(+)-O-(グアニジノ)-L-ホモセリン

The growth and development of final-stadium tobacco hornworm, manduca sexta (sphingidae) larvae fed a 2.5 mmole l-canavanine containing diet was markedly disrupted. Such canavanine-mediated disruption of larval growth was intensified greatly when these organisms were fed a canavanine-containing diet supplemented with a 1:10 molar ratio of l-arginine, l-citrulline, l-ornithine, or l-2,4-diaminobutyric acid; the larvae possess enhanced hemolymph volume (edema) and a significant mortality results from incomplete larval-pupal ecdysis.

The modulatory effects of a non-selective endothelin receptor antagonist, bosentan, were investigated together with those of relatively selective inducible nitric oxide synthase inhibitors, aminoguanidine and L-canavanine, on mesenteric blood flow decrease, liver and spleen injury elicited by endotoxemia. Swiss albino mice (20-40 g) were administered intraperitoneally bosentan (3, 10 or 30 mg/kg), aminoguanidine (15 mg/kg) or L-canavanine (20 or 100 mg/kg) 10 min before they received saline or Escherichia coli endotoxin (10 mg/kg). After 4 hr, the mice were anesthetized, mesenteric blood flow values were measured, spleen and liver weight/body weight ratios were determined and the organs were examined histopathologically. Endotoxin decreased mesenteric blood flow (mL/min), saline: 3.0 +/- 0.2; endotoxin: 2.2 +/- 0.2: n=10, p<0.05), increased the weight of liver (g per kg body weight, saline: 47.5 +/- 2.0; endotoxin: 60.8 +/- 1.9: n=10, p<0.05) and spleen (g per kg body weight, saline: 3.9 +/- 0.5; endotoxin: 8.6 +/- 0.9; n=10, p<0.01) while it inflicted significant histopathological injury to both organs. Bosentan was ineffective at 3 mg/kg but at 10 and 30 mg/kg doses, it abolished all the deleterious effects of endotoxin without exception. Aminoguanidine blocked most of the effects of endotoxin except those on spleen. In contrast, L-canavanine blocked only the endotoxin-induced increase in liver weight but itself increased spleen weight and failed to block any other effects of endotoxin. Thus, it can be speculated that the beneficial effects of aminoguanidine are produced largely by mechanisms other than selective inducible nitric oxide synthase inhibition since L-canavanine was not fully effective. The beneficial effects of endothelin inhibition by using bosentan in endotoxemia can be further exploited for the understanding and the therapy of sepsis-related syndromes.

The effects of L-canavanine, an inhibitor of nitric oxide synthase, on endotoxin-induced shock was investigated in the pentobarbitone anesthetized rat. Endotoxin infusion (2.5 mg kg-1 hr-1 over 6 hr) produced progressive and marked hypotension and hypoglycemia. Electron microscopy showed marked changes in the kidney, comprising severe endothelial cell disruption and the accumulation of platelets in the blood vessels. In the lung, there was marked accumulation of polymorphonuclear leukocytes in small blood vessels and endothelial disruption. Treatment with L-canavanine (10 mg kg-1 by bolus injection each hour starting 70 min after endotoxin or saline infusion) significantly reduced endotoxin-induced hypotension, without any effect on the hypoglycemia. This treatment markedly reduced the endotoxin-induced electron microscopical changes in the kidneys and lungs. Although L-canavanine, like L-NAME, inhibited both cerebellar constitute and splenic inducible nitric oxide synthase in vitro, in contrast to L-NAME it did not modify either arterial blood pressure or carotid artery blood flow in control rats. The data are consistent with L-canavanine being a selective inhibitor of inducible nitric oxide synthase, at least in vivo, and suggest that inhibitors of this enzyme may be beneficial in endotoxin-induced shock.

