CARCINOGENIC EFFECTS OF CYCAD MEAL AND CYCASIN, METHYLAZOXYMETHANOL GLYCOSIDE, IN RATS AND EFFECTS OF CYCASIN IN GERMFREE RATS.
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Cycadopsida↗
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[Cycasin carcinogenesis. (5). Carcinogenic properties of cycasin, methylazoxymethanol and methylazoxymethyl benzoate].
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Adenocarcinoma↗
Transport of cycasin by the intestinal Na+/glucose cotransporter.
The medicinal and food use of seed from the cycad plant (Cycas spp.), which contains the neurotoxin cycasin, is a proposed etiological factor for amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC), a prototypical neurodegenerative disease found in the western Pacific. Cycasin, the beta-D-glucoside of methylazoxymethanol might enter neurons and other cells via a glucose transporter. Since the intestinal brush-border Na+/glucose cotransporter plays a major role in the absorption of monosaccharides, the following studies were conducted to determine if cycasin, the beta-D-glucoside of methylazoxymethanol, is a substrate for the transporter. We measured the ability of cycasin to (i) inhibit Na+/glucose uptake into rabbit intestinal brush-border membrane vesicles, and (ii) to generate current by the cloned Na+/glucose cotransporter (SGLT1) expressed in Xenopus laevis oocytes. The results show that cycasin inhibits Na(+)-dependent sugar transport in the vesicles, and cycasin generates phlorizin-sensitive currents in oocytes. We conclude that cycasin is a substrate for the intestinal brush-border Na+/glucose cotransporter, albeit with a lower affinity than D-glucose. This suggests that cycasin may be absorbed from the gut lumen by the cotransporter, and as a result either cycasin or the aglycone is presented to the blood-brain barrier for uptake into the brain.
Animals↗
Na+-dependent and phlorizin-inhibitable transport of glucose and cycasin in brain endothelial cells.
Although cycasin (methylazoxymethanol beta-D-glucoside) is proposed to be a significant etiological factor for the prototypical neurodegenerative disorder Western Pacific amyotrophic lateral sclerosis and parkinsonism-dementia complex, the mechanism underlying transport of cycasin across the blood-brain barrier (BBB) is unknown. We examined cycasin transport in cultured bovine brain endothelial cells, a major element of the BBB. Cycasin was taken up into endothelial cells in a dose-dependent manner with maximal uptake observed at a concentration of 10 microM. Cycasin uptake was significantly inhibited by alpha-methyl-D-glucoside, a specific analogue for the Na+-dependent glucose transporter (SGLT), by the SGLT inhibitor phlorizin, by replacement of extracellular NaCl with LiCl, and by dinitrophenol (DNP), an inhibitor of energy metabolism. In addition, cycasin produced inward currents in a whole-cell voltage clamp configuration. Peak currents were observed at 10 microM with a trend toward reduction at higher concentrations, and currents were clearly blocked by alpha-methyl-D-glucoside, phlorizin, and DNP. In addition, cycasin never evoked currents in Na+-free extracellular solution. These results suggest that cycasin is selectively transported across brain endothelial cells, possibly across the BBB by a Na+/energy-dependent glucose transporter.
Animals↗
Similarities between the biochemical actions of cycasin and dimethylnitrosamine.
1. Rats were given the hepatotoxin and carcinogen cycasin by stomach tube. In one experiment, rats whose RNA had previously been labelled with [(14)C]-formate were given the acetate ester of the aglycone form of cycasin, methylazoxymethanol, by intraperitoneal injection. 2. Incorporation of (14)C from l-[U-(14)C]leucine into the proteins of some organs was measured in cycasin-treated rats. Cycasin inhibited leucine incorporation into liver proteins but not into kidney, spleen or ileum proteins. This inhibition was not evident until about 5hr. after cycasin administration, but once established it persisted for the next 20hr. 3. Methylation of nucleic acids was detected in some organs of rats treated with cycasin or methylazoxymethanol. The purine bases of RNA and DNA were isolated by acid hydrolysis followed by ion-exchange column chromatography. The resulting chromatograms showed an additional purine base that was identified as 7-methylguanine. It was shown that, in animals treated with the toxin, liver RNA was methylated to a greater extent than was either kidney or small-intestine RNA. Also, as a result of cycasin administration, liver DNA guanine was methylated to a greater extent than was RNA guanine. 4. These results are discussed in relation to comparable experiments with dimethylnitrosamine. It is suggested that cycasin and dimethylnitrosamine are metabolized to the same biochemically active compound, perhaps diazomethane, but that various tissues differ in their capacity to metabolize the two carcinogens.
Animals↗
Effect of phenobarbital on the development of neoplastic lesions in the liver of cycasin-treated rats.
