Susceptibility of Escherichia coli to the toxic L-proline analogue L-selenaproline is dependent on two L-cystine transport systems.

AIMS: L-Selenaproline (L-selenazolidine-4-carboxylic acid) is a toxic analogue of L-proline that inhibits the growth of the urinary tract pathogen Escherichia coli in both laboratory culture media and normal human urine. The aim of this study was to identify the transport systems involved in its uptake. METHODS AND RESULTS: Deletion mutants from the Keio collection were tested for their susceptibility to L-selenaproline (SCA) and L-selenocystine (SeCys) on minimal salts agar medium. All single-gene mutants were sensitive to both compounds, but double mutants with deletions in fliY and ydjN or in yecS and ydjN were resistant to SCA and SeCys. The YdjN transporter active in strain JW1905 (ΔfliY::kan yecC(+) yecS(+) ydjN(+)) was inhibited by both SCA and SeCys, but the FliY YecS YecC ABC transporter system active in strain JW1718 (fliY(+) yecC(+) yecS(+) ΔydjN::kan) was best inhibited by these compounds in the presence of dithiothreitol. CONCLUSIONS: L-selenaproline and L-selenocystine are accumulated by both the FliY YecC YecS and the YdjN L-cystine transporter systems in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Because susceptibility to selenium-containing analogues of L-proline and L-cystine is dependent on multiple transport systems, these compounds may be effective in the treatment of urinary tract infections.

Effects of selenium on development, survival, and accumulation in the honeybee (Apis mellifera L.).

Apis mellifera L. (Hymenoptera: Apidae) is an important agricultural pollinator in the United States and throughout the world. In areas of selenium (Se) contamination, honeybees may be at risk because of the biotransfer of Se from plant products such as nectar and pollen. Several forms of Se can occur in accumulating plants. In the present study, the toxicity of 4 compounds (selenate, selenite, methylselenocysteine, and selenocystine) to honeybee adult foragers and larvae was assessed using dose-response bioassays. Inorganic forms were more toxic than organic forms for both larvae (lethal concentration [LC50] selenate = 0.72 mg L(-1) , LC50 selenite = 1.0 mg L(-1) , LC50 methylselenocysteine = 4.7 mg L(-1) , LC50 selenocystine = 4.4 mg L(-1) ) and foragers (LC50 selenate = 58 mg L(-1) , LC50 selenite = 58 mg L(-1) , LC50 methylselenocysteine = 161 mg L(-1) , LC50 selenocystine = 148 mg L(-1) ). Inorganic forms of Se caused rapid mortality, and organic forms had sublethal effects on development. Larvae accumulated substantial amounts of Se only at the highest doses, whereas foragers accumulated large quantities at all doses. The present study documented very low larval LC50 values for Se; even modest transfer to brood will likely cause increased development times and mortality. The toxicities of the various forms of Se to honeybee larvae and foragers are discussed in comparison with other insect herbivores and detritivores.

Comparison of different selenocompounds with respect to nutritional value vs. toxicity using liver cells in culture.

The essential micronutrient selenium (Se) exerts its biological effects mainly through enzymatically active selenoproteins. Their biosynthesis depends on the 21st proteinogenic amino acid selenocysteine and thus on dietary Se supply. Hepatically derived selenoprotein P (SEPP) is the central selenoprotein in blood controlling Se transport and distribution. Kidney-derived extracellular glutathione peroxidase is another relevant serum selenoprotein depending on SEPP for biosynthesis. Therefore, secretion of SEPP by hepatocytes is crucial to convert nutritional sources into serum Se, supporting Se status and selenoprotein biosynthesis in other tissues. In order to compare the bioactivity of 10 different selenocompounds, their dose-dependent toxicities and nutritional qualities to support SEPP and glutathione peroxidase biosynthesis were determined in a murine and two human liver cell lines. Characteristic dose- and time-dependent effects on viability and SEPP production were observed. Incubations with 100 nM sodium selenite, l- or dl-selenocystine, selenodiglutathione or selenomethyl-selenocysteine increased SEPP concentrations in the culture medium up to 6.5-fold over control after 72 h. In comparison, sodium selenate, l- or dl-selenomethionine or methylseleninic acid was less effective and increased SEPP by 2.5-fold under these conditions. As expected, ebselen did not increase selenoprotein production, supporting its classification as a stable selenocompound. Methylseleninic acid, l-selenocystine, selenodiglutathione or selenite induced cell death in micromolar concentrations, whereas selenomethionine or ebselen was not toxic within the concentration range tested. Our results indicate that hepatic selenoprotein production and toxicity of selenocompounds do not correlate with and rather represent compound-specific properties. The favourable profile of selenomethylselenocysteine warrants its consideration as a promising option for supplementation purposes.

