Control of the steady-state concentrations of the nicotinamide nucleotides in rat liver

1. The effects of injecting nicotinamide, 5-methylnicotinamide, ethionine, nicotinamide+5-methylnicotinamide and nicotinamide+ethionine on concentrations in rat liver of NAD, NADP and ATP were investigated up to 5hr. after injection. 2. Nicotinamide induced three- to four-fold increases in hepatic NAD concentration even in the presence of 5-methylnicotinamide or ethionine, whereas 5-methylnicotinamide or ethionine alone did not cause marked changes in hepatic NAD concentration. 3. Nicotinamide alone also induced a twofold increase in hepatic NADP concentration. However, in the presence of 5-methylnicotinamide+nicotinamide, the NADP concentration decreased by 25% after 5hr., and in the presence of nicotinamide+ethionine by 30% in the same time. In the presence of 5-methylnicotinamide or ethionine alone hepatic NADP concentrations fell by 50% after 5hr. 4. 5-Methylnicotinamide inhibited the microsomal NAD(+) glycohydrolase (EC 3.2.2.6) by 60% at a concentration of 1mm and the NADP(+) glycohydrolase by 40% at the same concentration. 5. The rat liver NAD(+) kinase (EC 2.7.1.23) was found to have V(max.) 4.83mumoles/g. wet wt./hr. and K(m) (NAD(+)) 5.8mm. This enzyme was also inhibited by 5-methylnicotinamide in a ;mixed' fashion. 6. The results are discussed with respect to the control of NAD synthesis. It is suggested that in vivo the NAD(P)(+) glycohydrolases are effectively inactive and that the increased NAD concentrations induced by nicotinamide are due to increased substrate concentration available to both the nicotinamide and nicotinic acid pathways of NAD formation.     

Inhibition of glutathione efflux from isolated rat hepatocytes by methionine

A substantial inhibition (50-70%) of GSH efflux by methionine was demonstrated in hepatocytes isolated from fed rats. Concurrent measurements of intracellular GSH revealed maintenance of a higher concentration in methionine-supplemented cells over the 1-h incubation. Analysis of total GSH suggested that maintenance of higher intracellular GSH by methionine could be quantitatively accounted for by inhibition of GSH efflux rather than by net GSH synthesis. This conclusion was supported by studies with propargylglycine, a potent inhibitor of cysteine synthesis from methionine. Identical results were obtained in incubations containing either propargylglycine and methionine or methionine alone, thereby suggesting that net synthesis of GSH from methionine was minimal under the assay conditions. Similar decreases (40-60%) in the rate of extracellular accumulation of GSH were observed with ethionine and buthionine, two higher homologs of methionine, but not with a wide range of other naturally occurring and synthetic amino acids. The inhibition of GSH efflux by methionine was not dependent on the presence of sodium in the medium and did not correlate with metabolic consumption of ATP.     

Biochemical and developmental features of experimental phenylketonuria induced by L-ethionine in suckling rats

Suckling rats were injected subcutaneously with doses of L-ethionine (0.1 mumole/g body wt) at intervals of 12 hr. In the latter group, phenylalanine hydroxylase was effectively inhibited in vivo resulting in hyperphenylalaninemia and phenylketonuria. Due to the well-known sex-specific differences in L-ethionine metabolism female rats were much more affected by chronic administration of L-ethionine. The underlying mechanism of enzyme inhibition by ethionine could be disturbed protein synthesis and impaired protein phosphorylation, which was suggested by pronounced decreases in ATP content in liver. In the high dosage group depletions mainly of the branched-chain amino acids and lysine occurred in serum and brain, whereas the concentrations of methionine and tryptophan were increased. Tyrosine tended to be decreased in the course of hyperphenylalaninemia. Hyperphenylalaninemia and other resulting amino acid imbalances obviously impaired brain development during the early postnatal period. Concomitantly with reductions in protein concentrations, the activity of cathepsin D, a major intralysosomal acid proteinase, was increased in brain, suggesting also higher protein catabolism in brain. Side effects of this treatment, however, were higher mortality, loss of body weight, and a general impression of delayed development, resembling a state of undernutrition to some extent. These obvious side effects of ethionine limit the usefulness of ethionine as a suitable model for classic phenylketonuria in suckling rats.     

