In vitro detection of differential and cell-specific hepatobiliary toxicity induced by geldanamycin and 17-allylaminogeldanamycin in rats.

The differential toxicity of two anticancer agents is described using the in vitro rat liver slice culture model. Liver slices from F-344 rats were cultured for 5 days in Waymouth's-based medium with exposure to a range of geldanamycin (GEL) or 17-allylaminogeldanamycin (17-AAG) concentrations. GEL induced concentration-dependent reduction of alkaline phosphatase and of gamma-glutamyl transferase levels, which are indicators of biliary epithelial cell(s) (BEC) viability, and exhibited hepatocellular toxicity at higher concentrations. Histologically, BEC cell injury was evident at the lowest GEL concentration (0.1 microM) and progressed to overt bile duct necrosis at 5 microM, a level at which hepatocellular damage was also more prominent. Slices exposed to the same concentrations were more sensitive to toxic effects of GEL than of 17-AAG. 17-AAG at the lowest concentration had more slice biomarker retention than GEL, and histological analysis revealed minimal toxic effect on BEC. With increasing concentration, BEC were progressively lost, and BEC proliferation was completely inhibited at 5 microM 17-AAG. Hepatocellular injury was evident only at high dose exposures. This is believed to be the first use of an in vitro liver tissue model to accurately predict the differential and concentration-dependent toxicities of these compounds.

Cancer-protective properties of high-selenium broccoli.

Selenium (Se) from high-Se garlic reduces the incidence of chemically induced mammary tumors, and Se from high-Se broccoli reduces colon cancer. However, the ability of Se from high-Se broccoli to protect against mammary cancer has not been tested. Also, the sprout form of broccoli contains many secondary plant compounds that are known to reduce cancer risk, but the anticarcinogenic activity of broccoli sprouts has not been investigated. The present studies examined the ability of high-Se broccoli or high-Se broccoli sprouts to protect against chemically induced mammary or colon cancer. In one experiment, Sprague--Dawley rats that consumed diets containing 3.0 microg of Se/g supplied as high-Se broccoli had significantly fewer mammary tumors than rats fed 0.1 microg of Se as selenite with or without the addition of regular broccoli. In the second experiment, Fisher F-344 rats fed 2.0 microg of Se/g of diet supplied as either high-Se broccoli florets or high-Se broccoli sprouts had significantly fewer aberrant colon crypts than rats fed 0.1 or 2 microg of Se/g of diet supplied as selenite with or without the addition of low-Se broccoli. These data demonstrate that the cancer-protective effect of Se in high-Se broccoli extends to mammary cancer and the protective forms of broccoli against colon cancer include high-Se broccoli sprouts.

Characterization of the biological activity of gamma-glutamyl-Se-methylselenocysteine: a novel, naturally occurring anticancer agent from garlic.

Gamma-glutamyl-Se-methylselenocysteine (GGMSC) has recently been identified as the major Se compound in natural garlic and selenized garlic. Our working hypothesis is that GGMSC serves primarily as a carrier of Se-methylselenocysteine (MSC), which has been demonstrated in past research to be a potent cancer chemopreventive agent in animal carcinogenesis bioassays. The present study was designed to examine the in vivo responses to GGMSC or MSC using a variety of biochemical and biological end points, including (a) urinary Se excretion as a function of bolus dose; (b) tissue Se accumulation profile; (c) anticancer efficacy; and (d) gene expression changes as determined by cDNA array analysis. Our results showed that like MSC, GGMSC was well absorbed p.o., with urinary excretion as the major route for eliminating excess Se. When fed chronically, the profile of Se accumulation in various tissues was very comparable after treatment with either GGMSC or MSC. In rats that had been challenged with a carcinogen, supplementation with either GGMSC or MSC resulted in a lower prevalence of premalignant lesions in the mammary gland, and fewer mammary carcinomas when these early lesions were allowed to progress. More importantly, we found that a short term GGMSC/MSC treatment schedule of 4 weeks immediately after carcinogen dosing was sufficient to provide significant cancer protection, even in the absence of a sustained exposure past the initial 4-week period. With the use of the Clontech Atlas Rat cDNA Array, we further discovered that the gene expression changes induced in mammary epithelial cells of rats that were given either GGMSC or MSC showed a high degree of concordance. On the basis of the collective biology, biochemistry, and molecular biology data, we conclude that GGMSC is an effective anticancer agent with a mechanism of action very similar to that of MSC.

