Changes in organ nonprotein sulfhydryl and glutathione concentrations during acute and chronic administration of inorganic lead to chicks

The effects of dietary and injected lead (Pb) on organ nonprotein sulfhydryl (NPSH) and glutathione (GSH) concentrations in the chick were studied. Lead acetate·3H₂O was administered either in the diet for 3 wk at 2000 ppm Pb or by intraperitoneal (ip) injection of 3-wkold chicks with 52 mg Pb/100 g body wt. In Exp. 1, NPSH concentrations in liver and kidney were increased by both dietary and injected Pb in comparison to chicks not receiving Pb. Thigh muscle NPSH was decreased by injected Pb, whereas dietary Pb had no effect. In Expt. 2, whole blood and plasma NPSH were measured at 0, 0.5, 1.0, 2.0, and 4.0 h following ip Pb injection. Both whole blood and plasma NPSH were increased by 30 min. Whole blood NPSH concentrations plateaued at 30 min, and plasma NPSH continued to rise for 2 h. In Expt. 3, injected Pb increased hepatic NPSH, but not GSH concentrations. The ratio of GSH/NPSH was therefore lowered. The incorporation of [1-¹⁴C]glycine into hepatic GSH was stimulated by injected Pb. Buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, reduced hepatic NPSH and [¹⁴C]glycine incorporation in Pb-treated chicks to below control (non-Pb injected) values. In Expt. 4, dietary Pb fed for 3 wk increased the hepatic concentrations of both NPSH and GSH such that the ratio of GSH/NPSH was unchanged in comparison to chicks not fed Pb. The data suggest that the initial response to acute Pb intoxication may involve a mobilization of nonprotein thiols via the interorgan translocation system for GSH. Such a response would help to maintain adequate levels of GSH in organs crucial to detoxification.     

Advances in protein–amino acid nutrition of poultry

The ideal protein concept has allowed progress in defining requirements as well as the limiting order of amino acids in corn, soybean meal, and a corn–soybean meal mixture for growth of young chicks. Recent evidence suggests that glycine (or serine) is a key limiting amino acid in reduced protein [23% crude protein (CP) reduced to 16% CP] corn–soybean meal diets for broiler chicks. Research with sulfur amino acids has revealed that small excesses of cysteine are growth depressing in chicks fed methionine-deficient diets. Moreover, high ratios of cysteine:methionine impair utilization of the hydroxy analog of methionine, but not of methionine itself. A high level of dietary l-cysteine (2.5% or higher) is lethal for young chicks, but a similar level of dl-methionine, l-cystine or N-acetyl-l-cysteine causes no mortality. A supplemental dietary level of 3.0% l-cysteine (7× requirement) causes acute metabolic acidosis that is characterized by a striking increase in plasma sulfate and decrease in plasma bicarbonate. S-Methylmethionine, an analog of S-adenosylmethionine, has been shown to have choline-sparing activity, but it only spares methionine when diets are deficient in choline and(or) betaine. Creatine, or its precursor guanidinoacetic acid, can spare dietary arginine in chicks.     

Lead effects in the chick during selenium deficiency

1. Growing chicks (Gallus domesticus) were fed a selenium-deficient diet supplemented with 0 or 2000 ppm lead (Pb) and 0 or 0.1 ppm selenium (Se). 2. Selenium addition stimulated growth at 0 but not at 2000 ppm Pb, while Pb depressed growth at both levels of Se. 3. Selenium addition stimulated Se-dependent glutathione peroxidase (GSH-Px) activity in liver, but Pb was without effect on GSH-Px activity. 4. Lead addition increased non-protein sulfhydryl (NPSH) concentrations in liver, kidney and thigh muscle. NPSH levels were not altered by Se. 5. The reported antagonism between Pb and Se does not appear to be mediated through effects on GSH-Px or NPSH metabolism.     