Induction of hyperphenylalaninemia in mice by ethionine and phenylalanine

Female NMRI mice were fed diets containing l-ethionine (0.1 and 0.3% w/w) and phenylalanine (3% w/w), as well as respective control diets. Ethionine, the S-ethylated analog of methionine, was shown to inhibit phenylalanine hydroxylase in vivo, whereby in vitro kinetics remained unaffected. Treatment with ethionine resulted in fatty liver, reduced ATP content of liver, and alterations in serum amino acid concentrations. In the high dosage ethionine group, for instance, concentrations of Ala, Gly, Ser, Met, and Phe were increased whereas concentrations of Lys, Asp, and Pro were decreased. Applying ethionine together with phenylalanine resulted in hyperphenylalaninemia and phenylketonuria. Feeding phenylalanine alone also led to decreased activity of phenylalanine hydroxylase and increased concentration of Phe in serum. Ethionine only had a minimal effect on body weight gain; however, the hyperphenylalaninemic condition induced by application of the high dosage of ethionine and phenylalanine induced severe loss of body weight. A disturbed protein synthesis and protein phosphorylation might be the underlying mechanism of ethionine-induced suppression of phenylalanine hydroxylase.     

Nicotinamide–adenine dinucleotides in acute liver injury induced by ethionine, and a comparison with the effects of salicylate

1. The effects of single doses of ethionine or sodium salicylate on the nicotinamide-adenine dinucleotide content of rat liver have been studied. 2. There was no significant change in the sum of NAD+NADH(2) during the early period (0-2hr.) of the liver injury induced by ethionine but there was a decrease in this value of approx. 30% by 4hr. after administration. 3. Ethionine had no significant effect on the NADP+NADPH(2) during the first 2hr. period after administration. The sum then decreased to a value approx. 70% of the control by 3hr. after dosing but showed a partial recovery at the 4hr. period before decreasing again in later stages of the poisoning. 4. Salicylate produced a very rapid decrease in the NADP+NADPH(2) in the liver after intraperitoneal injection. After 1hr. the decrease was approx. 30% of the initial value; the sum slowly returned towards the normal range during the following 4hr. 5. A high parenteral dose of salicylate was found to cause only a small depression in the concentration of ATP in rat liver in contrast with the rapid depletion produced by ethionine. 6. These results are discussed in terms of the liver disturbances produced by ethionine and salicylate.     

Biochemical studies on the mechanism of action of liver poisons III. Depletion of liver glutathione in ethionine poisoning

Administration of ethionine to female rats was followed by rapid fall of the hepatic reduced glutathione content, concomitant with a progressive impairment of the cell-free protein-synthesizing capacity and a decline of the ATP level.In male rats and in adrenalectomized female rats, the onset of the first two alterations was considerably delayed. In contrast, the time course and the severity of the ethionine-induced ATP depletion were essentially the same in all instances.Treatment with CCl₄, despite the similarity of its hepatotoxic effects to those of ethionine, resulted in no appreciable decrease of the liver reduced glutathione level.Administration of methionine counteracted both the reduced glutathione decline and the other biochemical lesions induced by ethionine, whereas injection of adenine, though preventing the ATP depletion, failed to reverse the depression of reduced glutathione.The available evidence suggests that the decrease in the reduced glutathione level reflects a diminished capacity of the liver for oxidized glutathione reduction and its resultant inability to compensate the normal process of continuous reduced glutathione oxidation.The possible role of the altered reduced glutathione metabolism in the pathogenesis of ethionine hepatotoxicity is discussed.     

Conversion of 5-S-ethyl-5-thio-D-ribose to ethionine in Klebsiella pneumoniae. Basis for the selective toxicity of 5-S-ethyl-5-thio-D-ribose

