Mortality in mice infected with an amyocarditic coxsackievirus and given a subacute dose of mercuric chloride.

An amyocarditic strain of coxsackievirus B3 (CVB3/0) induces heart damage when inoculated into selenium (Se)-deficient mice. Mercury (Hg), an Se antagonist, is known to aggravate viral infections. The experiments reported here assessed the effect of prior Hg treatment in mice subsequently inoculated with an amyocarditic strain of coxsackievirus. A pilot study showed that under our conditions the maximum tolerated dose of HgCl2 in uninfected mice was 6 mg HgCl2/kg body weight. In the main study, doses of 0, 3 or 6 mg HgCl2/kg body weight were administered intraperitoneally (ip) to 7-wk-old male mice fed a standard chow diet. Two hours later, half the mice were inoculated ip with CVB3/0. Ten days postinoculation, no mortality was observed in mice given only virus. In mice not given virus, 10% injected with 6 mg HgCl2/kg body weight died. On the other hand, 64% of the mice given both virus and 6 mg HgCl2/kg body weight died. Fifteen percent of the hearts from virus-infected mice given 3 mg HgCl2/kg body weight and 33% of the hearts from virus-infected mice given 6 mg HgCl2/kg body weight exhibited a higher incidence of lesions than hearts from mice-given virus alone. Moreover, viral heart titers were elevated in infected mice injected with 6 mg HgCl2/kg body weight compared to infected mice receiving no Hg. Thus, an amyocarditic coxsackievirus given to mice after a nonlethal subacute dose of Hg results in mortality, increased incidence of heart lesions, and elevated viral heart titers. These results demonstrate the important role of toxic elements in determining the severity of viral infections.

Rapid determination of glutathione peroxidase and thioredoxin reductase activities using a 96-well microplate format: comparison to standard cuvette-based assays.

Gluthatione peroxidase and thioredoxin reductase are selenocysteine-containing enzymes that are constituents of the cellular antioxidant defense system. Conventional cuvette-based assays for glutathione peroxidase and thioredoxin reductase enzymes are laborious and time consuming. The ability to assay their activities rapidly in multiple samples would aid efforts focused on understanding the impact of these enzymes on the cellular antioxidant defense system. High throughput can be achieved with assays adapted to work in a clinical analyzer but require expensive equipment. Assays designed to work in a 96-well microplate reader provide an alternative methodology for high throughput with reduced instrumentation cost. However, due to differences in the light pathlength when using a 96-well format, the values obtained cannot be compared directly with those obtained using a 1-cm cuvette. Described here are assays for glutathione peroxidase and thioredoxin reductase modified to work in a 96-well format that incorporates light pathlength determinations into the assays. The values obtained using a high throughput 96-well format in conjunction with pathlength determinations are in agreement with those obtained using a standard 1-cm cuvette. While spectrophotometrically derived pathlengths are the most accurate, calculated pathlengths based on assay volume and well size can be used with only a small amount of error introduced. This method can also be applied to many other enzyme assays, thus allowing the rapid analysis of large numbers of samples without the need for expensive equipment.

Aurothioglucose inhibits murine thioredoxin reductase activity in vivo.

Gold (I)-containing compounds, including aurothioglucose (ATG), are potent in vitro inhibitors of several selenocysteine-containing enzymes. Gold compounds have also been shown to potentiate the virulence of several viruses in mice, including coxsackievirus, implicated as a possible infectious agent in Keshan disease. One possible mechanism by which gold compounds may be increasing the virulence of viral infections in mice is by acting as a selenium antagonist in vivo and inducing oxidative stress. To investigate the possible role of gold compounds in inducing oxidative stress in mice, we assessed the ability of ATG administered in vivo to inhibit the activity of the selenocysteine-containing enzymes thioredoxin reductase (TR) and glutathione peroxidase (GPX1). Doses as low as 0. 025 mg ATG/g body weight caused significant and prolonged inhibition of TR activity in all tissues examined. No such inhibition of GPX1 activity was seen, indicating differential in vivo sensitivity of the enzymes to inhibition by ATG. In liver and heart, some recovery of TR activity was observed after a 7-d period, but no recovery was observed in pancreas or kidney. Because TR is involved in several important cellular redox functions, its inhibition most likely will affect multiple cellular processes. These results indicate that in vivo administration of ATG results in significant and long-lasting inhibition of TR activity. Such inhibition of TR could lead to increased levels of oxidative stress in vivo, thereby increasing the virulence of several viruses including the coxsackievirus.

Vitamin E-deficient diets enriched with fish oil suppress lethal Plasmodium yoelii infections in athymic and scid/bg mice.