The effect of phenobarbital on the development of neoplastic lesions by cycasin was examined in inbred ACI rats. Tumor development in the liver and the kidney was observed in groups of rats treated with a single oral administration of cycasin (100 mg/kg body weight) and maintained on either a control diet or one supplemented with 0.05% phenobarbital. The feeding of phenobarbital diet after the application of cycasin significantly increased the incidence of liver tumors in female rats, but not in male rats. On the other hand, the administration of phenobarbital did not affect the incidence of kidney tumors in either sex. In addition, many large gamma-glutamyltranspeptidase (GGT)-positive foci, which were thought to be preneoplastic lesions, developed in the liver of rats treated with cycasin and then phenobarbital, whereas a small number of tiny foci were seen in rats treated with cycasin alone. Long-term feeding of the 0.05% phenobarbital diet without treatment of cycasin induced many GGT-positive foci which, however, were small-sized. These data indicated phenobarbital to possess a tumor-promoting effect in terms of induction of neoplastic lesions in the liver but not in the kidney.
Animals↗
Content of the neurotoxins cycasin (methylazoxymethanol beta-D-glucoside) and BMAA (beta-N-methylamino-L-alanine) in cycad flour prepared by Guam Chamorros.
Exposure to cycad seed kernel is an etiologic factor for the western Pacific amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC). Traditionally processed cycad flours (n = 17) obtained from Chamorro residents of Guam and the adjacent island of Rota at risk for neurodegenerative disease were extracted and analyzed by high-performance liquid chromatography for content of beta-N-methylamino-L-alanine (BMAA) and methyl-azoxymethanol beta-D-glucoside (cycasin). Cycasin (detection limit: picomole) was present in concentrations of 0.004 to 75.93 micrograms/g (mean, 12.45 +/- 5.0 micrograms/g), and levels of BMAA (detection limit: subpicomole) ranged from 0.00 to 18.39 micrograms/g (mean, 5.44 +/- 1.56 micrograms/g). On average, cycasin content was approximately 10 times higher than that of BMAA. The largest concentrations of cycasin were found in samples from villages with a high reported prevalence of ALS/PDC. Ingestion of cycad-derived food would result in estimated human exposure to milligram amounts of cycasin per day. The cytotoxic properties of cycasin merit consideration in relation to the etiology of western Pacific ALS/PDC.
Amino Acids, Diamino↗
Genotoxic activity in vivo of the naturally occurring glucoside, cycasin, in the Drosophila wing spot test.
Cycasin, methylazoxymethanol-beta-glucoside, is a naturally occurring carcinogenic compound. The genotoxicity of cycasin was assayed in the Drosophila wing spot test. Cycasin induced small single and large single spots on feeding at 10 mumol/g medium. The presence of these spots indicates that cycasin is genotoxic in Drosophila melanogaster. Microorganisms which showed beta-glucosidase activity for cleaving cycasin to toxic aglycon were isolated from gut flora of the Drosophila larvae. Consequently, the Drosophila wing spot test would be useful for mutagenicity screening of other naturally occurring glucosides.
Animals↗
Mutagenicity of the naturally occurring carcinogen cycasin and synthetic methylazoxymethanol conjugates in Salmonella typhimurium.
The aglycone methylazoxymethanol of the naturally occurring carcinogenic glucoside, cycasin, has previously been shown to be mutagenic, but cycasin per se has not. In this work, cycasin was demonstrated to be mutagenic using a modification of the Ames Salmonella test in which it was preincubated with beta-glucosidase and the tester strain in liquid medium. The mutagenicity of cycasin to six histine-depedent Salmonella strains varied considerably with strain HisG46 being the most susceptible. Methylazoxymethyl-beta-D-glucosiduronic acid, which also is nonmutagenic per se, similarly became mutagenic when preincubated with beta-glucuronidase. Methylazoxymethyl acetate, which is slightly mutagenic by the Ames standard pour plate method, became highly mutagenic on preincubation. The mutagenicity of free methylazoxymethanol was confirmed, and a linear dose-response relationship was observed. The common conditions required for activation of nonmutagenic methylazoxymethanol conjugates, the glucoside cycasin and methylazoxymethyl-beta-D-glucosiduronic acid, are 90-min preincubation at 30 degrees, pH 6.5, with an appropriate hydrolase and Salmonella typhimurium HisG46.
Azo Compounds↗
Carcinogenicity and hepatotoxicity of cycasin and its aglycone methylazoxymethanol acetate in nonhuman primates.
The carcinogenic potential of cycasin and methylazoxymethanol (MAM) acetate was investigated in nonhuman primates. Old-world monkeys (rhesus, cynomolgus, and African green monkeys) received cycasin and/or MAM acetate by oral or ip routes up to 11 years. Eighteen monkeys survived longer than 2 months after initiation of treatment with cycasin (50-75 mg/kg) or MAM acetate (1.5-3.0 mg/kg) given orally 5 days/week; 9 of the animals were necropsied. Histopathologic examination of a liver tumor from 1 of these monkeys revealed well-differentiated hepatocellular carcinoma. A second monkey had multiple tumors, including hepatocellular carcinoma, intrahepatic bile duct adenocarcinoma, renal carcinoma and adenomas, and adenomatous polyps of the colon. Although liver tumors were not observed in the other monkeys, all but 1 monkey had hepatic lesions such as toxic hepatitis and cirrhosis. These monkeys had received cycasin and/or MAM acetate for an average of 57 months (range, 2-133 mo). A group of 10 monkeys received MAM acetate by weekly ip injections (3-10 mg/kg). Six of these animals developed tumors after receiving an average of 6.14 g (range, 3.58-9.66 g) of MAM acetate for an average of 75 months (range, 50-89 mo). Four of the monkeys developed hepatocellular carcinomas, and 2 had multiple primary tumors including hepatocellular carcinomas, renal carcinomas, squamous cell carcinomas of the esophagus, and adenocarcinomas of the small intestine. Our results showed that long-term administration of cycasin and/or MAM acetate by oral and ip routes was hepatotoxic and carcinogenic in old-world monkeys.