Cysteamine prevents inhibition of adenylate kinase caused by cystine in rat brain cortex.

Cystinosis is a systemic genetic disease caused by a lysosomal transport deficiency accumulating cystine in the lysosomes of almost all tissues. Although tissue damage might depend on cystine accumulation, the mechanisms of tissue damage are still obscures. Adenylate kinase, along with creatine kinase, is responsible for the enzymatic phosphotransfer network, crucial for energy homeostasis. Taking into account that cystine is known to inhibit creatine kinase activity, the two enzymes have thiol groups, and the strong interaction between the two activities, our main objective was to investigate the effect of cystine on adenylate kinase activity in the brain cortex of Wistar rats. For the in vivo studies, the animals were injected twice a day with 1.6 micromol/g body weight of cystine dimethylester and/or 0.46 micromol/g body weight of cysteamine from the 25th to the 29th postpartum day and sacrificed after 12 h. Cystine inhibited the enzyme activity in vitro in a concentration dependent way, whereas cysteamine prevented the inhibition. Adenylate kinase activity was found diminished in the brain cortex of rats loaded with cystine dimethylester and co-administration of cysteamine prevented the diminution of the enzyme activity. Considering that adenylate kinase together with creatine kinase is crucial for energy homeostasis, the release of cystine from lysosomes with consequent enzymes inhibition could impair energy homeostasis, contributing to tissue damage in patients with cystinosis.

Cysteamine prevents inhibition of thiol-containing enzymes caused by cystine or cystine dimethylester loading in rat brain cortex.

Cystinosis is a systemic genetic disease caused by a lysosomal transport deficiency accumulating cystine in the lysosomes of all tissues. Although tissue damage might depend on cystine accumulation, the mechanisms of tissue damage are still obscures. Considering that thiol-containing enzymes are critical for several metabolic pathways, our main objective was to investigate the effects of cystine or cystine dimethylester load on the thiol-containing enzymes creatine kinase and pyruvate kinase, in the brain cortex of young Wistar rats. The animals were injected twice a day with 1.6 micromol/g body weight of cystine dimethylester or 1 micromol/g body weight of cystine and/or 0.46 micromol/g body weight of cysteamine from the 16th to the 20th postpartum day and sacrificed after 12 h. Cystine or cystine dimethylester administration inhibited the two enzyme activities. Co-administration of cysteamine, the drug used to treat cystinotic patients, normalized the two enzyme activities. Lactate dehydrogenase activity, a nonthiol-containing enzyme was not affected by cystine dimethylester administration. Cystine inhibits creatine kinase and pyruvate activities possibly by oxidation of the sulfhydryl groups of the enzymes. Considering that creatine kinase and pyruvate kinase, like other thiol-containing enzymes, are crucial for energy homeostasis and antioxidant defenses, the enzymes inhibition caused by cystine released from lysosomes could be one of the mechanisms of tissue damage in patients with cystinosis.

Cystine dimethylester model of cystinosis: still reliable?

The ability of cystine dimethylester (CDME) to load lysosomes with cystine has been used to establish the basic defect in cystinosis: defective cystine exodus from lysosomes. Using CDME loading, it has been postulated that cystine accumulation in cystinosis affects mitochondrial ATP production, resulting in defective renal tubular reabsorption. Recent studies in cystinotic fibroblasts, however, show normal adenosine triphosphate (ATP) generation capacity. To investigate the effect of CDME in more detail, mitochondrial ATP generation, reactive oxygen species production, and viability are compared in fibroblasts loaded with CDME with those of cystinotic cells with a defective cystine transporter. Intracellular cystine levels were comparable in fibroblasts loaded with CDME (1 mM, 30 min) and cystinotic fibroblasts. Intracellular ATP levels and mitochondrial ATP production were decreased in fibroblasts loaded with CDME, but normal in cystinotic fibroblasts. Superoxide production was increased with 300% after CDME loading, whereas no changes were observed in cystinotic fibroblasts. Exposure to CDME led to cell death in a time- and concentration-dependent manner. Our data demonstrate that CDME has a toxic effect on mitochondrial ATP production and cell viability. These effects are not observed in cystinotic cells, indicating that a more appropriate model is required for studying the pathogenesis of cystinosis.