Mice fed vitamin E-deficient diets containing omega-3 fatty acids survive infection with lethal Plasmodium yoelii. The current study sought to determine if antimalarial T- and B-cell responses were required for such dietary-mediated protection. In the first set of experiments, nu/nu mice (which lack alphabeta T-cell-receptor-positive T cells and do not produce antimalarial antibody) and nu/+ mice were fed casein-based diets containing 4% menhaden oil, with or without vitamin E supplementation, for 4 weeks prior to infection with lethal P. yoelii. All mice fed diets containing vitamin E developed fulminating parasitemias and quickly died, whereas both nu/nu and nu/+ mice fed diets deficient in vitamin E controlled their parasitemias for the first 18 days of infection. Thereafter, the nu/nu mice became anemic and died, whereas the nu/+ mice produced antimalarial antibodies and survived. In the second set of experiments, scid/scid.bg/bg mice (which lack B cells and alphabeta and gammadelta T cells and have reduced NK-cell activity) were fed the experimental diet for 6 weeks and then infected with the less virulent 17XNL strain of P. yoelii. Mice fed vitamin E-containing diets quickly died, whereas those fed the vitamin E-deficient diet survived without developing detectable parasitemias. Results from these experiments show that under prooxidant dietary conditions, mice were able to control and even survive malaria in the absence of malaria-primed T cells and antimalarial antibody. These results emphasize the importance of cellular oxidative processes in parasite elimination.

Association of lowered plasma carotenoids with protection against cecal coccidiosis by diets high in n-3 fatty acids.

A series of four experiments was run to assess the effectiveness of diets containing high amounts of n-3 fatty acids in reducing the pathological effects of cecal coccidiosis in chickens caused by Eimeria tenella. To determine whether the dietary effects were related to development of oxidative stress, plasma samples were analyzed for tocopherols and carotenoids. Plasma vitamin E (alpha-tocopherol) values were not consistent between experiments. Total plasma carotenoids, however, were significantly decreased by 2.5 to 20% diet supplementation with fish oil in several experiments. These decreases coincided with significant reductions in lesion scores. Under the experimental conditions, total plasma carotenoid concentrations may serve as a sensitive indicator for oxidative stress, which may be a factor in reducing cecal lesions in E. tenella infections.

Urinary malondialdehyde-equivalents during ingestion of meat cooked at high or low temperatures.

Excretion of malondialdehyde (MDA)-generating substances in the urine has been suggested as an indicator of in vivo lipid peroxidation. However, MDA in the urine also reflects the amount of lipid peroxidation products consumed in the diet. We determined MDA as the thiobarbituric acid (TBA)-MDA complex in urine of 19 healthy adults (10 male and 9 female) fed large quantities (3.6-4.1 g/kg body weight) of ground beef cooked at a low or a high temperature. Subjects ate a controlled diet with no alcohol or nutritional supplements. For 7 d they consumed ground beef cooked at 100 degrees C for 20 min (low-temperature meat) followed by 7 d with meat fried at 250 degrees C for 22 min (high-temperature meat). Prior to the study, subjects consumed their normal free choice diet with moderate amounts of meat. The concentration of MDA in urine at baseline was 2.1 +/- 0.3 mumol TBA-MDA equivalents/day (mean +/- SEM). After 7 d of low-temperature meat, urinary TBA-MDA equivalents increased to 23.1 +/- 1.4 mumol/d. Urinary TBA-MDA equivalents were consistently lower (6.9-8.0 mumol/d) 1, 2, 3, and 7 d after subjects changed to high-temperature meat. After 7 d of treatment, 97% of the MDA-equivalents in the meat was recovered in 24-h urine samples. The low temperature meat had 3-4 times more MDA than did the high-temperature meat. These data indicate that the amount of meat eaten and the cooking procedures used can dramatically alter urinary MDA. Dietary sources of MDA must be controlled if urinary MDA is to be used as an indicator of oxidative stress.

Rapid genomic evolution of a non-virulent coxsackievirus B3 in selenium-deficient mice results in selection of identical virulent isolates.

Previous work from our laboratory demonstrated that selenium deficiency in the mouse allows a normally benign (amyocarditic) cloned and sequenced Coxackievirus to cause significant heart damage. Furthermore, Coxsackievirus recovered from the hearts of selenium-deficient mice inoculated into selenium-adequate mice still induced significant heart damage, suggesting that the amyocarditic Coxsackievirus had mutated to a virulent phenotype. Here we report that sequence analysis revealed six nucleotide changes between the virulent virus recovered from the selenium-deficient host and the avirulent input virus. These nucleotide changes are consistent with known differences in base composition between virulent and avirulent strains of Coxsackievirus. To the best of our knowledge, this is the first report of a specific nutritional deficiency driving changes in a viral genome, permitting an avirulent virus to acquire virulence due to genetic mutation.

