l-Arginine and l-glutamine as immunonutrients and modulating agents for oxidative stress and toxicity induced by sodium nitrite in rats

Sodium nitrite (NaNO₂) is a flavoring, coloring and preservative agent in meat and fish products. The study aimed to evaluate the efficacy of l-arginine and l-glutamine supplementation as a potentially novel and useful strategy for the modulation of oxidative stress and toxicity induced by NaNO₂ in male rats. Rats were divided into six groups each of 10 rats and treated for 6 weeks: group 1 as normal control; group 2 fed standard diet containing 0.2% NaNO₂; group 3 and 4 fed the previous diet supplemented with 1% and 2% arginine, respectively; group 5 and 6 fed NaNO₂ diet supplemented with 1% and 2% glutamine, respectively. NaNO₂ treatment induced a significant increase in serum malondialdehyde, nitric oxide, arginase, glutathione-S-transferase activities, urea and creatinine as well as differential leucocytes%. However, a significant decrease was recorded in reduced glutathione, catalase activity, total protein, albumin and some hematological parameters as well as immunoglobulin G. On the other hand, arginine or glutamine showed a remarkable modulation of these abnormalities as indicated by reduction of malondialdehyde and improvement of the investigated antioxidant and hematological parameters. It can be concluded that arginine or glutamine supplementation may reduce oxidative stress and improve the hazard effects of NaNO₂.     

Protective effect of N-acetyl-l-cysteine against disulfiram-induced oxidative stress and apoptosis in V79 cells

This work investigated the effect of N-acetyl-l-cysteine (NAC) on disulfiram (DSF) induced oxidative stress in Chinese hamster fibroblast cells (V79). An increase in oxidative stress induced by DSF was observed up to a 200 μM concentration. It was evidenced by a statistically significant increase of both GSH_t and GSSG levels, as well as elevated protein carbonyl (PC) content. There was no increase in lipid peroxidation (measured as TBARS). DSF increased CAT activity, but did not change SOD1 and SOD2 activities. Analysis of GSH related enzymes showed that DSF significantly increased GR activity, did not change Se-dependent GPx, but statistically significantly decreased non-Se-dependent GPx activity. DSF showed also pro-apoptotic activity. NAC alone did not produce any significant changes, besides an increase of GSH_t level, in any of the variables measured. However, pre-treatment of cells with NAC ameliorated DSF-induced changes. NAC pre-treatment restored the viability of DSF-treated cells evaluated by Trypan blue exclusion assay and MTT test, GSSG level, and protein carbonyl content to the control values as well as it reduced pro-apoptotic activity of DSF. The increase of CAT and GR activity was not reversed. Activity of both GPx was significantly increased compared to their values after DSF treatment. In conclusion, oxidative properties are at least partially attributable to the cellular effects of disulfiram and mechanisms induced by NAC pre-treatment may lower or even abolish the observed effects. These observations illustrate the importance of the initial cellular redox state in terms of cell response to disulfiram exposure.     

l-cysteine as a regulator for arsenic-mediated cancer-promoting and anti-cancer effects

Previous studies have shown that activities of tyrosine kinases and secretion of the active form of matrix metalloproteinase-2 (MMP-2) are correlated with promotion of tumor growth, while apoptotic cell death in cancer cells is correlated with anti-cancer effects. Although arsenic has been reported to have both cancer-promoting and anti-cancer effects, the mechanisms of the arsenic-mediated bidirectional effects remain unknown. We examined the effects of arsenic on both proto-oncogene c-RET-transfected NIH3T3 cells with benign characters and oncogenic RET-MEN2A-transfected NIH3T3 cells with malignant characters. Arsenic promoted not only c-RET tyrosine kinase activity but also genetically activated RET-MEN2A kinase activity with promotion of dimer formation of RET proteins. Arsenic also increased secretion of the active form of MMP-2 in both RET-MEN2A-transfectants and c-RET-transfectants. On the other hand, arsenic promoted poly-(ADP-ribose) polymerase (PARP) degradation and cell death in both malignant and non-malignant cells. Interestingly, l-cysteine inhibited the arsenic-mediated tumor-promoting effects (activation of kinases and MMP-2 secretion) but not arsenic-mediated anti-cancer effects (PARP degradation and cell death). Our results suggest redox-linked regulation of arsenic-mediated activities of kinases and MMP-2 secretion but not arsenic-mediated cell death. Our results also suggest that l-cysteine is an ideal supplement that inhibits arsenic-mediated tumor-promoting effects without affecting arsenic-mediated anti-cancer effects.     

