fed the p-XSC diets and control diet were transferred, respectively, to control diet and p-XSC diets and continued on these diets until the termination of the study. All animals were necropsied during the 36th week after AOM treatment. Colonic mucosal prostaglandin E2 and selenium-dependent glutathione peroxidase were measured in animals fed the control and p-XSC diets at the termination of the study. The results indicate that 40 ppm p-XSC administered during the initiation phase significantly inhibited the colon tumor incidence (percentage of animals with tumors). Dietary p-XSC administered at 20 and 40 ppm levels during the initiation phase significantly inhibited colon tumor multiplicity (tumors/animal and tumors/tumor-bearing animal). Colon tumor incidence and multiplicity were significantly reduced in groups fed 20 and 40 ppm p-XSC diets at the postinitiation phase of carcinogenesis. Colonic mucosal selenium-dependent glutathione peroxidase activity was increased, and prostaglandin E2 was reduced in animals fed the p-XSC diet compared to animals fed the control diet. Whereas the precise mechanisms of p-XSC-induced inhibition of colon carcinogenesis remain to be elucidated, it is likely that the effect during the initiation and postinitiation phases may be due to alteration in carcinogen metabolism and to modulation of prostaglandin synthesis and selenium-dependent glutathione peroxidase activity.     

The effect of four chemical forms of selenium on mammary tumor incidence in BALB/c female mice treated with 7-12-dimethylbenz[a]anthracene

The study was conducted to determine the effect of four forms of selenium on inhibition of DMBA-induced mammary tumors. BALB/c virgin female mice were fed the AIN-76 diet containing 0.2 or 2.0 ppm Se as selenite, selenate, selenomethionine, or selenocystine prior to and for 6 months post DMBA-treatment. At necropsy, mammary glands were histologically treated for confirmation of adenocarcinomas and the livers were removed for analysis of glutathione-peroxidase (GSHPx) activity and selenium concentrations. Dietary levels or forms of selenium had no effect on body weights. Inorganic selenium fed at 2.0 ppm Se (selenite and selenate) decreased mammary tumor incidence, but organic selenium (selenomethionine and selenocystine) had no effect on mammary tumor incidence. Hepatic GSHPx activity was highest with the 2.0 ppm selenium as selenocystine diet, but hepatic selenium levels were highest with the 2.0 ppm selenium as selenite. This study showed that the dietary form of selenium affects inhibition of mammary tumorigenesis. Furthermore, the study suggested that the pathways for selenium incorporation into GSHPx and for tumor inhibition are different.     

Chemoprevention of colon cancer by a glutathione conjugate of 1,4-phenylenebis(methylene)selenocyanate, a novel organoselenium compound with low toxicity

We have consistently shown that several synthetic Organoselenium compounds are superior cancer chemopreventive agents and less toxic than selenite or certain naturally occurring selenoamino acids. 1,4-Phenylenebis(methylene)selenocyanate (p-XSC) is the lead Organoselenium compound in that it has been shown to be the most effective and the least toxic agent in several experimental cancer models. It is not known whether p-XSC or one of its metabolites is responsible for its chemopreventive efficacy. As an initial step, we synthesized one of its putative metabolites, i.e., the glutathione conjugate of p-XSC (p-XSe-SG), and determined its stability in the pH range from 2 to 8 and in the diet under normal feeding conditions. We also assessed its maximum tolerated dose and examined its chemopreventive efficacy against azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. p-XSe-SG proved to be very stable over the pH range tested. The maximum tolerated dose of p-XSe-SG determined in a 6-week subchronic toxicity study was found to be >210 ppm (>40 ppm selenium) when the compound was added to AIN-76A high-fat diet. To assess the efficacy of this agent in the postinitiation period of colon carcinogenesis, male F344 rats 6 weeks of age were fed the high-fat diet, and at beginning of weeks 7 and 8, all of the rats intended for carcinogen treatment were given AOM at a dose of 15 mg/kg body weight by s.c. injection. Two days after the carcinogen treatment, the groups of rats consuming the high-fat control diet began their respective high-fat experimental diet regimens with 0, 56, or 84 ppm p-XSe-SG (0.1, 10, and 15 ppm of selenium) supplementation. All animals continued on their respective diets for 38 weeks after the AOM-treatment and were then killed. Colon tumors were evaluated histologically using routine procedures and were also analyzed for cyclooxygenase (COX)-1 and COX-2 expression and enzymatic activities. The results indicate that p-XSeSG administered during the post-initiation stage significantly inhibited both the incidence (P < 0.05-0.01) and the multiplicity (P < 0.05-0.005) of AOM-induced colon adenocarcinomas. This agent also greatly suppressed the multiplicity (P < 0.01-0.001) of AOM-induced exophytic adenocarcinomas in a dose-dependent manner. Feeding of 56 or 84 ppm p-XSe-SG in the diet significantly suppressed total COX activity (P < 0.02 to -0.01) and COX-2 specific activity (P < 0.005-0.0005) but had minimal effect on the protein expression levels of COX-1 and COX-2. These results suggest that the newly developed synthetic Organoselenium compound, p-XSe-SG, is stable in the diet and at wide pH ranges, inhibits colon carcinogenesis when administered during the postinitiation stage, and inhibits COX activity. Compared with previous efficacy studies and considering the toxicity associated with selenium, p-XSe-SG seems to be the least toxic Organoselenium chemopreventive agent thus far tested in the experimental colon carcinogenesis. Studies are in progress to delineate whether p-XSe-SG is also effective when administered during the progression stage of colon carcinogenesis.     