5-S-Ethyl-5-thio-D-ribose (ethylthioribose) exhibits antiprotozoal activity against Plasmodium falciparum, Giardia lamblia, and Ochromonas malhamensis, but is nontoxic to cultured human and murine bone marrow cells (Riscoe, M. K., Ferro, A. J., and Fitchen, J. H. (1988) Antimicrob. Agents Chemother. 32, 1904-1906). We propose the following mechanism to account for the observed selective toxicity of ethylthioribose. 1) The cytocidal action of ethylthioribose against protozoa is a result of its conversion to ethionine, a well-known cytotoxic agent. 2) This transformation occurs through the pathway which normally converts 5-S-methyl-5-thio-D-ribose (methylthioribose) to methionine. 3) Conversion of ethylthioribose to ethionine cannot occur in mammalian cells since these cells cannot phosphorylate methylthioribose (ethylthioribose), a first step in the pathway to methionine (ethionine). To test this hypothesis, [5-3H]ethylthioribose has been synthesized and its metabolism by cell-free extracts of Klebsiella pneumoniae and rat liver was examined. The pathway by which methylthioribose is converted to methionine in K. pneumoniae is well characterized. When supplemented with ATP and L-glutamine, the bacterial extract efficiently converted [5-3H]ethylthioribose to [3H]ethionine. By contrast, ethionine was not produced upon incubation of [5-3H]ethylthioribose, ATP, and L-glutamine with rat liver homogenate. The mammalian cell extract lacks a kinase activity capable of converting ethylthioribose to 1-phospho-5-S-ethyl-5-thio-alpha-D-ribofuranoside, an obligate intermediate in the biosynthesis of ethionine from ethylthioribose in K. pneumoniae. These results support our hypothesis and provide a basis for understanding the apparently selective toxicity of ethylthioribose.     

Induction of morphogenesis by methionine starvation in Myxococcus xanthus: polyamine control

The induction of mycrocyst formation by methionine starvation was demonstrated in Myxococcus xanthus by several methods. Growing in a defined medium (M(1)), M. xanthus had a doubling time of 6.5 hr. Four amino acids-leucine, isoleucine, valine, and glycine-were required for growth under these conditions. When the concentration of several amino acids in the medium was reduced (M(2)), the doubling time increased to 10 to 12 hr, and a requirement for methionine was observed. Methionine starvation led to a slow conversion of the population to microcysts. Under conditions of methionine prototrophy (M(1)), microcyst formation could still be triggered in exponentially growing cells by the addition of either 5 mm ethionine or 0.1 m isoleucine plus 0.1 m threonine, feedback inhibitors of methionine biosynthesis. Vegetative growth in the absence of methionine was obtained in medium M(2) if the leucine concentration was raised to its level in medium M(1). Thus, methionine biosynthesis is controlled by the exogenous concentration of the required amino acid, leucine. During an examination of the effects of methionine metabolites on microcyst formation, the involvement of polyamines in morphogenesis was uncovered. Putrescine (0.05 m) induced the formation of microcysts; spermidine (2 to 5 mm) inhibited induction by methionine starvation, ethionine, or high isoleucine-threonine. Spermidine was the only polyamine detected in M. xanthus (16.0 mug/10(9) cells). Its concentration decreased by more than 50% shortly after microcyst induction by high isoleucine-threonine. It is postulated that spermidine is an inhibitor of microcyst induction; when spermidine formation is blocked by methionine starvation, morphogenesis is induced.     

Translational initiation regulators are hypophosphorylated in rat liver during ethionine-mediated ATP depletion

Administration of ethionine to female rats is known to inhibit hepatic protein synthesis by reducing the level of hepatic ATP. Administration of methionine and/or adenine rapidly restores the ATP levels and protein synthesis. The ethionine administration causes a progressive disaggregation of hepatic polysomes, suggesting that the initiation step of protein synthesis is inhibited. Recent studies indicate that changes in initiation are associated with alterations in the phosphorylation states of translational initiation regulators such as eukaryotic initiation factor (eIF) 4E, eIF4E-binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1). We found that these initiation regulators are hypophosphorylated in rat liver during ethionine-mediated ATP depletion (60% of the control value). Furthermore, the restoration of the ATP levels by the administration of methionine and adenine brought about a complete recovery of the phosphorylation states of all these regulators. The present data suggest that hypophosphorylation of various initiation regulators represents the primary event in the ethionine-induced breakdown of polysomes and inhibition of protein synthesis in the liver. Possible involvement of mammalian target of rapamycin (mTOR), as a sensor of intracellular ATP level, was also discussed.     

Methionine metabolism in BHK cells: selection and characterization of ethionine resistant clones.