Interaction of lead toxicity and riboflavin status in chicks (Gallus domesticus)

Chicks (Gallus domesticus) were fed diets containing 0 or 2000 ppm lead (Pb) and adequate, marginal or deficient levels of riboflavin. In comparison to adequate, marginal riboflavin depressed growth at 2000 ppm Pb but not at 0 ppm Pb. Deficient riboflavin depressed growth at both Pb levels, and Pb depressed growth at all riboflavin levels. Hepatic glutathione reductase activity was reduced by riboflavin deficiency, but Pb was without effect. Hepatic non-protein sulfhydryl concentrations were increased by Pb, and the increases were greater at the lower riboflavin levels. Dietary Pb appears to increase the level of riboflavin required in chick diets.     

The interaction of iron and ascorbic acid with lead in the chick

Three experiments were conducted with chicks to examine the effects of dietary iron and ascorbic acid on the accumulation of lead in various organs. Lead was fed as PbCl₂, 500 or 1000 ppm Pb, iron as FeSO₄·7H₂O, 1000 ppm Fe, and ascorbic acid at 0.5%. Iron was effective in reducing the accumulation of lead in the femur and kidneys at both levels of lead. Ascorbic acid reduced the lead level in the kidneys when the concentration of lead in the diet was 500 ppm, but not at 1000 ppm. The effects of ascorbic acid on bone accumulation was variable. In two experiments the lead concentration was increased and in one it was decreased. These findings may reflect two influences of ascorbic acid found by others, namely an increase in absorption and an increase in urinary excretion. The rapid accumulation of lead in chick bones suggests that it may be an excellent experimental animal for lead studies.     

Role of white adipose lipolysis in the development of NASH induced by methionine- and choline-deficient diet

Methionine- and choline-deficient diet (MCD) is a model for nonalcoholic steatohepatitis (NASH) in rodents. However, the mechanism of NASH development by dietary methionine/choline deficiency remains undetermined. To elucidate the early metabolic changes associated with MCD-NASH, serum metabolomic analysis was performed using mice treated with MCD and control diet for 3 days and 1  week, revealing significant increases in oleic and linoleic acids after MCD treatment. These increases were correlated with reduced body weight and white adipose tissue (WAT) mass, increased phosphorylation of hormone-sensitive lipase, and up-regulation of genes encoding carboxylesterase 3 and β2-adrenergic receptor in WAT, indicating accelerated lipolysis in adipocytes. The changes in serum fatty acids and WAT by MCD treatment were reversed by methionine supplementation, and similar alterations were detected in mice fed a methionine-deficient diet (MD), thus demonstrating that dietary methionine deficiency enhances lipolysis in WAT. MD treatment decreased glucose and increased fibroblast growth factor 21 in serum, thus exhibiting a similar metabolic phenotype as the fasting response. Comparison between MCD and choline-deficient diet (CD) treatments suggested that the addition of MD-induced metabolic alterations, such as WAT lipolysis, to CD-induced hepatic steatosis promotes liver injury. Collectively, these results demonstrate an important role for dietary methionine deficiency and WAT lipolysis in the development of MCD-NASH.     

Modification of lead toxicity and organ distribution by dietary sulfur amino acids in chicks (Gallus domesticus)

Chicks (Gallus domesticus) were fed a basal diet deficient in methionine and total sulfur amino acids with 0 or 1000 ppm added lead for 21 days. Methionine or methionine and cystine addition improved growth regardless of dietary lead level. Cystine addition alone improved growth only when lead was present. Relative inhibition of growth by lead was greater with diets containing no added methionine. Hepatic non-protein sulfhydryl concentration was increased by lead and all amino acid additions. Organ lead concentrations were generally lower with added amino acids. Dietary methionine appears to counteract lead toxicity more effectively than cystine.     

The relationship between iron status and lead absorption in rats

The absorption of lead from loops of small intestinein situ was investigated in rats in which iron absorption was increased by stimuli varying in type, intensity, or duration. Lead absorption was increased by a short period of severe iron restriction before any change in hematological indices became apparent. A period of hypoxia, which markedly increased iron absorption, did not influence absorption of lead. An extended period of moderate iron restriction resulted in a marked reduction in liver iron stores and increased iron absorption throughout the 17-wk experiment. Under these conditions lead absorption was initially also increased, but after 12 wk, when iron intake had become adequate to meet essential requirements, lead absorption was similar to that in iron-supplemented rats. These results are discussed in the light of evidence for a receptor-mediated absorption process for iron.     