Protection of l-methionine against H2O2-induced oxidative damage in mitochondria

Reactive oxygen species (ROS) is reported to be a critical pathogenic factor and mitochondria is one of the susceptible subcellular organs for oxidative damage. Methionine sulfoxide reductase A (MsrA) is a key anti-oxidant enzyme associated with cytoprotection and previous reports have revealed its importance in mitochondrial function. The anti-oxidation of MsrA is due to Met-centered redox cycle, suggesting that Met-centered redox cycle may play a critical role in mitochondrial protection. l-Methionine (l-Met), a natural amino acid with anti-oxidation activity, can mimic the effect of Met-centered redox cycle. Here, we investigated the protection of l-Met on H₂O₂-induced oxidative damage in mitochondria. Our study demonstrated that l-Met protected H₂O₂-induced injury in CHO cells. Cytoprotections of l-Met at low concentrations (1–5 mM) were abolished by dimethyl sulfoxide (DMSO), a competitive inhibitor of MsrA function, suggesting that these effects may involve the participation of MsrA. Overexpression of MsrA in CHO cells protected mitochondria from H₂O₂-induced downtrend of membrane potential and production of mitochondrial superoxide. Pre-treatment with l-Met (1 mM) produced a similar effect on the mitochondrial protection against H₂O₂. Furthermore, it was observed that topical application of l-Met can prevent 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced oxidative damage in the skin of mice. These results suggest that anti-oxidation activity of l-Met may promise a new strategy for the prevention of oxidative stress-induced damage.     

Protective role of l-carnitine supplementation against exhaustive exercise induced oxidative stress in rats

The objective of this study was to investigate temperature dependent effects of oral l-carnitine supplementation on exhaustive exercise induced oxidative damage in rats. 42 male Spraque Dawley rats were randomly divided into seven experimental groups. These groups were formed as three non-carnitine exercise groups, three carnitine-exercise groups and a sedentary group. l-carnitine was given intraperitoneally to the carnitine-exercise groups 1 h before the exercise in 100 mg/kg. Blood was collected to measure paraoxonase-1 (PON1) activity, plasma malondialdehyde (MDA), low-density lipoprotein (LDL) and cholesterol concentrations. These biomarkers were measured in venous blood samples collected before and after the rats swam in pools at different water temperatures (18 °C, 28 °C and 38 °C). In the non-carnitine group, exercise caused a significant decrease in PON1 activity and a significant elevation in MDA concentration at 28 °C compared to the sedentary group. No significant alterations were evidenced in LDL and cholesterol concentrations upon exercise. The decrease in PON1 activity became higher with increasing temperature whereas the elevation in MDA levels increased at 18 °C. In the l-carnitine supplementation group, recovery in PON1 activity was observed significant at 28 °C and very significant at 38 °C. MDA concentration was almost the same with that of the non-carnitine group at 18 and 38 °C, but it significantly decreased at 28 °C. Considering the recovery in PON1 and MDA levels at 28 °C, which is the temperature of the sedentary group; our results suggest that l-carnitine supplementation has a protective role on exhaustive exercise-induced oxidative stress. Findings of this study also demonstrate influences of thermal stress on these parameters during exhaustive exercise.     

Reduction of l-methionine selenoxide to seleno-l-methionine by endogenous thiols, ascorbic acid, or methimazole

Seleno-l-methionine (SeMet) can be oxidized to l-methionine selenoxide (MetSeO) by flavin-containing monooxygenase 3 (FMO3) and rat liver microsomes in the presence of NADPH. MetSeO can be reduced by GSH to yield SeMet and GSSG. In the present study, the potential reduction of MetSeO to SeMet by other cellular components and antioxidants was investigated. Besides GSH, other thiols (l-cysteine, or N-acetyl-l-cysteine) and antioxidants (ascorbic acid and methimazole) also reduced MetSeO to SeMet. This reduction is unique to MetSeO since methionine sulfoxide was not reduced to methionine under similar conditions. The MetSeO reduction by thiols was instaneous and much faster than the reduction by ascorbic acid or methimazole. However, only one molar equivalent of ascorbic acid or methimazole was needed to complete the reduction, as opposed to two molar equivalents of thiols. Whereas the disulfides produced by the reactions of MetSeO with thiols are chemically stable, methimazole disulfide readily decomposed at pH 7.4, 37 °C to yield methimazole, methimazole-sulfenic acid, methimazole sulfinic acid, methimazole S-sulfonate, 1-methylimidazole (MI) and sulfite anion. Collectively, the results demonstrate reduction of MetSeO to SeMet by multiple endogenous thiols, ascorbic acid, and methimazole. Thus, oxidation of SeMet to MetSeO may result in depletion of endogenous thiols and antioxidant molecules. Furthermore, the novel reduction of MetSeO by methimazole provides clear evidence that methimazole should not be used as an alternative FMO substrate when studying FMO-mediated oxidation of SeMet.     