Promotion of oxidative stress in kidney of rats loaded with cystine dimethyl ester.

Cystinosis is a systemic genetic disease caused by a lysosomal transport deficiency accumulating cystine in most tissues. Although tissue damage might depend on cystine accumulation, the mechanisms of tissue damage are not fully understood. Studies performed in fibroblasts of cystinotic patients and in kidney cells loaded with cystine dimethyl ester (CDME) suggest that apoptosis is enhanced in this disease. Considering that oxidative stress is a known apoptosis inducer, our main objective was to investigate the effects of CDME loading on several parameters of oxidative stress in the kidney of young rats. Animals were injected twice a day with 1.6 micromol/g body weight CDME and/or 0.26 micromol/g body weight cysteamine (CSH) from the 16th to the 20th postpartum day and killed after 1 or 12 h. CDME induced lipoperoxidation and protein carbonylation and stimulated superoxide dismutase, glutathione peroxidase (GPx), and catalase activities, probably through the formation of superoxide anions, hydrogen peroxide, and hydroxyl free radicals. Coadministration of CSH, the drug used to treat cystinotic patients, prevented, at least in part, those effects, possibly acting as a scavenger of free radicals. These results suggest that the induction of oxidative stress might be one of the mechanisms leading to tissue damage in cystinotic patients.

Excess dietary L-cysteine, but not L-cystine, is lethal for chicks but not for rats or pigs.

A comparative species investigation of the relative pharmacologic effects of sulfur amino acids was conducted using young chicks, rats, and pigs. Ingestion of excess Met, Cys, or Cys-Cys supplemented at 2.5-, 5.0-, 7.5-, or 10 times the dietary requirement in a corn-soybean meal diet depressed chick growth to varying degrees. Strikingly, ingestion of excess Cys at 30 g/kg Cys (7.5-times the dietary requirement) caused a chick mortality rate of 50% after only 5 d of feeding. Growth was restored and chick mortality was reduced by supplementing diets containing 25 g/kg excess Cys with KHCO3 at 10 g/kg. Additionally, mortality was prevented by supplementing the drinking water of chicks receiving 25 g/kg supplemental Cys with H2O2 (0.05% final concentration). After young rats and pigs consumed excess Cys or Cys-Cys up to 40 g/kg for 14 d, weight gain was severely depressed, but we observed no mortality. An excess of dietary Cys-Cys>or=48 g/kg caused some mortality in rats. Pigs exhibited rapid recovery from growth-depressing excesses of Cys or Cys-Cys. These results lend credence to the acute toxic effects associated with the ingestion of excess sulfur amino acids and highlight the potential for excess dietary cyst(e)ine to be more pernicious than Met in certain species.

Cystine inhibits creatine kinase activity in pig retina.

BACKGROUND: Cystinosis is an autosomal recessive disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Visual deficit is a possible consequence of cystine accumulation in cornea and retina. Fibroblasts from cystinotic patients present ATP deficit with intact mitochondrial energy-generating capacity by an unknown mechanism. Considering that creatine kinase is a thiol enzyme crucial for energy homeostasis in retina, and disulfides like cystine may alter thiol enzymes, the main objective of the present study was to investigate the effect of cystine and cysteamine, the drug used for treatment of cystinotic patients, on creatine kinase activity in cytosolic and mitochondrial fractions of the retina from adult pigs. METHODS: Retina was isolated from 6-month-old Landrace pigs, homogenized and mitochondrial and cytosolic fractions separated by centrifugation. Cytosolic and mitochondrial creatine kinase activities were determined in the presence of different concentrations of cystine and/or cysteamine. RESULTS: Cystine inhibited the enzyme activity in a dose- and time-dependent manner and cysteamine prevented and reversed the inhibition caused by cystine, suggesting that cystine inhibits creatine kinase activity by oxidation of the sulfhydryl groups of the enzyme. CONCLUSIONS: Considering that creatine kinase is a crucial enzyme for retina energy homeostasis, in case cystine leaves lysosome these results provide a possible mechanism for cystine toxicity and also another beneficial effect for the use of cysteamine in patients with cystinosis.