Thioredoxin reductase activity in rat liver is not affected by supranutritional levels of monomethylated selenium in vivo and is inhibited only by high levels of selenium in vitro.

Thioredoxin reductase is a selenoenzyme responsible for maintaining thioredoxin in the reduced form. Because thioredoxin is involved in many cellular processes, thioredoxin reductase is likely to be an important regulatory protein for both normal and transformed cells. Monomethylated selenium compounds inhibit carcinogenesis. In the present study, we investigated whether methylated forms of selenium would alter thioredoxin reductase activity in rats. The liver enzyme was used as a model system. Se-methylselenocysteine and methylseleninic acid consumed by rats at 2 microg Se/g diet for 3, 6, 10 or 22 wk did not affect activity compared with a basal diet containing 0.1 microg Se/g. The direct addition of 50 micromol dimethyl diselenide or dimethyl selenenylsulfide per L to liver extracts significantly inhibited thioredoxin reductase activity by approximately 60%. The magnitude of inhibition was dependent on the amount of thioredoxin in the assay and was reversible by dialysis, suggesting that a competitive type of inhibition occurs in vitro. Although thioredoxin reductase can be inhibited by high levels of selenium in a cell-free system, it should be noted that such a condition is unlikely to be attainable in vivo. Caution needs to be exercised in interpreting the in vitro results.

Tumorigenesis, metabolism, speciation, bioavailability, and tissue deposition of selenium in selenium-enriched ramps (Allium tricoccum).

Ramps (Allium tricoccum) were grown either in a mixture of vermiculite and peat moss or hydroponically with various concentrations of selenium as sodium selenate. The concentrations used were from 30 to 300 mg of selenium/kg of vermiculite-peat moss or from 10 to 120 mg/L in the hydroponic solutions. Levels as high as 784 mg of selenium/kg were obtained in the ramp bulbs when grown with high levels of selenium in the vermiculite-peat moss, and up to 600 mg of selenium/kg was obtained hydroponically. The predominant form of selenium in the ramp bulbs at all concentrations of selenium was Se-methylselenocysteine, with lower amounts of selenate, Se-cystathionine, and glutamyl-Se-methylselenocysteine. There was a approximately 43% reduction in chemically induced mammary tumors when rats were fed a diet with Se-enriched ramps. Dietary Se-enriched ramps for rats did not result in excessive tissue selenium accumulation or undesirable side effects. Bioavailability studies with rats indicated that selenium in ramps was 15-28% more available for regeneration of glutathione peroxidase activity than inorganic selenium as selenite. Therefore, Se-enriched ramps appear to have potential for the reduction of cancer in humans.

Chemical speciation influences comparative activity of selenium-enriched garlic and yeast in mammary cancer prevention.

A recent human intervention trial showed that daily supplementation with selenized yeast (Se-yeast) led to a decrease in the overall cancer morbidity and mortality by nearly 50%; past research has also demonstrated that selenized garlic (Se-garlic) is very effective in mammary cancer chemoprevention in the rat model. The goal of this study was to compare certain biological activities of Se-garlic and Se-yeast and to elucidate the differences based on the chemical forms of selenium found in these two natural products. Characterization of organic selenium compounds in yeast (1922 microg/g Se) and garlic (296 microg/g Se) was carried out by high-performance liquid chromatography with inductively coupled plasma mass spectrometry or with electrospray mass spectrometry. Analytical speciation studies showed that the bulk of the selenium in Se-garlic and Se-yeast is in the form of gamma-glutamyl-Se-methylselenocysteine (73%) and selenomethionine (85%), respectively. The above methodology has the sensitivity and capability to account for >90% of total selenium. In the rat feeding studies, supplementation of Se-garlic in the diet at different levels consistently caused a lower total tissue selenium accumulation when compared to Se-yeast. On the other hand, Se-garlic was significantly more effective in suppressing the development of premalignant lesions and the formation of adenocarcinomas in the mammary gland of carcinogen-treated rats. Given the present finding on the identity of selenomethionine and gamma-glutamyl-Se-methylselenocysteine as the major form of selenium in Se-yeast and Se-garlic, respectively, the metabolism of these two compounds is discussed in an attempt to elucidate how their disposition in tissues might account for the differences in cancer chemopreventive activity.