Comparative effect of inorganic and organic selenocyanate derivatives in mammary cancer chemoprevention

Recently El-Bayoumy and coworkers have reported that l, 4-phenylene-bis(methylene)setenocyanate(p-XSC) was very effective in inhibiting 7, 12-dimethylbenz(a)anthracene(DMBA)-induced mammary carcinogenesis and adduct formation duringthe initiation phase (Cancer Res., 52, 2402–2407, 1992).Furthermore, this compound was found to be well tolerated byrats at high doses. The present study was designed to extendthese earlier observations by investigating the response tolower levels of p-XSC given either before or after DMBA administration.At a level of 15 p.p.m. Se, p-XSC suppressed total mammary tumoryield by 80% and 52% in the initiation phase and post-initiationphase, respectively. A dose-response effect was evident in therange 5–15 p.p.m. Se. When p-XSC was given at a levelof 5 p.p.m. Se during the entire course of the experimentalperiod, total tumor yield was reduced by half. This dose isabout 4 x less than the maximum tolerable dose (MTD). Otherseleno-cyanate analogs were also examined in an attempt to obtaininformation on their respective chemopreventive index, whichis calculated as the ratio of MTD to the effective dose whichproduces approximately a 50% inhibition in total tumor yield(ED₅₀). The reagents studied included potassium seleno-cyanate,methyl selenocyanate and benzyl selenocyanate, as well as sodiumselenite (reference compound). Compared to p-XSC, which hasa chemopreventive index of 4.0, the other four compounds havea lower index ranging from 1.3 for sodium selenite and potassiumselenocyanate to 2.0 for methyl selenocyanate and 2.5 for benzylselenocyanate. A high chemopreventive index signifies that acompound is well tolerated at doses required for cancer suppression.The last component of the present study involved the repletionassay of liver glutathione peroxidase in selenium-deficientrats as a biomarker to estimate the metabolizability of theabove selenium compounds. The bioavailability data suggest thatthe selenium from p-XSC is not as efficiently incorporated intoglutathione peroxidase as the selenium from selenite or theother selenocyanate analogs. Currently, we are working underthe hypothesis that the chemical structure of the RSeCN compoundcould affect activity per se and also influence the rate ofrelease of selenium from the parent compound, thereby impactingon the anticarcinogenic efficacy, tolerance and bioavailabilityof the compound.     

Effects of 1,4-phenylenebis(methylene)selenocyanate and selenomethionine on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone- induced tumorigenesis in A/J mouse lung

We reported earlier that continuous feeding of 1,4- phenylenebis(methylene)selenocyanate (p-XSC) inhibited lung tumor induction by the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK) in the A/J mouse (El-Bayoumy et al., Carcinogenesis, 14, 1111-1113, 1993). The present investigation was designed to determine whether p-XSC inhibits pulmonary neoplasia induced by NNK in female A/J mice during the initiation phase of carcinogenesis or during the post-initiation phase. The naturally occurring selenomethionine was also included in this study. Doses higher than 4 p.p.m. of selenomethionine can induce toxic effects, therefore, dietary supplementation of this compound was selected at a dose level of 3.75 p.p.m. However, we were able to give p-XSC at selenium levels of 7.5 and 15 p.p.m., as mice can tolerate such doses in this form without any adverse effects. NNK was given by a single i.p. injection at dose of 10 micromol in 0.1 ml of saline. Selenomethionine did not show chemopreventive activity when administered in either phase of tumorigenesis. In contrast, p-XSC significantly reduced lung tumor multiplicity regardless of whether it was given during the initiation phase of tumorigenesis (P = 0.0009 at both levels of selenium) or post-initiation (P = 0.0009 at 15 p.p.m. and P = 0.036 for 7.5 p.p.m.). This is the first report describing that the synthetic organoselenium compound, p-XSC, can effectively block and suppress chemically (NNK)-induced lung tumor development in mice.      