Protection against murine cerebral malaria by dietary-induced oxidative stress.

Feeding 20% (w/w) menhaden-fish oil in a standard laboratory chow diet for 4 wk partially protected CBA/CaJ mice from the central nervous system consequences of infection with Plasmodium berghei (ANKA). Full protection (complete survival for 14 days postinfection) could be obtained by feeding a purified pro-oxidant vitamin E-deficient diet containing 4% (w/w) menhaden oil (MO - VE diet). The purified pro-oxidant MO - VE diet also exerted a pronounced suppressive effect against the parasite (depressed 6-day parasitemias). The anitmalarial effect of the MO - VE diet could be prevented by supplementing the diet with vitamin E or with either of 2 synthetic antioxidants, N,N'-diphenyl-p-phenylenediamine or probucol. These results suggest that the fish oil exerts its antimalarial effect by imposing a dietary-induced oxidative stress on the infected host erythrocyte, the parasite, or both. Nutritional manipulation of host oxidative stress status may be a useful adjunct therapy in patients undergoing treatment with pro-oxidant antimalarials such as drugs of the qinghaosu family.

Increased virulence of a human enterovirus (coxsackievirus B3) in selenium-deficient mice.

Coxsackievirus B3 (CVB3/20)-induced myocarditic lesions occurred more quickly and were more severe and virus titers in heart and liver were higher in selenium (Se)-deficient than Se-adequate mice. NK cell activity and serum neutralizing antibody titers were similar in both Se-adequate and -deficient CVB3/20-infected mice; however, lymphocyte proliferation to both mitogen and antigen was decreased in Se-deficient mice. CVB3/20 isolated from Se-deficient donor mice and inoculated into Se-adequate recipient mice induced severe myocarditis. In contrast, CVB3/20 isolated from Se-adequate donor mice and inoculated into Se-adequate recipient mice induced only moderate myocarditis, similar to that caused by the original virus stock. Thus, the general population of CVB3/20 virions, as a consequence of replicating in an Se-deficient host, underwent a phenotypic change to increased virulence. These results have important implications for the emergence of virulent viruses.

Benign human enterovirus becomes virulent in selenium-deficient mice.

Coxsackieviruses have been implicated as possible co-factors in the etiology of the selenium (Se)-responsive cardiomyopathy known as Keshan disease. Here we report that a cloned and sequenced amyocarditic coxsackievirus B3 (CVB3/0), which causes no pathology in the hearts of Se-adequate mice, induces extensive cardiac pathology in Se-deficient mice. CVB3/0 recovered from the hearts of Se-deficient mice inoculated into Se-adequate mice induced significant heart damage, suggesting mutation of the virus to a virulent genotype. We demonstrate the important role of host nutritional status in determining the severity of a viral infection.

Vitamin E deficiency intensifies the myocardial injury of coxsackievirus B3 infection of mice.

Feeding a vitamin E-deficient diet increases pathology in hearts of mice infected with a myocarditic coxsackievirus B3 (CVB3/20). Hearts from infected mice fed a vitamin E-deficient diet rich in highly unsaturated fat (menhaden oil) exhibited more severe pathology than hearts from infected mice fed a vitamin E-deficient diet based largely on saturated fat (lard). Furthermore, a cloned and sequenced amyocarditic coxsackievirus B3 (CVB3/0), which caused little or no pathology in the hearts of vitamin E-supplemented mice, induced extensive cardiac pathology in vitamin E-deficient mice. In infected mice, both mitogen and antigen responses were depressed by vitamin E deficiency, although neutralizing antibody responses were unaffected. Natural killer cell responses were comparable in infected mice fed a lard-based diet with or without supplemented vitamin E. However, a menhaden oil-based diet, whether supplemented with vitamin E or not, significantly depressed natural killer cell activity in infected mice compared with mice fed the lard-based diet. Coxsackievirus B3/0 recovered from the heart of a vitamin E-deficient donor mouse, passaged one time onto HeLa cells, caused significant heart damage when passed back into vitamin E-supplemented recipient mice, demonstrating that the amyocarditic CVB3/0 had changed to a virulent phenotype. Enhanced virulence was also seen with CVB3/20 virus similarly passaged in a vitamin E-deficient donor. Our work demonstrates the important role of host nutritional antioxidant status in determining the severity of certain viral infections.