Lead increases urinary zinc excretion in rats

The major purpose of this study was to determine whether acute or chronic Pb exposure would increase urinary excretion of zinc in the rat. Four groups of unanesthetized rats were given 0, 0.03, 0.3, or 3 mg Pb (as acetate) kg intravenously, and urinary excretion of zinc, sodium, and potassium was monitored for 6 h. Only at the highest dose was urinary Zn excretion significantly elevated; there were no significant changes in sodium and potassium excretion at any dose. Two other groups of rats were studied for 9 weeks in metabolism cages before and during administration of either 500 ppm Pb (as acetate) or equimolar Na acetate in the drinking water. Two days after Pb treatment and continuing through day 35, Zn excretion was elevated in the Pb-exposed animals; beyond this day, zinc excretion became similar in the two groups. The difference in Zn excretion was not the result of lower water intake by the Pb-treated animals. At sacrifice (70 days after starting Pb exposure), Pb-exposed animals had lower Zn content of the plasma and testis, but there was no difference in kidney Zn. Plasma renin activity was significantly higher in Pb-exposed animals. We conclude that chronic Pb exposure in rats can result in some degree of decreased tissue zinc, which is, at least in part, secondary to increased urinary losses of zinc.     

Effects of aurothioglucose and dietary se on glutathione S-Transferase activities and glutathione concentrations in chick tissues

Experiments were conducted to determine whether the increased glutathione S-transferase (GSH-T) activity associated with selenium (Se) deficiency is necessarily related to losses in the activity of Se-dependent glutathione peroxidase (SeGSHpx) in chicks. Nutritional Se status was altered in two ways: by treatment with an antagonist of Se utilization, aurothioglucose (AuTG), and by feeding diets containing excess Se. Chicks given AuTG (10–30 mg AU/kg, sc) had growth rates and hepatic GSH concentrations that were comparable to those of saline-treated controls; however, their plasma GSH levels exceeded those of either Se-deficient (6-fold) or-adequate (3-fold) saline-treated chicks. Hepatic SeGSHpx activities of AuTG-treated chicks were hals those of controls under conditions of Se-adequacy; however, this effect was not detected when Se was deficient. Hepatic GSH-T_CDNB (assayed with 1-chloro-2,4-dinitrobenzene) activities of AuTG-treated chicks were significantly greater than those of controls when Se was deficient (i.e., when SeGSHpx activity was 12% of the Se-adequate level); however, deprivation of Se did not affect GSH-T_CDNB activity in the absence of AuTG. chicks fed excess Se (6–20 ppm as Na₂SeO₃) in diets containing either low (2 IU/kg) or adequate (100 IU/kg) VE, showed hepatic GSH-T_CDNB activities and GSH concentrations greater than those of Se-adequate (0.2 ppm Se) chicks by 100% and 40%, respectively. That increased hepatic GSH-T_CDNB activity can occur because of either AuTG or excess Se status under conditions wherein SeGSHpx activity is not affected indicates that the transferase response is not directly related to changes in the peroxidase.     

Specific Contribution of Methionine and Choline in Nutritional Nonalcoholic Steatohepatitis: IMPACT ON MITOCHONDRIAL S-ADENOSYL-l-METHIONINE AND GLUTATHIONE*