The effects of L-canavanine and cadmium on the ribonucleoprotein constituents of HeLa S3 cells have been analyzed. Both chemicals induce a similar pattern of alterations in different RNP structures as well as in both RNA and protein synthesis. Pulse and chase autoradiographic experiments reveal that both canavanine and cadmium induce a preferential inhibition of nucleolar RNA synthesis and a slowdown in the transport or processing of nucleolar and extranucleolar RNA. Nucleoli become round and compact. Accumulation of perichromatin granules and fibrils occurs, there is a depletion of interchromatin fibrils, and nuclear formations appear which seem to be involved in the morphogenesis of perichromatin granules accumulated during the treatments. The appearance of clusters of 29- to 35-nm granules might be related with a deficient assembling of constituents of perichromatin granules. The effects of different inhibitors of the transcriptional processes on the accumulation of perichromatin granules suggest that these granules represent a particular subpopulation of hnRNP.

On the basis of several physiological properties of L-canavanine, we have tested the prediction that this analogue of arginine would enhance the cytotoxic effects of gamma-rays in mammalian cells. Using the human colonic tumor cell line, HT-29, time-dose studies were performed with log-phase cultures in order to determine conditions which maximize the incorporation of L-canavanine into cellular proteins while leaving a large fraction of the cells viable for subsequent gamma-ray survival measurements. At an input ratio of 2.5 (L-canavanine:arginine), the analogue exerted a cytostatic effect on the cells for at least 6 days following one cell division. Little cell killing (less than 20%) by clonogenicity was caused by L-canavanine during the first 12 hr of treatment of log-phase cells, even at a L-canavanine:arginine ratio of 20. A 24-hr exposure, however, produced an exponential decrease in survival as a function of L-canavanine concentration. The interaction between L-canavanine treatment and gamma-ray damage with respect to cell survival was examined under several conditions and times based on the above findings. Optimal enhancement of X-ray-induced cytotoxicity (assayed by loss of clonogenicity) was observed with a 48-hr exposure to the analogue at a L-canavanine:arginine ratio of 10. A marked increase in radiosensitivity was observed when L-canavanine was administered either before or after irradiation of the cells. In both protocols, enhancement was seen at all radiation doses. Together with our earlier findings showing the antitumor activity of L-canavanine in L1210 murine leukemia, these results suggest the potential usefulness of this amino acid analogue in the treatment of cancer.

TERRESTRIAL FATE: Based on a classification scheme(1), an estimated Koc value of 17(SRC), determined from a structure estimation method(2), indicates that L-canavanine is expected to have very high mobility in soil(SRC). The estimated pKa values of L-canavanine are 2.10 and 9.31(3), indicating that this compound will exist as a zwitterion in the environment and zwitterions generally adsorb more strongly to soils containing organic carbon and clay than their neutral counterparts(4). L-Canavanine is not expected to volatilize from dry soil surfaces(SRC) based upon an estimated vapor pressure of 3.2X10-8 mm Hg at 25 °C(SRC), determined from a fragment constant method(2). Biodegradation data in soil were not available(SRC, 2016).

AQUATIC FATE: Based on a classification scheme(1), an estimated Koc value of 17(SRC), determined from a structure estimation method(2), indicates that L-canavanine is not expected to adsorb to suspended solids and sediment(SRC). Estimated pKa values of 2.10 and 9.31(3) indicates L-canavanine will exist as a zwitterion at pH values of 5 to 9 and, therefore, volatilization from water surfaces is not expected to be an important fate process(SRC). According to a classification scheme(4), an estimated BCF of 3(SRC), from an estimated log Kow of -4.26(2) and a regression-derived equation(2), suggests the potential for bioconcentration in aquatic organisms is low(SRC). Biodegradation data in water were not available(SRC, 2016).

ATMOSPHERIC FATE: According to a model of gas/particle partitioning of semivolatile organic compounds in the atmosphere(1), L-canavanine, which has an estimated vapor pressure of 3.2X10-8 mm Hg at 25 °C(SRC), determined from a fragment constant method(2), is expected to exist solely in the particulate phase in the ambient atmosphere. Particulate-phase L-canavanine may be removed from the air by wet and dry deposition(SRC). L-Canavanine does not contain chromophores that absorb at wavelengths >290 nm(3) and, therefore, is not expected to be susceptible to direct photolysis by sunlight(SRC).