The urinary excretion of thiobarbituric acid reactive substances and malondialdehyde by normal adult males after consuming a diet containing salmon.

In this study we investigated the output of thiobarbituric acid reactive substances (TBARS) and malondialdehyde (MDA), as thiobarbituric acid (TBA)-MDA adduct, in the urine from subjects eating a diet in which the only source of n-3 long-chain, polyunsaturated fatty acids was fresh salmon. Nine healthy men, ages 30-65, were confined in the United States Department of Agriculture Western Human Nutrition Research Center, San Francisco, CA, for 100 d; food intake and exercise levels were controlled. All subjects were placed on a stabilization diet (StD) for 20 d, then six were fed the salmon diet for 40 d. The others remained on the StD. The groups switched diets for the last 40 d. Both diets were isocaloric (16% protein, 54% CHO and 30% fat by energy %). The salmon diet contained 7.5% of calories from n-6 fatty acids (FAs) and 2% from n-3 FAs, primarily eicosapentaenoic acid and docosahexaenoic acid in a 50:60 ratio, while the StD contained 7.5% from n-6 FAs and < 0.3% n-3 FAs (with presumably no significant amounts of C20 or C22 n-3 FAs). Twenty-four hour urinary output was collected, and 2% 3-d pool samples prepared for analysis of urinary TBARS and the TBA-MDA adduct. The total urinary output of each individual varied considerably, and on a daily basis the concentration of autoxidation products in an individual's urine varied also.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of aging and diet on proton NMR spectra of rat urine.

500 MHz 1H NMR studies of rat urine are used to assess metabolic changes resulting from aging (0.5-20 months) and changes in diet (casein versus chow). Aging rats fed chow diets decrease their excretion of citrate and 2-oxoglutarate, while the output of taurine and creatinine increases. Only young rats (1 month or less postweaning) excrete significant amounts of betaine and trimethylamine-N-oxide. Rats fed casein diets for 1 month postweaning do not excrete 2-oxoglutarate and excrete lower levels of hippurate, succinate, and citrate compared to rats fed chow diets. They also excrete N-methylnicotinamide. These high resolution proton NMR studies provide metabolic profiles which are not readily available by other techniques.

Plasmodium yoelii: comparative antimalarial activities of dietary fish oils and fish oil concentrates in vitamin E-deficient mice.

Feeding vitamin E-deficient diets containing either fish oils such as menhaden, salmon, or anchovy oil or fish oil concentrates based on n-3 ethyl esters or free fatty acids protected mice against Plasmodium yoelii as indicated by decreased parasitemia and improved survival. The fish oil concentrates depressed plasma tocopherol levels more strongly in vitamin E-supplemented mice than the menhaden oil. The free fatty acid concentrate appeared to suppress parasitemia in vitamin E-deficient mice better than the menhaden oil, although ultimate survival was similar in both groups. Dietary manipulation of host antioxidant status offers promise as a possible means of malaria control.

Menhaden-fish oil in a vitamin E-deficient diet: protection against chloroquine-resistant malaria in mice.

Feeding a vitamin E-deficient diet containing 5% menhaden oil to mice affords significant protection against both a chloroquine-sensitive and a chloroquine-resistant line of the malarial parasite. Nutritional manipulation may offer a new approach to the problem of drug-resistant malaria, a rapidly emerging global threat to public health.

Qinghaosu, dietary vitamin E, selenium, and cod-liver oil: effect on the susceptibility of mice to the malarial parasite Plasmodium yoelii.

Young female mice were fed torula-yeast-based diets deficient in vitamin E or selenium or supplemented with cod-liver oil to determine the effect of host antioxidant status on the therapeutic efficacy of the Chinese traditional antimalarial drug qinghaosu (QHS), a sesquiterpene endoperoxide. Vitamin E deficiency enhanced the antimalarial action of QHS against Plasmodium yoelii, both in terms of decreased parasitemia and improved survival but Se deficiency did not. A vitamin E-deficient diet containing 5% cod-liver oil had such strong antimalarial activity in itself that no additional therapeutic benefit of QHS could be demonstrated. Hematocrit values in parasitized mice treated with QHS or fed the cod-liver-oil-supplemented, vitamin E-deficient diet were normal. Nutritional manipulation of host antioxidant status may provide a promising prophylactic and/or therapeutic tool for the control of malaria.

Effect of dietary selenium on the development of Fusarium-induced tibial dyschondroplasia in broiler chickens.