In vitro and in vivo studies of methylseleninic acid: evidence that a monomethylated selenium metabolite is critical for cancer chemoprevention.

Previous research suggested that the beta-lyase-mediated production of a monomethylated selenium metabolite from Se-methylselenocysteine is a key step in cancer chemoprevention by this agent. In an attempt to affirm the concept, the present study was designed to evaluate the activity of methylseleninic acid, a compound that represents a simplified version of Se-methylselenocysteine without the amino acid moiety, thereby obviating the need for beta-lyase action. The in vitro experiments showed that methylseleninic acid was more potent than Se-methylselenocysteine in inhibiting cell accumulation and inducing apoptosis in TM12 (wild-type p53) and TM2H (nonfunctional p53) mouse mammary hyperplastic epithelial cells, and these effects were not attributable to DNA damage, as determined by the comet assay. In general, methylseleninic acid produced a more robust response at one-tenth the concentration of Se-methylselenocysteine. It is possible that these cell lines may have only a modest ability to generate a monomethylated selenium species from Se-methylselenocysteine via the beta-lyase enzyme. In contrast, methylseleninic acid already serves as a preformed active monomethylated metabolite, and this could be an underlying reason why methylseleninic acid acts more rapidly and exerts a more powerful effect than Se-methylselenocysteine in vitro. Interestingly, the distinction between these two compounds disappeared in vivo, where their cancer chemopreventive efficacies were found to be very similar to each other [in both methylnitrosourea and dimethylbenz(a)anthracene rat mammary tumor models]. The beta-lyase enzyme is present in many tissues; thus, animals have an ample capacity to metabolize Se-methylselenocysteine systemically. Therefore, Se-methylselenocysteine would be expected to behave like methylseleninic acid if beta-lyase is no longer a limiting factor. Taken together, the present in vitro and in vivo results provide strong evidence in support of our earlier hypothesis that a monomethylated selenium metabolite is important for cancer chemoprevention. Methylseleninic acid could be an excellent tool, especially for molecular mechanism studies in cell culture, and some of these attributes are discussed.

Chemoprevention with triphenylselenonium chloride in selenium-deficient rats.

Cancer chemoprevention by high levels of selenium, including compounds like sodium selenite or selenomethionine, is generally not accompanied by increases in known selenoenzymes. There has been no information on whether selenoenzymes are obligatory mediators of the anticarcinogenic effect of selenium. Our previous experience with triphenylselenonium chloride suggests that it might be an ideal agent for studying selenium chemoprevention while simultaneously precluding the synthesis of selenoenzymes. Triphenylselenonium chloride has excellent tumor inhibitory activity but does not support the repletion of selenoenzymes in animals that have been deprived of a bioavailable form of selenium. In the present experiments, we evaluated the efficacy of mammary cancer protection by this compound in rats fed either a selenite-deficient (< 0.01 ppm Se) or selenite-adequate (0.1 ppm Se) diet. We also measured the activities of liver glutathione peroxidase and thioredoxin reductase as markers of selenium bioavailability in these different treatment conditions. In carcinogen-treated control animals not receiving triphenylselenonium chloride, mammary tumor incidence and the total number of tumors were similar between the selenite-deficient and selenite-adequate groups. Thus the correction of selenium deficiency by the addition of 0.1 ppm Se as selenite did not have detectable anticarcinogenic effects. Supplementation of triphenylselenonium chloride at a level of 30 ppm Se suppressed mammary tumorigenesis by approximately 50% regardless of dietary selenium nutritional status. However, this supplement had little effect on tissue selenium levels and did not increase liver glutathione peroxidase or thioredoxin reductase activities. In contrast, a level of 0.1 ppm Se as selenite did not affect mammary tumorigenesis but markedly increased tissue selenium concentrations and selenoenzyme activities. It is concluded that triphenylselenonium chloride does not release inorganic selenium for selenoprotein synthesis and that its anticancer activity involves mechanisms that are probably intrinsic to the compound. This study also shows for the first time that selenium chemoprevention is possible in an environment of severely depressed selenoenzyme expression. Thus selenium chemoprevention efficacy can be separated experimentally from selenoprotein synthesis using this model system.