The selection of clones resistant to methionine antagonists was undertaken on baby hamster Kidney cells grown in a methionine free medium, supplemented with homocystine, folic acid and hydroxo-B12. Clones resistant to 30 mug/ml ethionine were isolated after mutagenesis at an induced mutation frequency of 2.3 X 10(-5). An ethionine resistant clone, ETH 304, was extensively studied. The resistant cells excreted methionine in the culture medium and the intracellular pools of methionine and SAM were two to five times greater in the resistant clone than in the wild type cells. A semidominant ethionine resistant phenotype was observed in hybrids between the wild type and this resistant clone. Measurement of the specific activity of menadione reductase, B12 methyltransferase and ATP: L-methionine S-adenosyl-transferase in crude extracts of the wild type showed a repressive action of methionine on the level of the three enzymes. However, the ethionine resistant clone ETH 304 was not modified in this function. Menadione reductase is feedback-inhibited by SAM in wild type cells. The enzyme of the ethionine resistant clone was significantly less sensitive to SAM. When a comparison of thermal stability was made between the wild type and ethionine resistant clone enzymes, it was found that the thermal stability of the latter was modified. Three other ethionine resistant clones, independantly isolated, were similarly affected in the properties of menadione reductase. These results suggest that the pathway of re-use of S-adenosyl homocysteine, produced during methylation reactions, is highly regulated by methionine and SAM.     

Effects of safrole and isosafrole pretreatment on N- and ring-hydroxylation of 2-acetamidofluorene by the rat and hamster

1. The effects of safrole and isosafrole pretreatment on both N- and ring-hydroxylation of 2-acetamidofluorene were studied in male rats and hamsters. 2. Isosafrole (100mg/day per kg body wt.) pretreatment of rats for 3 days did not have any effect on urinary excretion of hydroxy metabolites of 2-acetamidofluorene. However, similar pretreatment with safrole produced increased urinary excretion of N-, 3- and 5-hydroxy derivatives. 3. Similar treatment with these two chemicals for 3 days increased ring-hydroxylation activity by rat liver microsomal material. Increases in N-hydroxylation were much less than those in ring-hydroxylation. Isosafrole was twice as effective as safrole. 4. Increases in hydroxylating activity due to safrole or isosafrole treatment were inhibited by simultaneous administration of ethionine. Similarly, ethionine inhibition was almost completely reversed by the simultaneous administration of methionine. 5. Safrole or isosafrole (0.1mm and 1mm) inhibited 7-hydroxylation activity by liver microsomal material from control rats. At 1mm these two chemicals inhibited both 5- and 7-hydroxylation activity by liver microsomal material from 3-methylcholanthrene-pretreated rats. 3-Hydroxylation activity was not inhibited by 1mm concentrations of these two chemicals. 6. A single injection of safrole (50100 or 200mg/kg body wt.) 24h before assay had no appreciable effect on either N- or ring-hydroxylation activity by hamster liver microsomal material. However, isosafrole (200mg/kg body wt.) treatment inhibited N-, 3- and 5-hydroxylation activities by hamster liver microsomal material; it had no effect on 7-hydroxylation activity.     

Functionally impaired tRNA from ethionine treated rats as detected in injected Xenopus oocytes.

Treatment of rats with ethionine was found to cause severe impairment in the aminoacylation capacity of tRNA. This effect was only observed when assayed in injected oocytes, while invitro assays of aminoacylation failed to detect differences between normal tRNA and tRNA from ethionine treated animals. The effect of ethionine on the tRNA population was not uniform and differed for various amino acid specific tRNAs. Thus liver tRNA from ethionine treated rats showed a decreased capacity for phenylalanine aminoacylation, while no change was found in the case of leucine. On the other hand, the level of histidine aminoacylation was higher for tRNA from ethionine treated animals. An even more complex response was observed with methionine aminoacylation where tRNA from ethionine treated animals showed an initially faster rate than control tRNA. With more prolonged incubation periods, the methionyl-tRNA from ethionine treated animals was deacylated at an accelerated rate while the level of normal methionyl-tRNA remained almost constant.In addition to the aminoacylation reaction, the participation of aminoacyl-tRNA in protein synthesis was severely impaired. In this case, both the injected oocyte system and the cell-free wheat germ assay revealed these differences which were manifested with various mRNA and viral RNA preparations.     