Inhibition of 7,12-dimethylbenz(a)anthracene-induced mammary tumors and DNA adducts by dietary selenite

The present studies were designed to examine the influence of dietary selenite supplementation on the initiation phase of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis and to correlate selenite-induced changes in the binding of DMBA metabolites to rat mammary cell DNA with the ultimate tumor incidence. Diets formulated to contain selenium, as sodium selenite at 0.1, 0.5, 1, 2, or 4 micrograms/g were fed for 2 weeks prior to and 2 weeks following treatment with DMBA (5 mg/kg body weight). Food intake and weight gain did not differ among treatments. Tumor incidence correlated inversely to the quantity of selenium consumed (r = -0.99). Final tumor incidences were 52, 32, 24, 14, and 10% for rats fed 0.1, 0.5, 1, and 4 micrograms selenium/g, respectively. In a separate group of rats fed a diet containing 4 micrograms selenium/g during both the initiation and promotion stages the final tumor incidence was 4.8%. Selenite supplementation for 2 weeks markedly depressed the occurrence of individual and total DMBA-DNA adducts. The final mammary tumor incidence correlated positively with total DMBA-DNA adducts (r = 0.99). These studies clearly demonstrate that selenite can inhibit the initiation stage of mammary carcinogenesis. This reduction in tumor incidence is likely due to a reduction in carcinogen metabolism and ultimately adduct formation.     

The effects of 1-nitropyrene, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 7,12-dimethylbenz[a]anthracene on 8-hydroxy-2'-deoxyguanosine levels in the rat mammary gland and modulation by dietary 1,4-phenylenebis(methylene) selenocyanate

Humans are exposed to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 1-nitropyrene (1-NP) via several environmental sources and both are known mammary carcinogens in rodents, with the former being more potent (K. El-Bayoumy, Y.-H. Chae, P. Upadhyaya, A. Rivenson, K. Kurtzke, B. Reddy, S.S. Hecht, Comparative tumorigenicity of benzo[a]pyrene, 1-nitropyrene, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine administered by gavage to female CD rats, Carcinogenesis 16 (1995) 431-434). Following their metabolic activation, both carcinogens are known to bind covalently to DNA. However, it remains to be determined whether these carcinogens can also induce DNA-base oxidation. Our goal was to determine the effects of PhIP and 1-NP on the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG; a marker of oxidative DNA damage) in rat mammary glands and to evaluate the effect of the chemopreventive agent 1,4-phenylenebis(-methylene)selenocyanate (p-XSC) as an inhibitor of such damage. As an established potent mammary carcinogen, the synthetic 7,12-dimethylbenz[a]anthracene (DMBA) was included in this study. Female CD rats were fed a high-fat AIN-76A diet (23.5% corn oil) supplemented with p-XSC (10 ppm as selenium) or unsupplemented control diet for 1 week. At 50 days of age, each rat (12 rats/group) was gavaged with either PhIP (22 mg (100 micromol) per rat) or I-NP (20 mg (80 micromol) per rat) in trioctanoin (0.5 ml), DMBA (5 mg (20 micromol) per rat] in olive oil (0.2 ml), or the corresponding vehicle. Rats were sacrificed 6 and 24 h after carcinogen treatment (six rats per time point). Mammary fat pads were excised and DNA was isolated and enzymatically hydrolyzed. The hydrolysates were analyzed for 8-OHdG using HPLC with EC detection. PhIP significantly increased the levels of 8-OHdG by 83% after 6 h (P < 0.05), but the increase (47%) at the 24 h point was not significant. p-XSC alone had no effect on the levels of 8-OHdG. However, the elevation of 8-OHdG caused by PhIP at 6 h was significantly inhibited by p-XSC to levels similar to those measured in rats treated with the vehicle only (P < 0.05). p-XSC had no effect on PhIP-induced 8-OHdG at 24 h. I -NP had no effect on the levels of 8-OHdG at either time point. Levels of 8-OHdG were increased by 22% 6 h after DMBA administration and, significantly, rose to 84% at 24 h (P < 0.01); at either time point, this elevation was not inhibited by p-XSC. Although the mechanisms remain to be determined, to our knowledge, this is the first report demonstrating that PhIP and DMBA are capable of enhancing 8-OHdG levels in the rat mammary tissue in vivo.     

