John A. Milner

The effect of various seleno-compounds on ehrlich ascites tumor cells

The effect of various concentrations and forms of selenium on in vitro viability of Ehrlich Ascites Tumor Cells (EATC) was investigated. Sodium selenite, selenium dioxide, seleno-dl-cystine, and seleno-dl-methionine, dramatically decreased EATC viability as measured by dye exclusion. Sodium selenate only marginally decreased EATC viability. Cell viabilities decreased with increasing selenium in the incubation media and as a function of time. Viabilities determined by dye exclusion did not correlate with the inhibition of tumor growth observed after treatment with selenium. Intraperitoneal injections of selenite in mice previously inoculated with EATC significantly inhibited tumor development. Delaying intraperitoneal injections of selenite to 5 and 7 days after inoculation of mice with EATC reduced the effectiveness of this nutrient on the inhibition of EATC growth. Incubation of EATC in vitro with supplemental selenium prior to injection of mice completely inhibited EATC development in vivo before any appreciable alteration in cell viability was observed.

Antimutagenic effect of selenium on acridine orange and 7,12-dimethylbenz[a]anthracene in the Ames Salmonella/ microsomal system ☆

The antimutagenic effects of selenium as sodium selenite were investigated using the Ames Salmonella/microsome mutagenicity test. The compounds examined were acridine orange and 7,12-dimethylbenz[alpha]anthracene. Selenium (22 ppm) reduced the number of histidine revertants caused by 20 microgram acridine orange and 20 microgram 7,12-dimethylbenz[alpha]anthracene by 52 and 74%, respectively. Increasing the quantity of selenium added to the plates further suppressed the mutagenicity of the test compounds. The antimutagenic effects of selenium cannot be explained by lethality of Salmonella typhimurium.

Characteristics of human milk glutathione peroxidase.

Human milk glutathione peroxidase (GPx) was purified 4500-fold using acetone precipitation and purification by repetitive ion-exchange and gel filtration chromatography with an overall yield of 34%. Homogeneity was established by gel electrophoresis. Using gel filtration, the molecular weight (mol wt) of the enzyme was estimated to be 92 kdalton (kD). The monomeric molecular weight was estimated to b 23 kD from polyacrylamide gel electrophoresis, indicating that the native enzyme consists of four identical subunits. The molecular weight of each subunit was supported by amino acid analysis. Selenium (Se) content of the purified enzyme was 0.31%, in a stoichiometry of 3.7 g-atoms/mol. Data from these studies reveal that GPx provided approximately 22% of total milk Se, but only 0.025% of the total protein.

The effects of dietary selenium on the biotransformation of 7,12-dimethylbenz[a]anthracene☆

The influence of dietary selenium on the mutagenic activation of 7,12-dimethylbenz[a]anthracene (DMBA) by rat liver S9 was studied using the Ames test. Rats received supplemental selenium, as sodium selenite, in the drinking water or in the diet. All rats additionally received 0, 20, 50, 100, or 500 mg Aroclor 1254 per kg body weight. Revertant counts decreased 72 and 31% at the 20- and 100-mg/kg induction levels, respectively, with S9 preparations from rats given selenium supplementation, compared to controls. No significant effects of selenium on S9 preparations was observed in rats treated with 500 mg/kg Aroclor. Preparations of S9 from rats receiving 2.5 ppm Se in their diet produced 46, 84 and 70% less revertants than controls at the 20-, 50- and 100-mg/kg induction levels. Increasing the selenium concentration in the diet to 5 ppm reduced the revertant counts by 71, 68 and 65%, at the 20-, 50- and 100-mg/kg induction level of Aroclor, respectively. Dietary selenium supplementation was shown to decrease the mutagenic activation of DMBA by liver microsomes. These studies indicate that in vivo selenium supplementation may reduce susceptibility to the action of various carcinogens.

Inhibition of Chemical Carcinogenesis and Tumorigenesis by Selenium

Selenium is effective in inhibiting the incidence and total number of tumors resulting from treatment with various chemical carcinogens. This inhibition occurs both at the initiation and promotion phases of chemical carcinogenesis. At least part of the inhibition of the initiation stage is associated with changes in the metabolism of the parent carcinogen. Studies with 7,12-dimethylbenz(a)anthracene suggest that selenium specifically blocks the enzyme(s) responsible for the formation of anti-dihydrodiol epoxide adducts to DNA. Selenium is also effective in reducing the in vitro and in vivo growth of numerous neoplastic cells. However, differences in the sensitivity to selenium are evident in the various tumor cell lines that have been examined. Continuous selenium intake appears to be necessary to maximal inhibition in both models of carcinogenesis. Evidence suggests that selenodiglutathione or some other intermediate in selenium metabolism is responsible for the anticarcinogenic and antitumorigenic properties of this trace element. The mechanism by which selenium produces these effects is unknown, but it may relate to alterations in either RNA transcription or translation. These and other data strongly suggest that selenium is a naturally occurring anticarcinogenic and antitumorigenic agent.