The isoelectric fractionation of hen''s-egg ovotransferrin

1. ATP sulphurylase was assayed in various organs from vitamin A-deficient and pair-fed control rats at different stages of deficiency. Activity decreased slightly in the liver and markedly in the adrenal gland. Striking differences in liver activity were observed between pair-fed control and ad libitum-fed animals. This observation suggested that diet (apart from vitamin A) strongly influenced the activity of ATP sulphurylase. 2. Total starvation caused a severe decrease in activity in liver within 48hr. This was due to a lack of protein intake. 3. By feeding groups of vitamin A-deficient and pair-fed control rats on a diet containing 80% protein, the specific activity of the liver ATP sulphurylase was maintained in the pair-fed control group at the normal level of an ad libitum-fed rat, whereas it decreased by 25% (statistically significant at P<0.01) in the deficient rat. On a 20%-protein diet, there were no significant differences between vitamin A-deficient and pair-fed control rats. These relationships held also for enzyme activity expressed per g. of liver, per total liver and per g. of DNA. There were no differences in liver protein or DNA concentration between vitamin A-deficient and control rats on either protein intake. 4. Control rats on a 20%-protein diet had liver specific enzyme activities about one-half of those in control rats on an 80%-protein diet, as well as lower liver protein concentrations. 5. It is concluded that, when the effect of protein deprivation on ATP sulphurylase is separated from the effect of vitamin A deficiency, a lowering of the enzyme activity caused by the vitamin deficiency is demonstrable.     

Adaptation of adenosylmethionine metabolism and methionine recycling to variations in dietary methionine in the rat

Weanling rats were fed a casein-based diet supplemented to give dietary methionine (Met) concentrations of 0.41, 0.61, and 1.50%. After 2 weeks of feeding, the rats received intraperitoneally 800 nCi of 2-14C-labeled and/or methyl-3H-labeled L-Met. The animals were killed 20 min, 1 hr, or 2 hr after the isotope injection and the specific radioactivity of adenosylmethionine (AdoMet) as well as the total acid-soluble radioactivity was analyzed in the liver and skeletal muscle. Met concentrations of the liver and skeletal muscle were increased 20-fold by the diet containing 1.50% of Met. In the liver, but not in skeletal muscle, accumulation of AdoMet closely followed changes in Met concentration. Within 2 hr after intraperitoneal injection, the rate of disappearance of 3H label from the acid-soluble fraction was slow in both tissues; increasing in the liver and decreasing in skeletal muscle with increasing dietary Met concentration. At the same time, disappearance of 14C label was slow in both tissues in the rats fed the toxic Met diet, and also in the liver of the rats fed the Met-deficient diet. Decline of the specific radioactivity of the AdoMet pool with respect to 3H label was similar to that of 14C label in the skeletal muscle at all dietary Met concentrations. In the liver, the rate of disappearance of 14C label from the AdoMet pool was markedly increased and that of the 3H label slightly decreased with increasing dietary Met supply. Met deprivation resulted in rapid disappearance of 3H label from the hepatic AdoMet pool, whereas the disappearance of the 14C label was very slow. The results indicate that hepatic Met recycling is very effective with deficient or adequate dietary Met concentrations. In skeletal muscle, the capacity to catabolize extra Met is very limited and continuous flow of Met to liver takes place. Unlike in the liver, in skeletal muscle the transsulfuration route is not adaptable to changes in Met supply and plays a minor role in Met catabolism. The approach used to determine the efficacy and adaptation of methionine salvage pathways by following simultaneously the decline of the specific radioactivities of the methyl group and the methionyl carbon chain of AdoMet following intraperitoneal injection of double-labeled Met has several advantages over that used in literature reports. It offers a reliable means of observing these metabolic pathways in whole animals without disruption of metabolite fluxes.     