Selenium-induced inhibition of angiogenesis in mammary cancer at chemopreventive levels of intake.

The trace element nutrient selenium (Se) has been shown to possess cancer-preventive activity in both animal models and humans, but the mechanisms by which this occurs remain to be elucidated. Because angiogenesis is obligatory for the genesis and growth of solid cancers, we investigated, in the study presented here, the hypothesis that Se may exert its cancer-preventive activity, at least in part, by inhibiting cancer-associated angiogenesis. The effects of chemopreventive levels of Se on the intra-tumoral microvessel density and the expression of vascular endothelial growth factor in 1-methyl-1-nitrosourea-induced rat mammary carcinomas and on the proliferation and survival and matrix metalloproteinase activity of human umbilical vein endothelial cells in vitro were examined. Increased Se intake as Se-enriched garlic, sodium selenite, or Se-methylselenocysteine led to a significant reduction of intra-tumoral microvessel density in mammary carcinomas, irrespective of the manner by which Se was provided: continuous exposure (7-wk feeding) with a chemoprevention protocol or acute bolus exposure (3 d) after carcinomas had established. Compared with the untreated controls, significantly lower levels of vascular endothelial growth factor expression were observed in a sizeable proportion of the Se-treated carcinomas. In contrast to the mammary carcinomas, the microvessel density of the uninvolved mammary glands was not altered by Se treatment. In cell culture, direct exposure of human umbilical vein endothelial cells to Se induced cell death predominantly through apoptosis, decreased the gelatinolytic activities of matrix metalloproteinase-2, or both. These results indicate a potential for Se metabolites to inhibit key attributes (proliferation, survival, and matrix degradation) of endothelial cells critical for angiogenic sprouting. Therefore, inhibition of angiogenesis associated with cancer may be a novel mechanism for the anticancer activity of Se in vivo, and multiple mechanisms are probably involved in mediating the anti-angiogenic activity.

Chemoprevention of mammary cancer with Se-allylselenocysteine and other selenoamino acids in the rat.

The present study examined the mammary cancer chemopreventive activity of Se-methylselenocysteine, Se-propylselenocysteine and Se-allylselenocysteine in the rat methylnitrosourea (MNU) model. Each compound was supplemented in the diet at a level of 2 ppm Se for the entire duration of the experiment after MNU dosing. Se-Allylselenocysteine was the most active and caused a reduction in total tumor yield by 86%. Se-Methylselenocyteine and Se-propylselenocysteine were similar but less effective, and both produced a decrease of about 50% in tumorigenesis. All three compounds were very well absorbed through the gastrointestinal tract. However, more selenium was excreted in urine after gavaging with Se-propylselenocysteine or Se-allylselenocysteine compared with Se-methylselenocysteine. Analysis of selenium in the mammary gland and other organs showed that tissue selenium levels did not appear to be correlated with differences in chemopreventive activity. A lyase activity capable of catalyzing scission of the Se-alkyl group from the remainder of the amino acid was demonstrated. This activity was found to be high in liver and kidney, but relatively low in mammary gland and intestine. Minimal variations in enzyme activity towards each of the substrates were observed. Our results support the concept that Se-alkylselenoamino acids could be used as precursors for delivering the Se-alkyl moiety and that intrinsic chemical differences in the Se-alkyl substituent of the test compounds are likely to be important determinants of their biological effects.