Consequences of selenite supplementation on the growth and metabolism of cultures of canine mammary cells.

Previous studies with cultures of canine mammary cells revealed differences in the degree of growth inhibition caused by selenite supplementation, with canine mammary tumor cell line 13 > 11 >> non-neoplastic canine mammary cells. The present studies show this variation in growth retardation cannot be explained by selenium retention. Intracellular glutathione related inversely to the degree of growth inhibition resulting from the addition of selenite. Dimethyl selenide formation by S-9 preparations corresponded to the sensitivity of the culture to supplemental selenite. DL-buthionine-SR-sulfoximine, a specific inhibitor of glutathione biosynthesis, accentuated the growth inhibition and prevented the increase in intracellular glutathione caused by supplemental selenite. Treatment of canine mammary tumor cell line 13 cultures with DL-buthionine-SR-sulfoximine resulted in a persistent depletion of intracellular glutathione without affecting growth. Glutathione reductase activity, before and following selenite, was inversely related to the degree of growth inhibition, with canine mammary tumor cell line 13 > 11 > non-neoplastic canine mammary tumor cell line. Selenite addition increased the activity of gamma-glutamylcysteine synthetase in canine mammary tumor cell line 11 and non-neoplastic canine mammary cells, but not in canine mammary tumor cell line 13 cells. The present data suggest the differences in the growth inhibition caused by selenite among these mammary cells is related to glutathione regulation and ultimately to selenium detoxification.

Impact of ascorbic acid on selenium-induced growth inhibition of canine mammary tumor cells in vitro

Abstract Ascorbic acid (50 or 125 μmol/L) accentuated the in vitro growth inhibition of canine mammary tumor cell line 13 (CMT-13) induced by sodium selenite (12.6 μmol/L). While selenate was less effective than selenite in inhibiting growth of these cells, its inhibitory effect was also amplified by supplemental ascorbic acid. Ascorbic acid did not modify the anti-proliferative effects of selenomethionine. Both ascorbic and D-isoascorbic acid (an ascorbate analogue without anti-scorbutic properties) were equally effective in increasing the toxic effects of selenite against CMT-13 cells. Ascorbic acid (125 μmol/L) increased cellular selenium retention. These data show that the antitumorigenic effects of selenite and selenate can be enhanced by supplemental ascorbic acid or a related reducing compound.

In vitro effects of selenodiglutathione on canine mammary cells

Abstract Previous studies have shown that selenodiglutathione (SDG) is toxic to canine mammary tumor cell line 13 (CMT-13), reduces the growth of CMT-11, and does not modify the growth of primary non-neoplastic canine mammary (NCM) cells. In the present studies, approximately 6 times more SDG was required to inhibit by 50% the growth of CMT-11 cells compared with CMT-13 cells. Furthermore, these studies reveal that differences in sensitivity among these mammary cells cannot be explained by cellular retention of selenium from SDG. Data obtained using SDG (3.2 μ m ) double labelled with 75Se and 35S suggest that differences in the rate of selenium detoxification exist among the three cell cultures examined. The influence of SDG (3.2 μ m ) on intercellular glutathione depended on the cell examined, increasing in CMT-11, decreasing in CMT-13, and unchanged in NCM. Addition of GSH (100 μ m ) partially protected CMT-13 and CMT-11 cells against SDG toxicity. Preincubation of CMT-13 or CMT-11 cells with GSH for 48 hr before the addition of SDG completely prevented the growth inhibition caused by this selenocompound. These studies suggest glutathione is critical to the prevention of selenodiglutathione toxicity and accompanying growth depression.

Influence of ammonia and arginine upon orotic acid and urea biosynthesis in human liver

Abstract The in vitro rates of orotic acid and urea biosynthesis were determined in human liver obtained by biopsy. Increasing the quantity of ammonia added to the incubation medium from 0.0 mM to 5.0 mM resulted in an approximate 200% increase in liver urea biosynthesis and about a doubling of orotate biosynthesis. Supplementation of arginine to the medium doubled the rate of urea biosynthesis above that at saturated ammonia (5.0 mM) concentrations. Arginine supplementation to a medium containing 5mM ammonia reduced the rate of liver orotate biosynthesis to rates occuring with no supplemental ammonia. These studies show that the interrelationship between urea cycle activity and pyrimidine biosynthesis are similar metabolically to those observed previously in other mammals.

Effect of dietary protein concentration on rat-liver S9 bioactivation of 7,12-dimethylbenz[a]anthracene in the Salmonella/microsome assay.

These studies describe the influence of dietary protein concentration on the ability of rat-liver postmitochondrial fraction (S9) to mediate 7,12-dimethylbenz[a]anthracene (DMBA) mutagenicity in the Ames Salmonella/microsome assay. A negative correlation was observed between dietary protein content and DMBA mutagenicity. The effect of protein was not dependent on the quantity of Aroclor used for induction. Dietary protein did not influence the mutagenicity of DMBA when isolated microsomes were used in place of S9.