THE DISORGANIZATION OF HEPATIC CELL NUCLEOLI INDUCED BY ETHIONINE AND ITS REVERSAL BY ADENINE

The structure of nuclei and nucleoli of hepatic cells after short-term ethionine administration was investigated with the electron microscope. By 1½ hr after the injection, a distinct alteration occurred in the nucleoli which was characterized by the appearance of electron-opaque masses in the nucleolonema. After 6–8 hr, the nucleoli showed partial fragmentation into small, dense masses. Large aggregates of interchromatinic granules appeared in the nucleoplasm. Condensation of chromatin became prominent in the nucleoplasm particularly along the nuclear membrane. By 12 hr almost complete fragmentation of nucleoli had occurred. The administration of adenine or methionine at 4 hr prevented the development of nucleolar changes. Also, adenine administration at 8 hr after ethionine completely reversed the nucleolar lesion by 12 hr. After methionine administration at 8 hr, many nucleoli showed incomplete reconstruction with many twisted ropelike structures when viewed 4 hr later. Identical structures were found when adenine was given at 8 hr, and animals were sacrificed 2 hr later. On the basis of this observation, the simplified structures of nucleoli found 2 hr after adenine or 4 hr after methionine appeared to be precursors of the nucleolonema. It is suggested that nucleoli show at least two basic reaction patterns to inhibitors of RNA synthesis, one typified by actinomycin D and one by ethionine.     

Opposite effect of methionine-supplemented diet, a model of hyperhomocysteinemia, on plasma and liver antioxidant status in normotensive and spontaneously hypertensive rats

Hyperhomocysteinemia is often associated with an increase in blood pressure. However our previous study has shown that methionine supplementation induced an increase in blood pressure in Wistar-Kyoto (WKY) rats and a decrease in blood pressure in spontaneously hypertensive rats (SHR) with significant differences in plasma homocysteine (Hcy) metabolites levels. Previously liver antioxidant status has been shown to be decreased in SHR compared to WKY rats. It has been suggested that oxidative stress may predispose to a decrease in NO bioavailability and induce the flux of Hcy through the liver transsulfuration pathway. Thus the aim of this study was 1) to investigate the effect of methionine supplementation on NO-derived metabolites in plasma and urine 2) to investigate whether abnormalities in Hcy metabolism may be responsible for the discrepancies observed between WKY rats and SHR concerning blood pressure and 3) to investigate whether a methionine-enriched diet, differently modified plasma and liver antioxidant status in WKY rats an SHR. We conclude that the increase in blood pressure in WKY rats is related to high plasma cysteine levels and is not due to a decrease in NO bioavailability and that the decrease in blood pressure in SHR is associated with high plasma GSH levels after methionine supplementation. So GSH synthesis appears to be stimulated by liver oxidative stress and GSH is redistributed into blood in SHR. So the great GSH synthesis can be rationalized as an autocorrective response that leads to a decreased blood pressure in SHR.     

Significance of alterations in the metabolomics of sulfur-containing amino acids during liver regeneration

It has been known that liver regeneration is accompanied with a profound change in the metabolomics of sulfur-containing substances in liver. However, its physiological significance in the liver regenerative process is still unclear. Our previous work showed that buthioninesulfoximine and phorone, both widely used to deplete intracellular glutathione (GSH) in biological experiments, induced contrasting changes in the sulfur-containing amino acid metabolism in liver. In this study we employed these GSH-depleting agents to evaluate the role of sulfur-containing substances in the early phase of liver regeneration. Male rats treated with buthioninesulfoximine or phorone were subjected to two-thirds partial hepatectomy (PHx). At the doses used, the magnitude of GSH depletion after PHx was comparable, but buthioninesulfoximine administration inhibited the progression of liver regeneration as determined by liver weight increase, elevation of serum alanine aminotransferase activity, and cyclin D1 and proliferating cell nuclear antigen (PCNA) protein expressions, whereas liver recovery was significantly accelerated in the phorone-treated rats, suggesting that the role of GSH in this process is minimal. Hepatic concentrations of methionine, S-adenosylmethionine, cysteine, taurine and GSH were all elevated by PHx. Methionine adenosyltransferase activity was also induced in the remnant liver. Buthioninesulfoximine administration depressed the elevation of S-adenosylmethionine, but increased the catabolism of cysteine to taurine. In contrast, S-adenosylmethionine elevation was augmented whereas cysteine, hypotaurine and taurine were decreased in the phorone-treated rats. PHx elevated hepatic putrescine and spermidine, but lowered spermine concentrations. Buthioninesulfoximine administration increased putrescine further, but decreased spermidine and spermine concentrations. On the contrary, both spermidine and spermine concentrations were elevated in the rats treated with phorone. The results suggest that the availability of S-adenosylmethionine plays a critical role in the progression of liver regeneration via enhancement of polyamine synthesis. These findings raise the possibility that regulating hepatic transsulfuration reactions may be capable of modifying the recovery process after liver injury.     