The pathogenesis and treatment of nonalcoholic steatohepatitis (NASH) are not well established. Feeding a diet deficient in both methionine and choline (MCD) is one of the most common models of NASH, which is characterized by steatosis, mitochondrial dysfunction, hepatocellular injury, oxidative stress, inflammation, and fibrosis. However, the individual contribution of the lack of methionine and choline in liver steatosis, advanced pathology and impact on mitochondrial S-adenosyl-l-methionine (SAM) and glutathione (GSH), known regulators of disease progression, has not been specifically addressed. Here, we examined the regulation of mitochondrial SAM and GSH and signs of disease in mice fed a MCD, methionine-deficient (MD), or choline-deficient (CD) diet. The MD diet reproduced most of the deleterious effects of MCD feeding, including weight loss, hepatocellular injury, oxidative stress, inflammation, and fibrosis, whereas CD feeding was mainly responsible for steatosis, characterized by triglycerides and free fatty acids accumulation. These findings were preceded by MCD- or MD-mediated SAM and GSH depletion in mitochondria due to decreased mitochondrial membrane fluidity associated with a lower phosphatidylcholine/phosphatidylethanolamine ratio. MCD and MD but not CD feeding resulted in increased ceramide levels by acid sphingomyelinase. Moreover, GSH ethyl ester or SAM therapy restored mitochondrial GSH and ameliorated hepatocellular injury in mice fed a MCD or MD diet. Thus, the depletion of SAM and GSH in mitochondria is an early event in the MCD model of NASH, which is determined by the lack of methionine. Moreover, therapy using permeable GSH prodrugs may be of relevance in NASH.     

Iodothyronine deiodinase activity in methionine-deficient rats fed selenium-deficient or selenium-sufficient diets

We examined the effect of methionine deficiency on iodothyronine 5’-deiodinase activity in selenium-deficient rats or selenium-sufficient rats fed sodium selenate or selenomethionine. Forty-two weanling male Wistar rats were divided into six groups and pair fed the respective purifiedl-amino acid-based diets for 4 wk.l-methionine concentrations in the diet were 8.0 g/kg for sufficient rats, and 2.0 g/kg for deficient rats. Selenium concentrations in the diet were 0.5 mg/kg (as sodium selenate or selenomethionine) for selenium-sufficient rats and less than 0.005 mg/kg for selenium-deficient rats. Type I 5’-deiodinase activities were significantly lower in liver and higher in kidney of methionine-deficient rats than in those of methionine-sufficient rats fed either the selenium-sufficient or the selenium-deficient diets. The type I 5’-deiodinase activity in brain was significantly lower in the methionine-deficient rats than in the methionine-sufficient rats fed the selenium-deficient diet. Type II 5’-deiodinase activity in brain was significantly higher in the methionine-deficient rats than in the methionine-sufficient rats fed selenium-sufficient diet as sodium selenate. Both thyroxine and 3,3’,5-triiodothyronine concentrations in plasma were significantly higher in the methionine-deficient rats than in the methionine-sufficient rats. It is suggested that the methionine deficiency affects the 5’-deiodinase activity and thyroid hormones level in the rats.     

Monoamine oxidase A from human placenta and monoamine oxidase B from bovine liver both have one FAD per subunit.

Choline and C1 metabolism pathways intersect at the formation of methionine from homocysteine. Hepatic S-adenosylmethionine (AdoMet) concentrations are decreased in animals ingesting diets deficient in choline, and it has been suggested that this occurs because the availability of methionine limits AdoMet synthesis. If the above hypothesis is correct, changes in hepatic AdoMet concentrations should relate in some consistent manner to changes in hepatic methionine concentrations. Rats were fed on a choline-deficient or control diet for 1-42 days. Hepatic choline concentrations in control animals were 105 nmol/g, and decreased to 50% of control after the first 7 days on the choline-deficient diet. Hepatic methionine concentrations decreased by less than 20%, with most of this decrease occurring between days 3 and 7 of choline deficiency. Hepatic AdoMet concentrations decreased by 25% during the first week, and continued to decrease (in total, by over 60%) during each subsequent week during which animals consumed a choline-deficient diet. Hepatic S-adenosylhomocysteine (AdoHcy) concentrations increased by 50% when animals consumed a choline-deficient diet. AdoHcy is formed when AdoMet is utilized as a methyl donor. In summary, choline deficiency can deplete hepatic stores of AdoMet under dietary conditions that only minimally decrease the availability of methionine within liver. Thus decreased availability of methionine may not have been the only mechanism whereby choline deficiency lowers hepatic AdoMet concentrations. We suggest that increased utilization of AdoMet might also have occurred.     