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.      

Efficacy of cancer prevention by high-selenium garlic is primarily dependent on the action of selenium

We reported previously that garlic cultivated with selenitefertilization showed powerful chemopreventive activity in therat dimethylbenz[a]anthracene (DMBA)-induced mammary tumor model(Carcinogenesis 15, 573–576, 1994). In order to ascertainthat the efficacy of the high-selenium garlic in cancer protectionis primarily dependent on the action of selenium we comparedthe effects of two batches of garlic powder with marked differencesin their level of selenium enrichment, 112 or 1355 p.p.m. Sedry weight. Both products were added to the diet to achievethe same final concentration of 2 p.p.m. Se. The supplementationprotocol was designed to evaluate the efficacy during eitherthe initiation phase or post-initiation phase of DMBA mammarycarcinogenesis. Significant tumor reduction was observed witheither treatment protocol. Furthermore, the magnitude of tumorsuppression, as well as the extent of DMBA-DNA adduct inhibition,were very similar with the two batches of garlic, even thoughthe amounts of garlic in the diet varied considerably betweenthem (1.8% for the 112 p.p.m. Se garlic versus 0.15% for the1355 p.p.m. Se garlic). This suggests that the anti-cancer activityof the high-selenium garlic was likely to be accounted for bythe effect of selenium, rather than the effect of garlic perse. A continuous feeding of the high-selenium garlic produceda modest increase in total selenium in various tissues. In generalthe profile of selenium accumulation was comparable in ratsingesting either the 112 or the 1355 p.p.m. Se garlic. Thus,based on the results of several biological responses, it appearsthat the ability of the high-selenium garlic to protect againsttumorigenesis is primarily dependent on increased intake ofselenium provided by the vegetable. Future research will befocused on the chemical form of selenium in the garlic.     

Chemoprevention of familial adenomatous polyposis development in the APC(min) mouse model by 1,4-phenylene bis(methylene)selenocyanate

Epidemiological and experimental studies have suggested that dietary supplementation with selenium can inhibit the development of cancers at several organ sites. We have consistently shown that 1, 4-phenylene bis(methylene) selenocyanate (p-XSC) is a highly effective cancer chemopreventive agent against the development of chemically induced cancers in several laboratory animal species. This is the first report describing the preventive effects of p-XSC in an animal model of familial adenomatous polyposis (FAP) containing a germline mutation of the APC gene. Six-week old male (heterozygous) C57BL/6J-APC(min) or wild-type mice were fed high fat diets containing 0, 10 or 20 p.p.m. p-XSC. After 80 days, the mice were killed and their intestines were excised and evaluated for polyps. Multiple samples were also harvested from normal appearing small intestine and colon for molecular analysis. Both the mucosa and polyps from the intestine and colon were assayed for beta-catenin, cyclooxygenase (COX)-2 expression and COX isoform activities. Administration of p-XSC in the diet significantly decreased the rate of formation of small intestinal tumors (P < 0. 0001) and colon tumors (P < 0.002) in APC(min) mice. p-XSC produced a dose-dependent inhibition of tumors in both small intestine (P < 0. 0001) and colon (P < 0.035). Mice fed 20 p.p.m. p-XSC had significantly lower levels of beta-catenin expression and COX-2 activity in polyps. These observations demonstrate for the first time that the synthetic organoselenium compound p-XSC possesses antitumor activity against genetically predisposed neoplastic lesions, such as FAP. While the exact mechanism(s) for this antitumor activity of p-XSC remains to be elucidated, it appears that modulation of beta-catenin expression and COX-2 activity is associated with inhibition of intestinal polyps.     

Inhibition of nuclear factor-kappaB DNA binding by organoselenocyanates through covalent modification of the p50 subunit

Most known chemopreventive agents including certain selenium compounds suppress the activation of the nuclear factor kappaB (NF-kappaB), but the mechanisms remain largely elusive. Toward this end, we initially showed that the inhibition of NF-kappaB DNA binding by benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) was reversed by the addition of DTT; this suggests the formation of DTT-reducible selenium-sulfur bonds between selenocyanate moieties and cysteine residues in NF-kappaB (p50) protein. Furthermore, the inhibitory effect of selenocyanates on NF-kappaB was not altered in the presence of physiologic level of reduced glutathione (1 mmol/L), suggesting that selenocyanates can also inhibit NF-kappaB in vivo. Using both matrix-assisted laser desorption/ionization-time of flight and tandem mass spectrometry fragmentation, we showed for the first time that the Cys(62) residue in the active site of NF-kappaB (p50) protein was modified by BSC through the formation of a selenium-sulfur bond. In addition, p-XSC-bound NF-kappaB (p50) protein was also detected by a radiotracer method. To provide further support, molecular models of both BSC and p-XSC positioned in the DNA binding pocket of the p50 were constructed through the covalent modification of Cys(62); the models reveal that DNA substrate could be hindered to enter its DNA binding region. This study shows for the first time that BSC and p-XSC may exert their chemopreventive activity, at least in part, by inhibiting NF-kappaB through covalent modification of Cys(62) of the p50 subunit of NF-kappaB.     