Lessons from basic research in selenium and cancer prevention.

The article reviews the progress in basic research of selenium and cancer prevention during the past decade. Special emphasis is placed on the following four major areas of discussion: 1) chemical forms of selenium and anticarcinogenic activity; 2) selenium-enriched food; 3) in vitro effects of selenite vs. monomethylated selenium; and 4) aromatic selenium compounds. It is clear that basic research has contributed new knowledge to our understanding of selenium biochemistry, anticancer efficacy and regulation of cell growth. Some of this information could be ready for incorporation into the design of a second-generation selenium trial in humans.

Activities of structurally-related lipophilic selenium compounds as cancer chemopreventive agents.

The present study compared the effects of four lipophilic forms of selenium with regard to cancer chemopreventive activity, tissue selenium accumulation, and bioavailability for synthesis of a selenoprotein. These reagents included methylphenyl selenide, diphenyl selenide, triphenyl-selenonium chloride, and p-xylylbis(methylselenide). The maximum tolerable dose (added in the diet) for each of these compounds was 5, 30, > 200, and 5 ppm Se, respectively. Because of differences in their tolerance, the cancer chemopreventive activities (in a methylnitrosourea-induced mammary tumor model in rats) of all 4 compounds were assessed at the 5 ppm Se level. Methylphenyl selenide was the most effective--79% inhibition, followed by p-xylylbis-(methylselenide)--66% inhibition, triphenylselenonium chloride--27% inhibition, and diphenyl selenide--10% inhibition. With respect to tissue selenium levels, p-xylylbis(methylselenide) produced the highest accumulation of selenium (approximately 3-fold increase in liver and kidney, 14-fold increase in mammary gland); methylphenyl selenide and diphenyl selenide showed more modest increases (1.5-fold or less in liver and kidney, 2.5-fold or less in mammary gland); while triphenylselenonium chloride resulted in no change. Highest bioavailability of selenium was observed for p-xylylbis(methylselenide), which was followed closely by methylphenyl selenide. Bioavailability was very low with diphenyl selenide, and undetectable with triphenylselenonium chloride. The chemical reactivities of these different selenium compounds are discussed in relation to the biological effects reported here.

Review of the effects of trans fatty acids, oleic acid, n-3 polyunsaturated fatty acids, and conjugated linoleic acid on mammary carcinogenesis in animals.

I review the effects of trans fatty acids, oleic acid, n-3 polyunsaturated fatty acids, and conjugated linoleic acid on mammary carcinogenesis in animals. The goal is not to provide an exhaustive survey of all the publications on these topics; such a Herculean effort has been accomplished by previous reviews, which are cited in the text. Instead, the emphasis is on the consistency or lack of consistency of information regarding each of the above fatty acids, confounding factors that may help to reconcile discrepancies in the database, a perspective of the history of the research, and certain unique or exciting opportunities that are worthy of special attention in evaluations of the relations between specific fatty acids and cancer. This review arrives at four conclusions: 1) There is little evidence that trans fatty acids have an adverse effect on carcinogenesis. 2) The data on cancer protection by oleic acid are not convincing. An inhibitory effect attributed to an increased intake of oleic acid could be due to an inadequate supply of linoleic acid. 3) Although a suppressive response to n-3 polyunsaturated fatty acids is observed in most cases, the availability of linoleic acid is likely to be a confounding factor in determining the final outcome. 4) Conjugated linoleic acid is unique in the sense that concentrations < or = 1% are sufficient for producing significant cancer protection and that this effect seems to be independent of the other fatty acids.

Triphenylselenonium and diphenylselenide in cancer chemoprevention: comparative studies of anticarcinogenic efficacy, tissue selenium levels and excretion profile.