IDENTIFICATION AND USE: L-canavanine is a solid. It is a potentially toxic antimetabolite of L-arginine that is stored by many leguminous plants. It has demonstrative antineoplastic activity against a number of animal-bearing carcinomas and cancer cell lines. L-canavanine has been used as an experimental medication. HUMAN EXPOSURE AND TOXICITY: L-Canavanine is a naturally occurring L-amino acid that interferes with L-arginine-utilizing enzymes owing to its structural analogy with this L-amino acid. In macrophages and polymorphonuclear leukocytes, which express inducible nitric oxide synthase (iNOS), L-canavanine is able to prevent the L-arginine-derived synthesis of nitric oxide (NO). L-canavanine exerts differential effects on human platelets in relation to the concentrations: at low levels, it exerts antiaggregating effects by actions independent of NOS inhibition, whereas, at high levels, it inhibits NO synthesis and does not exert antiaggregating effects. L-canavanine was cytotoxic to human peripheral blood mononuclear leucocytes (PBMCs) in culture. The results of these studies suggest that the mechanism of L-canavanine toxicity is mediated through L-arginine-utilizing mechanisms and that the L-canavanine metabolite, L-canaline, is toxic to human PBMCs by disrupting polyamine biosynthesis. ANIMAL STUDIES: It was only slightly toxic to rats following a single sc injection: the LD50 was 5.9 +/- 1 8 g/kg in adult rats and 5.0 +/- 1.0 g/kg in 10-day-old rats. Repeated sc administration of canavanine resulted in more severe toxicity. Weight loss and alopecia were observed in rats given daily sc canavanine injections for 7 days. Food intake was decreased by 80% in adult rats subjected to this dosing regimen, but returned to normal after canavanine injections were terminated. Histological studies of tissues from adult rats treated with 3.0 g/kg canavanine daily for 6 days revealed pancreatic acinar cell atrophy and fibrosis. Serum amylase and lipase levels were elevated following one sc injection of 2.0 g/kg canavanine; after three daily injections both serum enzymes were depleted. Elevations in serum glucose and urea nitrogen, and depletion of cholesterol, were observed. The most significant changes were severe attenuations of serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activity. Eighteen female mice were fed a diet containing 1.56% canavanine sulphate (1% base) and eighteen others were fed a control diet from day 84 to day 477 of age. Four g/d/mouse diet were fed from day 84 to day 164 of age and 5 g/d/mouse were fed thereafter. Only 6 of 10 canavanine-fed mice with copulatory plugs (vs 5 of 5 controls) carried any pups to 17d of gestation. Counts of corpora lutea, embryos and resorption sites indicate that these significant effects on pregnancy may have been due to failure of implantation. Only 50% of control mice and a full 89% of canavanine-fed mice survived to 477 days of age. These results indicate that canavanine may extend the life of mice, but interferes with their reproduction. Mutagenic activities of l-canavanine and metabolite l-canaline on Salmonella typhimurium TA100 and Bacillus subtilis h 17 rec+ & M 45 rec- were investigated in order to elucidate the mechanism of cytotoxicity of each compound. Both compounds and their metabolites obtained from rat liver homogenate did not cause base-pair substitutions and frameshift activities on DNA structure. Apparently, the compounds do not act on DNA directly, but other mechanisms, such as formation of l-canavanine-containing proteins, appear to influence DNA metabolism. Canavanine induced marked growth inhibition of the rat colon carcinoma.

L-Canavanine occurrence plants(1).[Table#4617]

Canavanine occurrence plants(1). /Canavanine/[Table#4618]

Using a structure estimation method based on molecular connectivity indices(1), the Koc of L-canavanine can be estimated to be 17(SRC). According to a classification scheme(2), this estimated Koc value suggests that L-canavanine is expected to have very high mobility in soil. The estimated pKa values of 2.10 and 9.31(3) indicate that this compound will exist as a zwitterion in the environment and zwitterions generally adsorb more strongly to soils containing organic carbon and clay than their neutral counterparts(4).

L-Canavanine, a basic amino acid, was isolated from jack beans (Canavalia ensiformis (L), family: Leguminosae). The compound constitutes about 1.5% of the dry weight of alfalfa seed and sprouts(1).

L-Canavanine was isolated and identified as the active principle in Astragalus roots that inhibited metamorphosis of the silkworm Bombyx mori.