Berberine exerts nephroprotective effect against cisplatin-induced kidney damage through inhibition of oxidative/nitrosative stress,inflammation, autophagy and apoptosis

The aim of this study was to investigate the therapeutic activity of isoquinoline alkaloid berberine against cisplatin (CP)-induced nephrotoxicity in mice. Berberine was administered at daily doses of 1, 2 and 3 mg/kg by gavage for two successive days, 48 h after intraperitoneal CP injection (13 mg/kg). Mice were sacrificed 24 h after the last dose of berberine. Histopathological changes and the increase in serum creatinine and blood urea nitrogen (BUN) induced by CP were significantly ameliorated by berberine in a dose-dependent manner. Additionally, oxidative/nitrosative stress, evidenced by the increase in renal 4-hydroxynonenal (4-HNE), 3-nitrotyrosine (3-NT), cytochrome P450 E1 (CYP2E1) and heme oxygenase (HO-1) expression, was significantly reduced. The expression of nuclear factor-kappaB (NF-κB), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was markedly suppressed by berberine, indicating the inhibition of inflammatory response. Treatment of CP-intoxicated animals with berberine also significantly reduced the expression of p53, active caspase-3 as well as autophagy marker light chain 3B (LC3B) in the kidneys. The results of the current study showed the nephroprotective activity of berberine against CP-induced renal injury, which could be attributed to the inhibition of oxidative/nitrosative stress, inflammation, autophagy and apoptosis.     

Chronic administration of troxerutin protects mouse kidney against d-galactose-induced oxidative DNA damage

Troxerutin, a natural bioflavonoid, has been reported to have many benefits and medicinal properties. In this study, we evaluated the protective effect of troxerutin against d-gal-induced oxidative DNA damage in mouse kidney, and explored the potential mechanism of its action. Our data showed that troxerutin significantly decreased levels of urea, uric acid and creatinine in serum and the renal histological injury in d-gal-treated mice. Troxerutin markedly restored Cu/Zn-SOD, CAT and GPx activities in the kidney of d-gal-treated mouse. Furthermore, the increase of 8-hydroxydeoxyguanosine (a marker of oxidative DNA damage) induced by d-gal was effectively suppressed by troxerutin. Internucleosomal DNA ladder fragmentation and the number of terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labeling (TUNEL)-positive cells in d-gal-treated mice were inhibited by troxerutin, which might be attributed to its antioxidant property by decreasing activities of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) and levels of reactive oxygen species (ROS). In conclusion, these results suggested that troxerutin could protect the mouse kidney against d-gal-induced injury by improving renal function, attenuating histopathologic changes, reducing ROS production, renewing the activities of antioxidant enzymes and decreasing DNA oxidative damage. This study provided novel insights into the protective mechanisms of troxerutin in d-gal-induced kidney injury.     

Effects of oxidative stress on apoptosis in manganese-induced testicular toxicity in cocks

Manganese (Mn) is a trace element known to be essential for maintaining the proper function and regulation of many biochemical and cellular reactions. However, little is known about the reproductive toxicity of Mn in birds. To investigate the toxicity of Mn on male reproduction in birds, 50-day-old cocks were fed either a commercial diet or a Mn-supplemented diet containing 600, 900, and 1800 mg/kg MnCl₂. After being treated with Mn for 30, 60, and 90 d, the following were determined: Mn content; histological and ultrastructural changes in the testes, apoptosis; the malondialdehyde (MDA) level; the activities of superoxide dismutase (SOD); the inhibition ability of hydroxyl radicals (OH); the levels of nitric oxide (NO), nitric oxide synthase (NOS), and protein carbonyl in the testes; the DNA–protein crosslinks (DPC); and the activity of the ATP enzyme. Exposure to Mn significantly lowered the activity of SOD and glutathione peroxidase (GPx) and the inhibition ability of OH. Mn exposure also increased the levels of MDA, NO, NOS, DPC, and protein carbonyl; the number of apoptotic cells; and the Mn content and caused obvious histopathological changes in the testes. These findings suggested that Mn exposure resulted in the oxidative damage of cock testicular tissue by altering radical formation, ATP enzyme systems, apoptosis, and DNA damage, which are possible underlying reproductive toxicity mechanisms induced by Mn exposure.     