Inhibition of creatine kinase activity by cystine in the kidney of young rats.

Nephropathic cystinosis is a lethal genetic disease caused by a lysosomal transport disorder leading to intralysosomal cystine accumulation in all tissues. Cystinosis is the most common inherited cause of Fanconi syndrome, but the mechanisms by which cystine causes tissue damage are not fully understood. Thiol-containing enzymes are critical for renal energy metabolism and may be altered by disulfides like cystine. Therefore, in the present study our main objective was to investigate the in vivo and in vitro effects of cystine on creatine kinase, which contains critical thiol groups in its structure, in the kidney of young Wistar rats. We observed that cystine inhibited in vivo and in vitro the enzyme activity and that this inhibition was prevented by cysteamine and glutathione. The results suggest oxidation of essential sulfhydryl groups necessary for creatine kinase function by cystine. Considering that creatine kinase and other thiol-containing enzymes are crucial for renal energy metabolism, and programmed cell death occurs in situations of energy deficiency, the enzyme inhibition caused by cystine released from lysosomes might be a mechanism of tissue damage in patients with cystinosis.

Cytisine for the treatment of nicotine addiction: from a molecule to therapeutic efficacy.

Cytisine, a natural plant alkaloid, has been marketed in Central and Eastern Europe for over 40 years for the clinical management of smoking cessation. Despite the fact that cytisine has been used by millions of smokers, its characteristics have not been reviewed in scientific literature in English, and presently existing clinical studies on its effectiveness and safety are insufficient to warrant licensing by modern standards. Understanding of the mechanism of cytisine action as a smoking cessation aid provides a necessary basis for conducting clinical trials to confirm its efficacy as an optimal antismoking therapy. Hereafter, we present a review of current knowledge about the pharmacokinetics, pharmacodynamics, toxicity, therapeutic efficacy and safety of cytisine, and about its derivatives that are under development. Recent pharmacological research has elucidated that the drug is a low efficacy partial agonist of alpha4beta2 nicotinic acetylcholine receptors, which are believed to be central to the effect of nicotine (NIC) on the reward pathway. The drug reduces the effects of NIC on dopamine release in the mesolimbic system when given alone, while simultaneously attenuating NIC withdrawal symptoms that accompany cessation attempts. Clinical studies on cytisine as a smoking cessation aid have demonstrated that the drug is effective and safe. Our recent uncontrolled trial has shown that a 12-month carbon monoxide-verified continuous abstinence rate following a standard course of treatment with cytisine with minimal behavioral support is similar (13.8%; N = 436) to that observed following treatment with NIC replacement therapy. Since cytisine exhibits a desirable pharmacological profile which makes it an attractive smoking cessation drug, it should be advanced to randomized clinical trials. However, more detailed preclinical studies on its pharmacokinetics and safety profile are required.

Interés toxicológico de las microcistinas / Toxicological interest of Microcystins

Las Microcistinas (MCs) son toxinas de estructura heptapeptídica producidas por floraciones de cianobacterias tóxicas de aguas superficiales eutróficas. Las MCs al igual que la nodularina son hepatotóxicas en humanos, aunque también dan lugar a alteraciones gastrointestinales, reacciones alérgicas o irritación y sintomatología similar a la neumonía. El principal riesgo tóxico deriva de su actividad promotora tumoral, y capacidad genotóxica. Diversos estudios epidemiológicos sugieren una mayor incidencia de cáncer de hígado en zonas cuya población está expuesta de forma prolongada a MCs, por consumo de aguas de bebida contaminadas, advirtiéndose la necesidad de conocer otras posibles fuentes de exposición humana, tales como alimentos. La Organización Mundial de la Salud ha adoptado (WHO, 1998) un valor guía provisional de 1,0 ug/L de MCLR en aguas de bebida, comprendiendo tanto las MCs intra como las extracelulares. En la Reglamentación técnico-sanitaria vigente en España para el abastecimiento y control de la calidad de las aguas potables de consumo público (RD 1138/1990 de 14 de Septiembre) no se hace referencia a la determinación y control de MCs; sin embargo en el nuevo proyecto de reglamentación, anexo D, que hace referencia a las sustancias tóxicas, se fija una concentración máxima admisible de MCs de 1 ug/L en el caso de aguas eutróficas. En esta revisión se consideran los efectos tóxicos agudos, crónicos y el mecanismo de acción de las Microcistinas (AU)

Floraciones tóxicas de cianobacterias detectadas en el suroeste de la península ibérica

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[Selenium methylation and toxicity mechanism of selenocystine].