Studies on the role of iron in the reversal of vanadium toxicity in chicks

The interaction of dietary iron levels on vanadium toxicity was studied in chicks. Dietary iron levels ranged from a deficiency, ca. 10 ppm, to an adequacy, 100 ppm supplemental iron. to an excess, 1000 ppm supplemental iron. Vanadium was fed at 10, 20, and 40 ppm. Vanadium toxicity as measured by chick growth was more severe in the iron-deficient animals than in those receiving supplemental iron. The increase in degree of toxicity in the iron-deficient animals was accompanied by an increase in the liver vanadium, both total and concentration. The addition, of vanadium to the diet did not influence the iron concentration of the liver or kidney. Radioisotope, studies with⁴⁸V revealed that the absorption of vanadium was not influenced by the iron concentration of the diet, but that the iron-deficient animals retained more vanadium in the blood and liver and less in the bone than did the iron supplemented animals. It is proposed that the degree of iron saturation of transferrin and ferritin to which vanadium can bind is a possible explanation for the results obtained. Paper No. 10687 of the Journal Series of the NC Agricultural Research Service, Raleigh, NC 27695-7601. The use of trade names implies neither endorsement of the products named nor criticism of similar products not mentioned by the NCARS.     

酵母菌促进铅和镉胁迫下麻疯树生长的研究

为了筛选对铅和镉具有抗性和吸附性的酵母菌,构建麻疯树根系-酵母菌联合修复体系,促进高浓度铅和镉胁迫下麻疯树的生长。该研究分别从麻疯树的根段、珙桐的茎段、珙桐的根段分离到3株具有铅、镉抗性的酵母菌,分别命名为Jc、Di1、Di2,测定了三者对铅、镉的抗性和吸附性,并将筛选出的2株能吸附铅、镉的酵母菌菌株接种到麻疯树幼苗,研究接种两种酵母菌的麻疯树植株对铅、镉胁迫的响应。结果表明:经形态学和生理生化特征观察,Jc初步鉴定红酵母属(Rhodotorula sp.),Di1为假丝酵母属(Candida sp.),Di2为德巴利酵母属(Debaryomyces sp.)。三种酵母菌对铅、镉都有一定的抗性,其抗性能力的大小为JcDi2Di1。Di1和Jc对铅和镉都具有一定的吸附性将其用于接种麻疯树幼苗。与不接种酵母菌(CK)的麻疯树植株相比,接种Di1和Jc的麻疯树植株在根、茎、叶、全株干重方面显著增加,叶绿素、全株氮、全株磷浓度显著增加,SOD、POD、CAT的活性提高,丙二醛(MDA)浓度显著下降。从综合接种效应来看,Jc、Di1作为铅、镉的钝化剂,是铅、镉胁迫下促进麻疯树生长的备选菌株,这对于提高麻疯树对铅、镉污染土壤修复效率具有重要的意义。     