/CASE REPORTS/ For the past 2 decades there has been vigorous disagreement over the purported toxicity of Hedysarum alpinum seeds, and whether the consumption of such seeds was a factor in the 1992 death of Chris McCandless, the subject of the book Into the Wild. Our objective was to confirm or disprove the presence of L-canavanine (a nonprotein amino acid known to induce systemic lupuslike symptoms in humans) in H. alpinum seeds. Liquid chromatography-tandem mass spectrometry analysis of H. alpinum seeds was performed. Our analysis confirmed the presence of L-canavanine in H. alpinum seeds and demonstrated that it is a significant component of the seeds, with a concentration of 1.2% (weight/weight), roughly half of that found in Canavalia ensiformis. The data led us to conclude it is highly likely that the consumption of H alpinum seeds contributed to the death of Chris McCandless.

/ALTERNATIVE and IN VITRO TESTS/ L-Canavanine and its arginase-catalyzed metabolite, L-canaline, are two novel anticancer agents in development. Since the immunotoxic evaluation of agents in development is a critical component of the drug development process, the antiproliferative effects of L-canavanine and L-canaline were evaluated in vitro. Both L-canavanine and L-canaline were cytotoxic to peripheral blood mononucleocytes (PBMCs) in culture. Additionally, the mononucleocytes were concurrently exposed to either L-canavanine or L-canaline and each one of a series of compounds that may act as metabolic inhibitors of the action of L-canavanine and L-canaline (L-arginine, L-ornithine, D-arginine, L-lysine, L-homoarginine, putrescine, L-omega-nitro arginine methyl ester, and L-citrulline). The capacity of these compounds to overcome the cytotoxic effects of L-canavanine or L-canaline was assessed in order to provide insight into the biochemical mechanisms that may underlie the toxicity of these two novel anticancer agents. The results of these studies suggest that the mechanism of L-canavanine toxicity is mediated through L-arginine-utilizing mechanisms and that the L-canavanine metabolite, L-canaline, is toxic to human PBMCs by disrupting polyamine biosynthesis. The elucidation of the biochemical mechanisms associated with the effects of L-canavanine and L-canaline on lymphoproliferation may be useful for maximizing the therapeutic effectiveness and minimizing the toxicity of these novel anticancer agents.

/ALTERNATIVE and IN VITRO TESTS/ L-Canavanine (CAV) is a higher plant nonprotein amino acid and a potent L-arginine antimetabolite. CAV can inhibit the proliferation of tumor cells in vitro and in vivo, but little is known regarding the molecular mechanisms mediating these effects. We demonstrated that the treatment of human lung adenocarcinoma A549 cells with CAV caused growth inhibition; G1 phase arrest is accompanied by accumulation of an incompletely phosphorylated form of the retinoblastoma protein, whose phosphorylation is necessary for cell cycle progression from G1 to S phase. In addition, CAV induces the expression of p53 and subsequent expression of a cyclin-dependent kinase inhibitor, p21/WAF1. The p53-dependent induction of p21/WAF1 and the following dephosphorylation of the retinoblastoma protein by CAV could account for the observed CAV-mediated G1 phase arrest.

An estimated BCF of 3 was calculated in fish for L-canavanine(SRC), using an estimated log Kow of -4.26(1) and a regression-derived equation(1). According to a classification scheme(2), this BCF suggests the potential for bioconcentration in aquatic organisms is low(SRC).

The estimated pKa values of L-canavanine are 2.10 and 9.31(1), indicating that this compound will exist as a zwitterion at pH values of 5 to 9; therefore, volatilization from water surfaces is not expected to be an important fate process. L-Canavanine is not expected to volatilize from dry soil surfaces(SRC) based upon an estimated vapor pressure of 3.2X10-8 mm Hg(SRC), determined from a fragment constant method(2).