A trial was conducted to determine the effects of dietary level of selenium on the pathogenesis of Fusarium-induced tibial dyschondroplasia (FITD) in broiler chicks, and to assess the applicability of FITD as an animal model of Kashin-Beck disease of humans. Day-old female broilers were fed diets that were deficient in selenium (0.02 ppm Se), adequate in selenium (0.15 ppm Se), or generous in selenium (0.50 ppm Se). TDP-1, the toxic component of the fungus, was administered to 15 of 26 chicks in each dietary group starting at 1 week of age and continuing until the chicks were killed at 24-30 days of age. Plasma selenium levels and hepatic glutathione peroxidase activity were significantly lower in the selenium-deficient group than in other dietary groups; these parameters were not affected by treatment with TDP-1. The mortality rate of the TDP-1-treated selenium-generous group was significantly less than that in the other TDP-1-treated groups, but there were no differences in the incidence, severity, or character of the FITD lesions among the groups. Thus, the interaction of selenium and TDP-1 did not include an effect on FITD.

Dietary selenium intake and selenium concentrations of plasma, erythrocytes, and breast milk in pregnant and postpartum lactating and nonlactating women.

The selenium status of a group of 23 lactating and 13 nonlactating women was assessed from 37-wk gestation through 6-mo postpartum. The mean overall dietary Se intake of both groups of women was 80 +/- 37 micrograms/d. Plasma and erythrocyte Se levels were lower in the lactating than in the nonlactating mothers both before and after parturition. Breast-milk Se concentrations fell from 20 micrograms/L (0.25 mumol/L) at 1-mo postpartum to 15 micrograms/L (0.19 mumol/L) at 3- and 6-mo postpartum. A weak (r = 0.38) but statistically significant (p less than 0.025) relationship was observed between maternal plasma Se level and breast-milk Se concentration. The dietary Se intake of these lactating North American women appears sufficient to maintain satisfactory Se nutriture in their breast-fed infants during the first 6 mo of lactation.

Relative nutritional availability to rats of selenium in Finnish spring wheat (Triticum aestivum L.) fertilized or sprayed with sodium selenate and in an American winter bread wheat naturally high in Se.

A Finnish national programme to fertilize crops with sodium selenate led us to compare the nutritional availability to rats of selenium in two Finnish spring wheats (Triticum aestivum L.), either fertilized or sprayed with sodium selenate, with that in an American winter bread wheat naturally high in Se. Weanling male rats were given a Se-deficient Torula yeast diet for 4 weeks followed by either continued depletion or repletion for 4 weeks with graded levels of Se as sodium selenite (standard) or wheat (test food). Plasma and liver Se levels and plasma and liver glutathione peroxidase (EC 1.11.1.9; GSH-Px) activities were used as criteria of body Se status. The availability of Se under these conditions was calculated with the point-slope technique at two dietary levels of Se (Expt 1) and with the slope-ratio method (Expt 2). In the point-slope assay, the level of dietary Se fed had a considerable effect on the apparent availability values obtained which made interpretation of the results difficult. In the slope-ratio assay, no difference in the availability of Se from the various wheats was observed when plasma or liver Se levels were used as the response criteria. The Se in the fertilized wheat was somewhat more available than that in the sprayed wheat when plasma or liver GSH-Px activities were the response criteria. Overall, availability values (%) derived by averaging all four response criteria were 86, 77 and 73 for the fertilized and sprayed Finnish wheats and the American wheat respectively (sodium selenite 100). These results show that wheat is a relatively available source of Se to rats regardless of whether its Se content is naturally high or is increased by fertilization or spraying.

Effect of selenium deficiency on the chronic toxicity of adriamycin in rats.

The effect of selenium deficiency on the chronic toxicity of adriamycin was examined in rats fed diets adequate in vitamin E. Selenium-deficient and selenium-supplemented diets were fed to rats for 10 wk, after which groups of 10 rats fed each diet were given weekly intravenous injections of adriamycin in saline at doses of 0, 0.5 or 1.0 mg/kg body weight for 12 wk. All rats were killed at 24 wk. Even though the cardiac glutathione peroxidase activity in the selenium-deficient group was less than 1% of that of the selenium-supplemented group, the severity of the adriamycin-induced cardiomyopathy was similar in both groups. However, the selenium-deficient rats were more sensitive to the growth-inhibiting effect of the higher dose of adriamycin than the selenium-supplemented rats. Moreover, the lower dose of adriamycin caused a mild nephropathy in 70% of the deficient rats but affected only 10% of the supplemented rats. Selenium status may have to be considered when adriamycin is used as a chemotherapeutic agent.