Effects of temporary selenium supplementation on the genesis of spontaneous mammary tumors in inbred female C3H/St mice

Female inbred C₃H/St mice infected with the Bittner Milk Particle,develop mammary adenocarcinoma with 27% incidence if maintainedon a Torula Yeast diet supplemented with 1 ppm of selenium (organicallybound, in yeast). Animals switched from the 1 ppm Se diet toa diet containing only 0.15 ppm Se after reaching the age of13.8 months develop mammary tumors rapidly during their remaininglife span, the overall tumor incidence reaches 69%, not statisticallydifferent from the 77% incidence of tumors observed in animalsmaintained on the 0.15 ppm Se diet over their entire post-weaninglife span. Conversely, animals changed from the 0.15 ppm Sediet to that containing 1.0 ppm Se at the age of 13.8 monthsdevelop mammary tumors with a total incidence of only 46%, significantlylower (P<0.05) than in the 0.15 ppm Se control group. Thisstudy demonstrates that dietary selenium prevents and retardstumor development only as long as it is supplied in adequateamounts, consistent with its role as a non-accumulative tracenutrient.     

Mechanisms in the chemoprevention of colon cancer: modulation of protein kinase C, tyrosine protein kinase and diacylglycerol kinase activities by 1,4-phenylenebis-(methylene)selenocyanate and impact of low-fat diet

Epidemiological and experimental studies suggest an inverse relationship between the intake of dietary selenium and/or low fat-intake and colon cancer risk. Efficacy studies in rodents suggest that the organoselenium compound 1, 4-phenylenebis(methylene)selenocyanate (p-XSC), is a more effective and less toxic chemopreventive agent than other organic or inorganic selenium compounds such as selenomethionine and Na2SeO3. The efficacy of p-XSC against colon cancer is significantly augmented by a low-fat diet. To explore the mechanisms by which this combined inhibiting effect against colon carcinogenesis comes about, we have investigated protein kinase C (PKC), tyrosine protein kinase (TPK), diacylglycerol kinase (DGK) activities and 8-isoprostane levels in colonic mucosa and tumor tissues in an azoxymethane (AOM)-induced rat colon cancer model. Weanling male F344 rats were fed the semipurified AIN-76A diet until seven weeks of age. Then various experimental groups were fed the low- or high-fat diets containing 0 or 20 ppm p-XSC (10 ppm as selenium). At seven weeks of age, groups of rats were injected s.c. with azoxymethane (AOM; 15 mg/kg body wt., once weekly for 2 weeks) and continued on their respective experimental diets until 38 weeks after the second AOM treatment. They were then sacrificed and colonic mucosal and tumor samples were evaluated for PKC, TPK, DGK and 8-isoprostane levels. Administration of p-XSC along with a low-fat diet significantly inhibited Ca+2-dependent and -independent PKC (P<0.05-0.01) activities in colonic mucosa and tumors. Administration of p-XSC either low-fat or high-fat diet significantly suppressed both colonic mucosal and tumor TPK activity (P<0.05-0.01). Suppression of TPK activity was more pronounced in rats maintained on a low-fat diet containing p-XSC. In contrast, rats receiving p-XSC with either low- or high fat diet showed significantly increased DGK activity (P<0.01-0.0001). Rats fed low-fat or high-fat plus p-XSC had lower-levels of 8-isoprostane in the colonic tumors than animals who had been given low- or high-fat diets without the organoselenium compound. Interestingly, 8-isoprostane levels were lower in the colon tumors of the rats fed the low-fat diet than those fed the high-fat diet. Our findings suggest that p-XSC induced down-regulation of PKC and TPK activities and up-regulation of DGK activity. These events may in part be responsible for the chemopreventive activity against colon carcinogenesis. Further, this study implies that p-XSC with a low-fat dietary regimen will augment regulation of PKC, TPK and DGK activities in the colon.