The objectives of the present study were to evaluate the cancer chemopreventive activity of triphenylselenonium chloride and diphenylselenide and to investigate the pharmacology of these two compounds with respect to their tissue accumulation and excretion profile. Although both phenyl selenide derivatives are related to each other structurally, they differ substantially in their intrinsic chemical properties. Triphenylselenonium is positively charged and amphiphilic, while diphenylselenide is uncharged and lipophilic. With the use of either the DMBA- or MNU-induced mammary tumor model in rats, triphenylselenonium was found to have superior chemopreventive efficacy compared to diphenylselenide. Both reagents were present at 30 ppm Se in the diet. At the time of sacrifice (22 weeks post-carcinogen), triphenylselenonium produced only minimal accumulation of selenium in the liver, kidney, mammary gland and plasma. In contrast, diphenylselenide caused a 2- to 3-fold elevation in selenium concentration depending on the tissue examined. Thus even though diphenylselenide was able to increase total selenium in tissues, it was less active in cancer protection. Fecal excretion following a single oral dose of triphenylselenonium (equal to the amount consumed in 1 day by an animal fed a diet containing 30 ppm Se) was approximately 78% and 8% of the dose during the first and second day, respectively, suggesting that the bulk of the dose was not absorbed. With diphenylselenide, fecal excretion was about 6% and 30% of the dose during the first and second day, and about 20% of the dose was excreted in the urine in each of the 2 days. This observation suggests that a large proportion of the diphenylselenide dose was absorbed and that urinary excretion was a major route of elimination for diphenylselenide once it was absorbed. Further studies are needed to clarify the basis for the differential effects of these phenyl selenide derivatives.

Morphological and biochemical status of the mammary gland as influenced by conjugated linoleic acid: implication for a reduction in mammary cancer risk.

Previous research showed that treatment with conjugated linoleic acid (CLA) during the period of active mammary gland morphogenesis was sufficient to confer a lasting protection against subsequent mammary tumorigenesis induced by methylnitrosourea. The present study was designed to characterize certain morphological and biochemical changes of the mammary gland that might potentially render it less susceptible to cancer induction. Female Sprague Dawley rats were fed a 1% CLA diet from weaning until about 50 days of age. The mammary gland parameters under investigation included (a) the deposition of neutral lipid, (b) the identification and quantification of CLA and its metabolites, (c) the density of the epithelium, and (d) the proliferative activity of various structural components. Our results showed that CLA treatment did not affect total fat deposition in the mammary tissue nor the extent of epithelial invasion into the surrounding fat pad but was able to cause a 20% reduction in the density of the ductal-lobular tree as determined by digitized image analysis of the whole mounts. This was accompanied by a suppression of bromodeoxyuridine labeling in the terminal end buds and lobuloalveolar buds. The recovery of desaturation and elongation products of CLA in the mammary gland confirmed our prior suggestion that the metabolism of CLA might be critical to risk modulation. The significance of the above findings was investigated in a mammary carcinogenesis bioassay with the use of the dimethylbenz[a]anthracene model. When CLA was started at weaning and continued for 6 months until the end of the experiment, this schedule of supplementation produced essentially the same magnitude of mammary tumor inhibition in the dimethylbenz[a]anthracene model as that produced by 1 month of CLA feeding from weaning. The observation is consistent with the hypothesis that exposure to CLA during the time of mammary gland maturation may modify the developmental potential of a subset of target cells that are normally susceptible to carcinogen-induced transformation.

Modulation of phase I and phase II xenobiotic-metabolizing enzymes by selenium-enriched garlic in rats.