Aromatic l-amino acid decarboxylase activity in central and peripheral tissues and serum of rats with l-dopa and l-5-hydroxytryptophan as substrates

This paper describes the distribution of aromatic l-amino acid decarboxylase (AADC) activities in seventeen tissues (eleven peripheral and six brain tissues) of rats, using both l-DOPA and l-5-hydroxytryptophan (l-5-HTP) as substrates. The ratios of the activities of the enzyme using both substrates in the same homogenates were also determined. Rat pineal gland had the highest activity with the substrates followed by the liver, kidney and adrenals. Activity in the adrenals was not only high, but the ratio of the activities for l-DOPA and l-5-HTP was the highest. AADC activity was detected in rat serum by a new and highly sensitive high-performance liquid chromatography-voltammetric assay, using l-DOPA and l-5-HTP as substrates; the ratio of the activities for the two substrates was the lowest. K_m values, measured for the two substrates using the homogenates of the pineal, liver, and adrenals of rats, were found to be similar, but K_m values for l-DOPA were about 3-fold higher than those for l-5-HTP in the three tissues.     

Long-term treatment with l-isoleucine or l-leucine in AIN-93G diet has promoting effects on rat bladder carcinogenesis

In the present study, effects of l-leucine and l-isoleucine on rat bladder carcinogenesis were investigated using AIN-93G and MF basal diet. In Experiment 1, N-butyl-N-(4-hydroxybutyl)-nitrosamine was used as an initiator of bladder carcinogenesis. In the AIN-93G diet groups, a significantly higher incidence and multiplicity of bladder tumors, accompanied by decreased final body weight, was observed in the l-leucine-supplemented group and a significantly higher incidence of papillomas and total tumors was observed in the l-isoleucine-supplemented group. In the MF diet groups, the multiplicity of papillary and nodular hyperplasia was significantly increased in the l-isoleucine-supplemented group. Urinary pH values were not affected by supplementing either type of diet with l-leucine or l-isoleucine. In Experiment 2, the amino acid was administered in the basal diets for 2 weeks without initiator. No pathological lesions were observed in the bladder urothelium in any of the groups, and no significant differences in urinary pH values, microcrystals or aggregates were observed between the amino acid-supplemented groups and their respective control groups. In conclusion, long-term treatment with l-leucine or l-isoleucine has a promoting effect on rat bladder carcinogenesis; therefore, their long-term use as a dietary supplement for bladder cancer patients should be avoided until more is known.     

Nonylphenol induces apoptosis via mitochondria- and Fas-l-mediated pathways in the liver of adult male rat

Nonylphenol (NP) is an environmental contaminant known to possess estrogenic properties. Humans are constantly exposed to NP by contaminated water and food products. In the present study we sought to investigate whether treatment with low doses of NP induces apoptosis in the liver of adult rats. Rats were administered with NP by oral gavage at the doses of 15,150 and 1500 μg/kg body weight per day for 45 days. Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assayed. Apoptosis-related proteins namely cytochrome c, caspase-3, caspase-8, caspase-9, Fas and Fas-l, and expression of bcl-2 mRNA and bax mRNA were examined in the liver. Levels of AST and ALT were increased in the treated rats. Western blot analysis revealed elevation in the levels of cytochrome c, caspase-3, caspase-8, caspase-9, Fas and Fas-l in the liver of NP-treated rats. Decreased expression of bcl-2 mRNA (anti-apoptotic) and increased expression of bax mRNA (apoptotic) were observed in the liver of treated rats. Increased localization of caspase-3 in the hepatocytes and DNA damage were observed in the liver of treated rat. It is concluded that NP induces apoptosis in liver involving both mitochondria-dependent and Fas–Fas-l pathways and thereby, leading to hepatic damage in rats.     

l-Cysteine reduces oral ethanol self-administration and reinstatement of ethanol-drinking behavior in rats