/LABORATORY ANIMALS: Acute Exposure/ The toxicity of L-canavanine was investigated because of its demonstrated potential as an antitumor drug. This natural product was only slightly toxic to Sprague-Dawley rats following a single sc injection: the LD50 was 5.9 +/- 1 8 g/kg in adult rats and 5.0 +/- 1.0 g/kg in 10-day-old rats. Following a single dose of 2.0 g/kg, the systemic clearance value for canavanine in adult rats was 0.114 liter/hr, the volume of distribution at steady state was 0.154 liter, and the half-life was 1.56 hr. Forty-eight percent of the dose was excreted unaltered in the urine following an iv injection, and 16% of a sc dose was recovered in the urine. Bioavailability of a 2.0 g/kg sc dose was 72%. Single oral doses of canavanine were less toxic to adult rats than sc injections. Bioavailability of a 2.0 g/kg po dose was 43%, and only 1% of the administered canavanine was recovered in the urine. Twenty-one percent of the administered canavanine remained in the gastrointestinal tract 24 hr after an oral dose. Less than 1% of a 2.0 g/kg dose of L-[guanidinooxy-(14)C]canavanine was incorporated into the proteins of adult and neonatal rats 4 or 24 hr following administration. Repeated sc administration of canavanine resulted in more severe toxicity. Weight loss and alopecia were observed in rats given daily sc canavanine injections for 7 days. Food intake was decreased by 80% in adult rats subjected to this dosing regimen, but returned to normal after canavanine injections were terminated. Histological studies of tissues from adult rats treated with 3.0 g/kg canavanine daily for 6 days revealed pancreatic acinar cell atrophy and fibrosis. Serum amylase and lipase levels were elevated following one sc injection of 2.0 g/kg canavanine; after three daily injections both serum enzymes were depleted. Elevations in serum glucose and urea nitrogen, and depletion of cholesterol, were observed. The most significant changes were severe attenuations of serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activity.

/LABORATORY ANIMALS: Subchronic or Prechronic Exposure/ L-Canavanine [2-amino-4-(guanidinooxy) butyric acid], a non-protein amino acid that is structurally analogous to arginine, has been proposed as a major antinutritional factor responsible for the toxic effects induced by raw Canavalia ensiformis (L.) seeds in chicks. We investigated the effects of L-canavanine on performance and select metabolic responses of growing chicks. Canavanine was added to a control diet, in an amount equivalent to that provided by 300 g raw canavalia seeds/kg diet (10 g free base canavanine/kg diet). Growth, plasma basic amino acids and kidney arginase, activity were measured. The incorporation of canavanine into a nutritionally balanced diet for growing chicks depressed feed intake and growth by approximately 25% (p<0.01) compared with the control diet. Performance was unaffected by equimolar amounts of arginine. Canavanine exerted its growth-depressing effect exclusively by reducing feed intake, because this effect was not observed in a pair-feeding experiment. Chicks fed a diet containing 473 mmol canavanine sulfate/kg for 11 d were given an intracrop dose of 946 mmol of canavanine sulfate or arginine hydrochloride. In both cases, plasma histidine and lysine concentrations were significantly decreased compared with a placebo group dosed with water. Plasma arginine concentration was unaffected by the canavanine sulfate dose but, as expected, was significantly increased by the arginine hydrochloride dose. Free base canavanine significantly (p<0.05) reduced kidney arginase activity. No overt toxic effects were observed at any point during the study. These data indicate that, although canavanine is not the principal antinutritional factor in Canavalia ensiformis seeds, its presence in the diet precludes optimum performance of chicks.

Non-protein amino acid found in leguminous plants; [Merck Index] Used in biochemical research; [HSDB]

Predominantly found in leguminous plants; serves as nitrogen storage compound and defensive mechanism. Cytotoxic antimetabolite; substrate for arginyl-tRNA synthetase.

Biochemical research

MEDICATION

L-Canavanine, a potentially toxic antimetabolite of L-arginine that is stored by many leguminous plants, has demonstrative antineoplastic activity against a number of animal-bearing carcinomas and cancer cell lines. This investigation evaluated the natural abundance of this anti-cancer compound in commercially available sprouts, and in ten varieties of the seed of alfalfa, Medicago sativa (L.). Canavanine abundance in commercially grown sprouts varied according to the source; the young plant stored appreciable canavanine that ranged from 1.3 to 2.4% of the dry matter. Alfalfa seeds were also rich in this nonprotein amino acid as the canavanine content varied from 1.4 to 1.8% of the dry matter. On average, the tested seeds contained 1.54 =/- 0.03% canavanine. Alfalfa seed canavanine content was comparable to the levels found in the seeds of representative members of the genus Canavalia, which are amongst the more abundance sources of this antimetabolite.