Early alteration in liver of rats fed 2-fluorenylacetamide investigated by L-ethionine probe

The metabolic activity of liver of rats fed a diet containing 0.03% 2-N-fluorenylacetamide (FAA) was investigated using the probe of L-[ethyl-14C]ethionine (613 mumol L-E/100 g body wt.). Shortly after the onset of the carcinogenic regimen, the capacity of liver to accumulate S-adenosylethionine (SAE) began to decline, reaching its minimum (30% of the concentration in control rats) within 3 weeks. This decreased capacity to accumulate SAE results from the FAA-induced decrease in activity of ATP-L-methionine adenosyltransferase. The concentration of hepatic ATP assayed without L-ethionine (L-E) probe also declined during the first 2 weeks of the carcinogenic regimen, but then increased, attaining the normal values within 2 more weeks. Administering the L-E probe to the FAA-fed rats produced an even greater drop in hepatic ATP concentration during the first 2 weeks; however from the third week on, the L-E dose produced no depressing effect, despite the SAE accumulation remaining at its same depressed levels and, therefore, trapping the same amount of ATP as in the previous weeks. The results show that the modification of L-E metabolism and of ATP turnover, observed previously in DL-E fed rats, need not be specific for the carcinogen fed and can occur even when the carcinogens are metabolized by different enzymatic systems.     

Effects of Dietary Glutathione on Plasma and Liver Lipid Levels in Rats Fed on a High Cholesterol Diet

The effects of dietary glutathione (GSH) on plasma and liver lipid concentrations were investigated with rats fed on a high cholesterol diet. When graded levels of GSH, 0.75 to 5.0%, were added to the 25% casein basal diet, the plasma total cholesterol level was significantly decreased and the HDL-cholesterol level was inversely increased in all addition levels without influence on the growth of animals except for the 5% addition level; the dietary addition of 5% GSH markedly depressed the growth and food consumption of rats and caused a slight diarrhea. Plasma triglyceride and phospholipid levels were decreased by the dietary addition of GSH. The contents of cholesterol and triglyceride in the liver were decreased as the dietary addition level of GSH was increased. The dietary addition of a mixture of glutamic acid, cysteine and glycine, or cysteine alone corresponding to 2.5% GSH resulted in a cholesterol-lowering effect which could not be distinguished from the effect of GSH in rats fed on the 25% casein diet. When 1.5% GSH was added to a low (10%) casein diet, the plasma cholesterol-lowering effect of GSH was also observed and the effect was comparable to that of cysteine. These results indicate that dietary-added GSH has a plasma and liver cholesterol-lowering efficacy and that this effect is largely attributable to the cysteine residue of GSH rather than to the tripeptide itself or the other amino acid residues.     

The concentration and biosynthesis of nicotinamide nucleotides in the livers of rats treated with carcinogens

1. The oxidoreduction state and concentration of both NAD and NADP as well as the maximum potential activities of NMN adenylyltransferase and NAD(+) kinase have been measured in the livers of rats treated for 14-28 days with 4-dimethylamino-3'-methylazobenzene, 4-dimethylamino-4'-fluoroazobenzene, alpha-naphthyl isothiocyanate or ethionine and in primary hepatomas induced by 4-dimethylamino-3'-methylazobenzene. 2. The total NAD and total NADP both decreased in the livers of rats treated with either azo-dyes or alpha-naphthyl isothiocyanate but not in those treated with ethionine. The activities of NMN adenylyltransferase and NAD(+) kinase did not alter appreciably after such treatments. 3. In the primary hepatomas the concentrations of both NAD and NADP fell drastically and the activities of NMN adenylyltransferase and NAD(+) kinase fell to about 50% of the control activities. 4. No correlation could be established between the concentrations of the nucleotides and the activities of the enzymes synthesizing them. It appears, however, that a relationship exists between the NAD content of the tissue and the amount of NADP present. 5. The results are discussed with respect to the control of NAD and NADP synthesis by ATP. At the concentrations of NAD normally present in the cell it is suggested that NAD may be a rate-limiting substrate in NADP synthesis.