Our previous findings have shown that l-cysteine, a non essential amino acid, prevented ethanol (EtOH) induced conditioned place preference. The aim of the present study was to examine the effect of l-cysteine on the acquisition and maintenance of oral EtOH self-administration and on the reinstatement of EtOH-drinking behavior in Wistar rats. Rats were pretreated intraperitoneally with saline or l-cysteine (20 and 40 mg/kg) 30 min before each acquisition trial, in an operant nose-poking paradigm where they were given the opportunity to orally self-administer tap water or EtOH (5-10% v/v). Further, to evaluate if l-cysteine reduces the acquired oral EtOH self-administration, we carried out an independent experiment in which rats were trained to self-administer EtOH (10%); after all groups of rats developed similarly stable oral EtOH self-administration, the effect of l-cysteine (0, 40, 60, 80 and 100 mg/kg) was tested. An additional group of rats was pretreated with saline or l-cysteine (80 mg/kg) and tested on reinstatement after EtOH extinction and, at the end of last reinstatement session, were utilized to measure blood and brain EtOH levels. The animals that had access to EtOH solution discriminated between the active and inactive nose-pokes and showed rates of active nose-pokes significantly higher than the tap water group. Furthermore, rats self-administering EtOH (10%) also demonstrated extinction behavior and gradually reinstated active nose-poke responding when EtOH was reintroduced. l-cysteine reduced both the acquisition and maintenance of oral EtOH self-administration. The reduced reinstatement of EtOH-drinking behavior was paralleled by a significant reduction of EtOH intake and correlated with blood and brain EtOH levels. The efficacy of l-cysteine on the various phases of alcohol drinking in rats, could represent an interesting pharmacological approach and could open a new line of research for the development of therapies to reduce EtOH intake in alcoholic patients.     

Allethrin induces oxidative stress,apoptosis and calcium release in rat testicular carcinoma cells (LC540)

Over the years, pyrethroids, including D-allethrin, are widely used for domestic and agricultural purposes and are found to be toxic to many organ systems including the male reproductive system. However, the molecular mechanisms of allethrin toxicity are not well understood. In this study, we demonstrate that allethrin exhibited a dose-dependent cytotoxicity on Leydig cell carcinoma cells (LC540) and isolated primary Leydig cells with an IC₅₀ of 125 μM and 59 μM respectively. Cytotoxicity was associated with generation of reactive oxygen species, increased lipid peroxidation and alterations in antioxidant enzyme status. Morphological analyses of LC540 cells treated with allethrin revealed the presence of apoptotic bodies. Using flow cytometry, we observed that the number of cells that displayed early apoptotic features and entering into G₀ phase of cell cycle increased significantly with loss of mitochondrial membrane potential. The levels of p53 mRNA and cleaved PARP-1 protein were increased, whereas BCL-2, pro-Caspase-3 and PARP-1 were decreased. Allethrin induced apoptosis was associated with voltage gated calcium channel mediated intracellular calcium release. Results of our study demonstrate that allethrin toxicity in the male reproductive tract may involve Leydig cell apoptotic death.     

Allethrin induces oxidative stress,apoptosis and calcium release in rat testicular carcinoma cells (LC540)

Over the years, pyrethroids, including D-allethrin, are widely used for domestic and agricultural purposes and are found to be toxic to many organ systems including the male reproductive system. However, the molecular mechanisms of allethrin toxicity are not well understood. In this study, we demonstrate that allethrin exhibited a dose-dependent cytotoxicity on Leydig cell carcinoma cells (LC540) and isolated primary Leydig cells with an IC₅₀ of 125 μM and 59 μM respectively. Cytotoxicity was associated with generation of reactive oxygen species, increased lipid peroxidation and alterations in antioxidant enzyme status. Morphological analyses of LC540 cells treated with allethrin revealed the presence of apoptotic bodies. Using flow cytometry, we observed that the number of cells that displayed early apoptotic features and entering into G₀ phase of cell cycle increased significantly with loss of mitochondrial membrane potential. The levels of p53 mRNA and cleaved PARP-1 protein were increased, whereas BCL-2, pro-Caspase-3 and PARP-1 were decreased. Allethrin induced apoptosis was associated with voltage gated calcium channel mediated intracellular calcium release. Results of our study demonstrate that allethrin toxicity in the male reproductive tract may involve Leydig cell apoptotic death.     