Previous research showed that treatment with selenium-enriched garlic (Se-garlic) was able to inhibit the initiation phase of mammary carcinogenesis in the dimethyl-benz[a]anthracene (DMBA) model in rats. The present study was designed to investigate the following parameters: 1) DMBA-DNA adduct formation in liver and mammary gland, 2) urinary excretion of DMBA metabolites, 3) phase I and phase II xenobiotic-metabolizing enzymes, and 4) tissue selenium levels as a function of Se-garlic supplementation. Prior feeding with an Se-garlic-containing diet (at 3 ppm Se) for two weeks resulted in a consistent reduction of all DMBA adducts in liver and mammary gland. This was accompanied by a 40% increase in urinary excretion of DMBA metabolites over a two-day period. Several liver P-450 enzymes were examined in rats fed a diet supplemented with 1, 2, or 3 ppm Se. Compared with controls receiving 0.1 ppm Se, no significant alteration in activity was detected with respect to P-450 1A1 (responsible for DMBA activation), 1A2, 2B1, 2E1, and 3A4. In contrast, glutathione S-transferase and uridine 5'-diphosphate-glucuronyltransferase activities were elevated to a maximum of 2- to 2.5-fold in liver and kidney. As expected, there was a dose-dependent elevation of selenium concentrations in liver, kidney, mammary gland, and plasma as a function of the level of Se-garlic supplementation. Our data seem to suggest that an increased detoxification of carcinogen via the phase II conjugating enzymes might represent a mechanism of tumor suppression by Se-garlic.

Chemoprevention of mammary cancer by diallyl selenide, a novel organoselenium compound.

Previous research has demonstrated that structurally distinctive organoselenium compounds are superior to the corresponding sulfur analogs in cancer prevention. The present study was designed to extend this observation to diallyl selenide (DASe), a volatile synthetic compound, and diallyl sulfide (DAS), a flavor component of garlic. Their anticarcinogenic activities were evaluated using the 7,12-dimethylbenz(a)-anthracene (DMBA)-induced mammary tumor model. Rats were gavaged three times with DASe (6 or 12 mumol/kg body wt) or DAS (300, 900 or 1,800 mumol/kg) at 96, 48 and 24 hours before DMBA treatment. Significant tumor inhibition was found with the two doses of DASe and the highest dose of DAS. Based on these results, DASe appears to be at least 300 times more active than DAS. Analysis of total DMBA-DNA binding and individual DNA adducts in the mammary gland and liver showed that DASe had no effect on these parameters, suggesting that DASe might influence some unknown risk-associated events other than carcinogen activation/detoxification. Although the mechanism of action of DASe remains to be elucidated, its potential relevance to natural products will be discussed in the context of the chemistry of selenium-enriched garlic which has been reported to be effective in cancer protection in several studies.

Effect on an aqueous extract of selenium-enriched garlic on in vitro markers and in vivo efficacy in cancer prevention.

Previous work has shown that the efficacy of cancer prevention by selenium-enriched garlic (Se-garlic) is primarily dependent on the action of selenium. An aqueous extract containing 43 micro Se/ml was prepared from lyophilized Se-garlic powder by the Soxhlet method. The activity of this Se-garlic extract was evaluated in a transformed mammary epithelial cell culture model for its effect on cell morphology, cell growth, cell cycle progression and the induction of single and double stranded breaks in DNA. Comparisons were also made with a similarly prepared extract from regular garlic, Se-methylselenocysteine (a major water-soluble seleno-amino acid identified in Se-garlic) and selenite (used for fertilizing Se-garlic). In contrast to the regular garlic extract which produced little or no modulation of the above parameters, treatment with the Se-garlic extract resulted in growth inhibition, GI phase cell cycle arrest and apoptotic DNA double strand breaks in the absence of DNA single strand breaks. This pattern of cellular responses was duplicated with exposure to Se-methylselenocysteine. Selenite, on the other hand, induced cell cycle blockage in the S/G2-M phase, and a marked increase in DNA single strand breaks (a measure of genotoxicity) in addition to growth suppression. The chemopreventive efficacy of the two garlic extracts was also investigated in the rat methylnitrosourea mammary tumor model. Both extracts were supplemented in the diet for 1 month immediately following carcinogen administration. Significant cancer protection was observed with treatment by the Se-garlic extract (at 3 p.p.m. Se in the diet), while little benefit was noted with treatment by the regular garlic extract. Based on the above in vitro and in vivo findings, it is hypothesized that the Se-garlic extract, in part via the action of Se-methylselenocysteine, is able to inhibit tumorigenesis by suppressing the proliferation and reducing the survival of the early transformed cells. Furthermore, the data also support the concept that the modulation of certain in vitro markers may be of value in predicting the effectiveness of novel forms of selenium for cancer prevention.