Lead-induced tissue fatty acid alterations and lipid peroxidation

Previous work showed that dietary lead (Pb) increases the relative concentration of arachidonic acid (20∶4) as a percentage of total fatty acids, and decreases the relative proportion of linoleic acid (18∶2) to arachidonic acid (18∶2/20∶4) in chick liver, serum, and erythrocyte membranes. The present investigation was undertaken to examine the time-course and magnitude of the fatty acid alterations with increasing dietary Pb levels. We also examined the effects of Pb on the fatty acid composition and lipid peroxide content of hepatic subcellular organelles. In Exp. 1, chicks were fed diets containing 0, 62.5, 125, 250, 500, or 1000 ppm added Pb (as Pb acetate trihydrate) from 1 to 21 d of age. After 21 d, no growth effects were observed; however, Pb lowered the 18∶2/20∶4 ratio and increased 20∶4 concentration in total liver and serum lipids, and in total hepatic phospholipids in a dose-dependent manner. Hepatic mitochondrial membrane fatty acids were not altered, nor was there any increase in hepatic lipid peroxidation. In Exp. 2, chicks were fed diets containing 0, 500, 1000, or 2000 ppm added Pb from 1 to 21 or 22 d of age. Pb depressed growth in a dose-dependent manner. In addition, Pb lowered the 18∶2/20∶4 ratio and increased 20∶4 concentration in total liver lipids and in hepatic mitochondrial and microsomal membranes in a dose-dependent manner. Total hepatic lipid peroxidation was increased over control values by 1000 ppm Pb, and hepatic microsomal lipid peroxidation was increased by dietary Pb levels of 1000 and 2000 ppm. In Exp. 3, body weight, hepatic microsomal lipid peroxidation, and fatty acid composition were determined in 4-, 9-, 14-, 18-, and 23-d-old chicks fed 0 or 1500 ppm added Pb. Body weights of Pb-treated chicks were significantly lower than those of control chicks by day 18. Microsomal 20∶4 concentration and peroxidation increased, and the 18∶2/20∶4 ratio decreased with age in both groups, but the changes were of greater magnitude in the Pb-treated chicks. The results suggest that some of the manifestations of Pb toxicity may be a reflection of increased concentration of 20∶4 in specific membranes. Further, since the Pb-induced alterations in fatty acid composition were noted in the absence of any growth depression, we propose that fatty acid composition is more sensitive than growth rate to the presence of lead in the diet.     

Formation and utilization of methionine by rat liver cells in culture

Summary The enzymeN ⁵-methyltetrahydrofolate: homocysteine methyltransferase (methionine synthetase) catalyzes the synthesis of methionine from homocysteine. Methylcobalamin is a cofactor for the reaction. The effects of methionine deprivation and methylcobalamin supplementation on the growth of normal and transformed rat liver epithelial cell lines were determined using growth constants to quantitate cell proliferation. No marked specific requirement by the transformed cell lines for methionine relative to leucine was observed. A sigmoidal relationship, however, was found to exist between growth constants and the logarithms of the amino acid concentrations for both normal and transformed cells. Methylcobalamin stimulated the growth rates of the normal and transformed liver cells in methionine-deficient, homocysteine-containing medium. Growth on methionine was not increased by the addition of methylcobalamin. The growth constants for two normal, two spontaneously transformed, one chemically transformed, and one tumor cell line grown in medium in which methionine was replaced by homocysteine were found to be proportional to the level of methionine synthetase. The results demonstrate the utility of growth quantitation to study the methionine dependency of transformed cells. Presented in part at the Conference on Differentiation and Carcinogenesis in Liver Cell Cultures sponsored by the New York Academy of Sciences, October 11, 1979 (see reference 1).     

Effect of different heavy-metal concentrations on Drosophila melanogaster larval growth and development

The survival percentage of Drosophila melanogaster larvae on synthetic media containing different concentrations of heavy metals, including Cd, Cu, Pb, and Zn, in the first generation indicated no significant reduction in their growth and development up to 500 ppm for all tested heavy-metal concentrations. At 500 ppm, results showed that there was a significant reduction in pupa and adult stages: 65% and 25% for Cd, 50% and 25% for Cu, 100% and 95% for Pb, and 85% and 75% for Zn, respectively. The survival percentages at 1000 ppm were further significantly reduced: 15% and 0% for Cd, 35% and 15% for Cu, 45% and 90% for Pb, and 65% and 35% for Zn, respectively for pupa and adult stages. For the second generation, there was no significant reduction in survival growth and development up to 100 ppm, but above 500 ppm, there was a significant reduction. For most of these heavy-metal concentrations, the survival percentages of the second generation at the pupa stage was higher than the first generation, whereas for the adult, there was a lower survival percentage, indicating some effect on metamorphosis of these heavy-metal concentrations on Drosophila melanogaster Comparing the survival percentage between first and second generations at 500 ppm for pupa indicate a significant increase on Cu only, and for the adult, there was a significant reduction for Cd.