Brain targeting with surface-modified poly(d,l-lactic-co-glycolic acid) nanoparticles delivered via carotid artery administration

In this study, we investigated surface-modified nanoparticles (NP) formulated using a biodegradable polymer, poly(d,l-lactide-co-glycolide) (PLGA), for targeting central nervous system (CNS) diseases. Polysorbate 80 (P80), poloxamer 188 (P188), and chitosan (CS) were used to modify the surfaces of PLGA NP to improve the brain delivery of NP. Surface-modified PLGA NP were formulated using an emulsion solvent diffusion method. 6-Coumarin was used as a fluorescent label for NP. The different formulations of 6-coumarin-loaded PLGA NP were injected into rats via carotid arteries. NP remaining in the brain were evaluated quantitatively, and brain slices were observed using confocal laser scanning microscopy (CLSM). Carotid artery administration was more effective for delivering NP into the brain compared to intravenous administration. After administration, NP concentrations in the brain were increased by NP surface modification, especially CS- and P80-PLGA NP. CLSM observations indicated that P80-PLGA NP could cross the blood-brain barrier and thus serve as a drug delivery system for the CNS. These results indicate that surface-modified PLGA NP have a high potential for use in CNS delivery systems.     

The fate of [C]l-3,4-dihydroxyphenylalanine in isolated perfused rat hearts

The fate of [¹⁴C]l-3,4-dihydroxyphenylalanine (¹⁴C-levodopa) in isolated perfused rat hearts has been studied. Seventy per cent of the perfused [¹⁴C]levodopa was recovered intact, while 15 per cent was recovered as dopa metabolites. Only 2 per cent of the perfused radioactivity was retained by the heart tissue. Deaminated derivatives accounted for the majority of the metabolites (7.2 per cent of perfused dose) although 3-O-methyl dopa was found in substantial quantities (1.6 per cent). The absence of significant tissue accumulation of norepinephrine-¹⁴C leads to the speculation that amine synthesis from perfused dopa occurs at an extraneuronal site.     

Interaction of l-alanosine (NSC 153, 353) with enzymes metabolizing l-aspartic acid, l-glutamic acid and their amides

A comprehensive analysis has been made of the manner in which l-alanosine [l-2-amino-3-(N-nitroso, N-hydroxy) aminopropionic acid] interacts with the enzymes responsible for the metabolism of the dicarboxylic amino acids and their amides. The drug impedes the transport of l-aspartic acid and, to a lesser degree, of l-glutamic acid, l-asparagine and l-glutamine by lymphoblasts. in vitro; in each of these instances, inhibition is apparently competitive in type. Of the enzymes involved in the metabolism of l-aspartic acid, adenylosuccinate synthetase (EC 6.3.4.4). SAICAR synthetase (5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase) (EC 6.3.2.6). l-aspartyl tRNA synthetase (EC 6.1.1.12), l-aspartate transcarbamylase (EC 2.1.3.2) and l-aspartate aminotransferase (EC 2.6.1.1) were inhibited by l-alanosine; moreover, each of these enzymes except l-aspartyl tRNA synthetase accepted the antibiotic as substrate, although at substantially diminished rates. Of the enzymes involved in the metabolism of l-glutamic acid, l-alanosine inhibited only l-glutamine synthetase (EC 6.3.1.2) and l-glutamate decarboxylase (EC 4.1.1.15) to a prominent degree; this last enzyme was also capable of decarboxylating l-alanosine. Of the enzymes metabolizing l-asparagine and/or l-glutamine. only the l-glutaminase activity of l-asparagine amidohydrolase (EC 3.5.1.1) (with l-glutamine as substrate) and, to a lesser degree, carbamyl phosphate synthetase II (EC 2.7.2.9) were inhibited by the antibiotic. Although l-alanosine provokes a rise in the concentration of inosinic acid (IMP) in vitro, pointing to the conclusion that the drug is capable of inhibiting adenylosuccinate synthetase under these circumstances, no such rise was seen in vivo either in tumor or liver. However, 1 and 5 hr after administration l-alanosine depressed hepatic ATP and NAD pools, an effect which indicates that the drug is. in fact, restricting the intracellular concentration of adenine nucleotides. Of the metabolites of l-alanosine formed in vitro, α-decarboxy alanosine, α-keto alanosine, a-hydroxy alanosine. alanosyl IMP and N-carbamyll-alanosine did not inhibit adenylosuccinate synthetase to any prominent degree, whereas the metabolite generated by SAICAR synthetase powerfully inhibited this enzyme, with a K_i, of 0.3μM. Parenteral therapeutic doses of l-alanosine produced striking increases in the concentrations of l-aspartic acid in tumor and liver as well as of l-aspartic and l-glutamic acids in urine. It is concluded that the N-hydioxy, N-nitroso functionality of l-alanosine is analogous in structure to the β-carboxyls of l-aspartic and l-glutamic acids, respectively; this analogy permits l-alanosine to be anabolized and catabolized via several of the enzymatic routes which ordinarily operate on these dicarboxylic amino acids.