Selenium is an essential trace element and a toxicant for animals. Selenocystine, a selenium-containing amino acid, is one of the chemical forms in which selenium exists in food. This review summarized recent studies on the toxicity mechanism of selenocystine in experimental animals. Hepatotoxicity is caused by repeated oral administration of selenocystine. Selenocystine is metabolized by reduced glutathione and/or glutathione reductase to hydrogen selenide via selenocysteine-glutathione selenenyl sulfide. The hydrogen selenide is a key intermediate in the selenium methylation metabolism of inorganic and organic selenium compounds. Accumulation of the hydrogen selenide resulting from inhibition of the selenium methylation metabolism, detoxification metabolic pathway of selenium, is found in animals following repeated administration of a toxic dose of selenocystine. The excess of the hydrogen selenide produced by inhibition of the selenium methylation metabolism contributes to the hepatotoxicity caused by selenocystine.

Melatonin attenuates L-cysteine-induced seizures and lipid peroxidation in the brain of mice.

The effect of melatonin, a potent free radical scavenger, on L-cysteine-induced seizures and lipid peroxidation was investigated in mice. When L-cysteine (1.25, or 5.0 mumol/animal) was injected intracerebroventricularly (i.c.v.) into mice, severe tonic seizures were observed for over 20 sec in 75% and 100% of the treated mice, respectively. However, when melatonin (20 or 100 mg/kg) was injected subcutaneously (sc) into mice 15 min before L-cysteine injection (1.25 mumol/animal, i.c.v.), the incidence of seizures was observed in only 35% and 20% of the treated mice, respectively. Furthermore, when L-cysteine (1.25 or 5.0 mumol/animal, i.c.v.) was injected into mice, lipid peroxidation in whole brain 20 min after injection was significantly increased by 56% or 67% as compared to that of the control. However, when the seizures induced by L-cysteine (1.25 mumol/animal) were abolished by preadministration of melatonin, the increased lipid peroxidation induced by L-cysteine was prevented. These results suggest that there may be a positive correlation between free radical formation and seizures induced by L-cysteine and that melatonin affords protection against the seizures as well as against the associated lipid peroxidation.

Intracellular distribution of cystine in cystine-loaded proximal tubules.

Cellular cystine loading with cystine dimethyl ester has been shown to inhibit transport in proximal convoluted tubules perfused in vitro and decrease the rate of oxygen consumption in suspensions of proximal tubules. The present study was designed to examine the intracellular distribution of cystine in this model of the Fanconi syndrome of cystinosis and to determine whether cystine or its degradation product, cysteine, is the cytotoxic agent in cystine-loaded rabbit proximal tubules. Tubules were incubated with 2 mmol/L cystine dimethyl ester for 10 min at 37 degrees C and subjected to cellular fractionation. The intralysosomal cystine content (272 +/- 125 nmol/mg protein) was significantly higher than that measured in the nucleus (8.7 +/- 2.0 nmol/mg protein) and cytosol (9.8 +/- 4.0 nmol/mg protein (p < 0.05). Electron micrographs of tubules loaded with cystine depicted large swollen lysosomes. To determine whether cystine or its breakdown product, cysteine, was the cytotoxic agent in tubules incubated with cystine dimethyl ester, the intracellular cystine and cysteine contents were measured and found to be 86.5 +/- 14.8 and 5.7 +/- 1.7 nmol/mg protein, respectively. These tubules had a 50% decrease in the rate of O2 consumption. To examine whether the increased level of intracellular cysteine played a role in this decrease in O2 consumption, we loaded tubules with 2 mmol/L cysteine methyl ester for 10 min. Despite an intracellular cysteine concentration of 59.6 +/- 5.8 nmol/mg protein, cysteine-loaded tubules had a rate of O2 consumption equal to that measured in control tubules. Thus, intracellular cystine loading significantly increases intralysosomal cystine content.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicity and chemical form of selenium in the liver of mice orally administered selenocystine for 90 days.

