L-Canavanine (CAV) is an arginine (ARG) analog isolated from the jack bean, Canavalia ensiformis. CAV becomes incorporated into cellular proteins of MIA PaCa-2 human pancreatic cancer cells and the aberrant, canavanyl proteins are not preferentially degraded. Hydrolytic cleavage of CAV to canaline (CAN) and urea is mediated by arginase. CAN is a potent metabolite that inactivates vitamin B6-containing enzymes and may inhibit cell growth. To determine the presence of arginase and its specificity for ARG and CAV in MIA PaCa-2 cells, a radiometric assay, which quantifies the (14)C released from the hydrolytic cleavage of L-[guanidino-(14)C]ARG or L-[guanidinooxy-(14)C]CAV mediated by arginase, was employed. Insignificant amounts of (14)CO2 were released when cells were exposed to [(14)C]CAV or to [(14)C]ARG. Pancreatic cancer cells secrete a negligible amount of arginase. Cytotoxic effects of CAN and CAV were compared on cells exposed to varying concentrations of ARG. These studies provide evidence that CAV's cytotoxic effects on MIA PaCa-2 cells cannot be attributed to conversion to the active metabolite CAN. A slower and decreased hydrolysis of CAV by arginase allows CAV to persist and increases its chances of incorporating into proteins in these cells. Lack of considerable amounts of arginase in the pancreas makes CAV a worthy candidate for further studies in pancreatic cancer.
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.
The metabolism of L-canavanine, a nonprotein amino acid with significant antitumor effects, was investigated. L-Canavanine, provided at 2.0 g/kg, was supplemented with 5 uCi of L-[guanidinooxy-(14)C]canavanine (58 uCi/mumol) and administered iv, sc, or orally to female Sprague-Dawley rats weighing approximately 200 g. 14C recovery in the urine at 24 hr was 83, 68, or 61%, respectively, of the administered dose. Another 5-8% of the (14)C was expired as (14)CO2. The gastrointestinal tract contained 21% of orally administered (14)C. Serum, feces, tissues, and de novo synthesized proteins only accounted for a few percent of the original dose by any administrative route. Analysis of the (14)C-containing urinary metabolites revealed that [(14)C] urea accounted for 88% of the urinary radioactivity for an iv injection, 75% for sc administration, and 50% following an oral dose. By all routes of administration, [(14)C]guanidine represented 5% of the radioactivity in the urine and [(14)C]guanidinoacetic acid accounted for 2%. Serum and urine amino acid analysis showed a markedly elevated ornithine level. Basic amino acids such as histidine, lysine, and arginine were also higher in the urine. Plasma ammonia levels were determined following oral canavanine doses of 1.0, 2.0, and 4.0 g/kg. A rapid but transient elevation in plasma ammonia was observed only at the 4.0 g/kg dose. This indicates that elevated plasma ammonia is not a likely cause of canavanine toxicity at the drug concentrations used in this study.
It was observed previously that hydroxyguanidine is formed in the reaction of canavanine(2-amino-4-guanidinooxybutanoate) with amino acid oxidases. The present work shows that hydroxyguanidine is formed by a nonenzymatic beta,gamma-elimination reaction following enzymatic oxidation at the alpha-C and that the abstraction of the beta-H is general-base catalyzed. The elimination reaction requires the presence in the alpha-position of an anion-stabilizing group--the protonated imino group (iminium ion group) or the carbonyl group. The iminium ion group is more activating than the carbonyl group. Elimination is further facilitated by protonation of the guanidinooxy group. The other product formed in the elimination reaction was identified as vinylglyoxylate (2-oxo-3-butenoate), a very highly electrophilic substance. The product resulting from hydrolysis following oxidation was identified as alpha-keto-gamma-guanidinooxybutyrate (ketocanavanine). The ratio of hydroxyguanidine to ketocanavanine depended upon the concentration and degree of basicity of the basic catalyst and on pH. In the presence of semicarbazide, the elimination reaction was prevented because the imino group in the semicarbazone derivative of ketocanavanine is not significantly protonated. Incubation of canavanine with 5'-deoxypyridoxal also yielded hydroxyguanidine. Since the elimination reactions take place under mild conditions, they may occur in vivo following oxidation at the alpha-C of L-canavanine (ingested or formed endogenously) or of other amino acids with a good leaving group in the gamma-position (e.g., S-adenosylmethionine, methionine sulfoximine, homocyst(e)ine, or cysteine-homocysteine mixed disulfide) by an L-amino acid oxidase, a transaminase, or a dehydrogenase. Therefore, vinylglyoxylate may be a normal metabolite in mammals which at elevated concentrations may contribute to the in vivo toxicity of canavanine and of some of the other above-mentioned amino acids.
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.
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.
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.