Selenium-enriched garlic inhibits the early stage but not the late stage of mammary carcinogenesis.

Previous work has shown that the efficacy of cancer prevention by selenium-enriched garlic (Se-garlic) is primarily dependent on the action of selenium. Additionally, supplementation of Se-garlic inhibited the post-initiation phase of mammary carcinogenesis when it was given continuously to the animals. In this report, experiments were carried out in which treatment with the Se-garlic was started after carcinogen dosing (DMBA or MNU) but was restricted to either the early or late stage of neoplastic progression. The results from these two models showed that a short-term exposure to the Se-garlic for 1 month immediately following carcinogen administration was just as effective in cancer prevention as the continuous exposure regimen (5 months), suggesting that the Se-garlic may irreversibly alter the process of clonal expansion and/or selection of transformed cells during their early stage of development. Plasma and mammary tissue selenium levels essentially returned to basal levels at 1 month after withdrawal of supplementation. These observations imply that the outcome of cancer protection by short-term Se-garlic intervention was not due to a slow turnover, and therefore a lingering presence, of selenium in the target organ or in the circulation. The above finding was in contrast to that of a second study in which Se-garlic was supplemented starting at 13 weeks after carcinogen treatment. With this protocol, the number of new tumors and the number of new tumor-bearing rats found during the intervention period (weeks 13 to 22) were not statistically different between the control and supplemented groups, suggesting that Se-garlic had a minimal effect on the later stages of mammary carcinogenesis.

The efficacy of conjugated linoleic acid in mammary cancer prevention is independent of the level or type of fat in the diet.

The objective of the present study was to investigate whether the anticarcinogenic activity of conjugated linoleic acid (CLA) is affected by the amount and composition of dietary fat consumed by the host. Because the anticancer agent of interest is a fatty acid, this approach may provide some insight into its mechanism of action, depending on the outcome of these fat feeding experiments. For the fat level experiment, a custom formulated fat blend was used that simulates the fatty acid composition of the US diet. This fat blend was present at 10, 13.3, 16.7 or 20% by weight in the diet. For the fat type experiment, a 20% (w/w) fat diet containing either corn oil (exclusively) or lard (predominantly) was used. Mammary cancer prevention by CLA was evaluated using the rat dimethylbenz[a]anthracene model. The results indicated that the magnitude of tumor inhibition by 1% CLA was not influenced by the level or type of fat in the diet. It should be noted that these fat diets varied markedly in their content of linoleate. Fatty acid analysis showed that CLA was incorporated predominantly in mammary tissue neutral lipids, while the increase in CLA in mammary tissue phospholipids was minimal. Furthermore, there was no evidence that CLA supplementation perturbed the distribution of linoleate or other fatty acids in the phospholipid fraction. Collectively these carcinogenesis and biochemical data suggest that the cancer preventive activity of CLA is unlikely to be mediated by interference with the metabolic cascade involved in converting linoleic acid to eicosanoids. The hypothesis that CLA might act as an antioxidant was also examined. Treatment with CLA resulted in lower levels of mammary tissue malondialdehyde (an end product of lipid peroxidation), but failed to change the levels of 8-hydroxydeoxyguanosine (a marker of oxidatively damaged DNA). Thus while CLA may have some antioxidant function in vivo in suppressing lipid peroxidation, its anticarcinogenic activity cannot be accounted for by protecting the target cell DNA against oxidative damage. The finding that the inhibitory effect of CLA maximized at 1% (regardless of the availability. of linoleate in the diet) could conceivably point to a limiting step in the capacity to metabolize CLA to some active product(s) which is essential for cancer prevention.