The subacute oral toxicity of selenocystine and chemical form of selenium in the liver following exposure to this compound were assessed in ICR male mice. Animals were dosed 6 days/week for 30, 60 or 90 days with 0, 5, 10 or 15 mg/kg per day. Body weight gain decreased with dosage. The activities of aspartate aminotransferase and alanine aminotransferase in plasma were significantly elevated at the highest dose level after 60 days and at the two higher dose levels after 90 days of exposure. However, the level of selenium content in the liver was the same at the two higher dosages at both 60 and 90 days of exposure. The subcellular distribution of selenium in the liver from mice treated with selenocystine showed that the major part of the total selenium content, 68.3-72.1%, existed in the cytosolic fraction. Sephadex G-150 chromatograms of liver cytosol of the animals administered selenocystine revealed three selenium-containing fractions which involve glutathione peroxidase (molecular weight 80,000) high molecular (molecular weight 55,000-60,000) and low molecular (molecular weight < 10,000) substances. Selenium content and acid-volatile selenium content in the high molecular weight fraction increased with exposure time to selenocystine. Thus, in a subacute toxicity study selenocystine given for 90 days caused hepatic damage in mice, depending on the acid-volatile selenium content in the liver cytosol.

Description of a selection method highly cytotoxic for cystinotic fibroblasts but not normal human fibroblasts.

Nephropathic cystinosis is an inherited disorder characterized by a high intralysosomal accumulation of cystine due to a defect in lysosomal cystine transport. Cystine can be specifically loaded into the lysosomal compartment of intact cells by incubating cells with cystine dimethyl ester (CDME). We have applied this methyl ester loading technique to develop a selection method that is highly cytotoxic for cystinotic fibroblasts but not normal human fibroblasts and that is based on the inherent differences in lysosomal cystine transport activity of normal and cystinotic fibroblasts. Thus, only 0-0.03% of fetal cystinotic fibroblasts survive exposure to 2 mM CDME for 20 min whereas 70-80% of normal fetal fibroblasts survive these same conditions. Following transfection of cystinotic fibroblasts with normal human genomic DNA or cDNA, this CDME selection method can be used to select for those cells that have been transformed to the normal phenotype and thus aid in the identification of the gene coding for the lysosomal cystine transport protein.

Alterations in the immune system in cats fed diets with excess cystine.

As part of an ongoing biochemical study in nutrition we examined blood profiles, serum chemistry, lymphocyte transformation and lymphoid pathology in cats fed a diet containing 5% cystine with and without taurine. Automated blood counts of whole blood samples showed a decrease in red blood cell counts accompanied by a significant decrease in hemoglobin and hematocrit in cats fed 5% cystine in the absence of taurine compared to cats fed 0.05% taurine (control). A significant increase was noted in serum cholesterol in cats fed cystine and cystine/taurine compared to cats fed control diets. There were no significant differences in lymphocyte transformation using leukocytes isolated from the spleen and blood with the mitogens, phytohemagglutinin and pokeweed. However, lymphocyte transformation of both spleen and blood without mitogen from the excess cystine group were significantly higher than leukocytes from the 0.05% taurine group (control). Pathological examination of regional lymph nodes, livers, and spleens showed histological abnormalities in cats fed the excess cystine diet. These results indicate that there are alterations in the immune system of cats fed a diet containing 5% cystine with and without dietary taurine.

Transient hypothermia and hyperphagia induced by selenium and tellurium compounds in mice.

The effects of sublethal doses of selenite, selenate, selenocystine (Se-Cys) and selenomethionine (Se-Met) as well as of tellurite on body temperature and feeding behavior were examined in male ICR mice. Ten or 30 mumol/kg of chemicals were injected subcutaneously and body temperature was measured up to 4 h. In a separate experiment, the gastric content was weighted 4 h after injection. All chemicals except Se-Met induced both hypothermia and hyperphagia, suggesting that: (a) these two effects are related to each other; (b) among the chemicals tested, Se-Cys appears to be the most potent hypothermia inducer; (c) Se-Met is unique in that it has neither effect.