Animals↗
On the distribution of genotoxic factors in various organs of mice treated with cycasin.
The distribution of genotoxic factors in various organs of mice treated orally with methylazoxymethanol-beta-D-glycoside (cycasin) was investigated using the DNA-repair host mediated assay. Indicator of genotoxic activity was a pair of streptomycin dependent Escherichia coli strains differing vastly in DNA repair capacity; uvrB/recA vs. uvr+/rec+. The animal-mediated assays were performed by injecting mixtures of the two strains i.v. and orally into mice, which were subsequently treated with the test chemical and from which the differential survival of the indicator bacteria present in several organs was determined. The same strains and selection procedures were also used for assessing the DNA-damaging activity in vitro. In the animal-mediated assays in which cycasin was applied orally, significant effects were observed at doses of 100 and 500 mg/kg body weight. The organ distribution of genotoxic factors in the host animal was as follows: the highest genotoxic activity was observed in the liver, followed by intestine and stomach; a clear effect was also observed in the kidneys and, to a lower extent, in the blood stream and in the lungs at the highest dose administered (500 mg/kg body weight). Under in vitro conditions a marginal genotoxic effect was observed even in the absence of liver homogenate, indicating that the test compound is possible activated (hydrolysed) by the E. coli cells. Therefore the genotoxic activity of cycasin observed in the gastrointestinal tract was not unexpected, since the substance was applied orally, thereby exposing the indicator bacteria in these organs to high levels of unmetabolised compound, especially in the stomach. In the intestine members of the microbial flora probably contribute to the metabolic activation of the test compound. The occurrence of genotoxic factors remote from the gastrointestinal tract shows that the present compound or active metabolites thereof penetrate through the intestinal barrier. The extraordinarily high genotoxic activity observed in the liver suggests that the compound is additionally activated in this organ. In compliance with previous in vitro findings this second activation step might lead to the formation of the highly reactive aldehydic form of methylazoxymethanol (MAMAL) mediated by dehydrogenases. Comparison with carcinogenicity studies indicates a good correlation between the distribution of genotoxic effects as determined in the present studies and the localisation of tumors in various organs of rodents treated with cycasin.
Animals↗
DNA fragmentation in some organs of rats and mice treated with cycasin.
Cycasin (methylazoxymethanol-beta-D-glucoside) is carcinogenic in several animal species. It produces a variety of malignant tumours, mainly in the liver of mice, and in the liver, kidney and large intestine in rats. It does not appear to be mutagenic in the Ames test, even in the presence of liver microsome fraction, and it is among those carcinogens (less than 10%) ranked as "false negatives" in this test. The ability of cycasin to damage in vivo liver, kidney, lung and colonic DNA of Wistar rats and C57BL/L mice was investigated by means of alkaline elution technique. Oral single-dose administration of cycasin, in the range of 50-400 mg/kg body weight, produced in the rat a clearly evident dose-dependent DNA fragmentation in the liver, and less marked damage to DNA from kidney and colon mucosa. In mice, the same treatment produced dose-dependent DNA damage only in the liver. DNA repair up to 18 h appeared to be incomplete both in mice and rats. Methylazoxymethanol acetate is considered to be an active form of cycasin. While in vivo methylazoxymethanol acetate caused DNA damage, in vitro it appeared inactive and required metabolic activation, possibly consisting in its hydrolysis by esterase activity, to be able to cause DNA fragmentation.
Animals↗
Genotoxicity of cycasin in the hepatocyte primary culture/DNA repair test supplemented with beta-glucosidase.
The genotoxicity of cycasin was examined in the standard hepatocyte primary culture (HPC)/DNA repair test and in the test supplemented with beta-glucosidase. Generally, no DNA repair was elicited by cycasin in the standard test except for one assay which showed a strong response. With the addition of beta-glucosidase to the test medium, cycasin elicited DNA repair with clear dependence on both dose and amount of beta-glucosidase. These results indicate that supplementation of the HPC/DNA repair test with the appropriate should be useful in detecting potentially genotoxic glucosides and suggests that supplementation with other specific enzymes could compensate for extrahepatic biotransformation processes required prior to final activation by hepatocytes.
Animals↗
Activation of cycasin to a mutagen for Saccharomyces cerevisiae by rat intestinal flora.
Genetic test systems involving microorganisms and liver enzyme preparations may be insufficient to detect compounds that require breakdown by enzymes provided by the microbial flora of the intestinal tract. A method is described for providing such activation and for simultaneously testing the potential genetic activity of breakdown products in an indicator organism. Parabiotic chambers containing Saccharomyces cerevisiae genetic test organisms in one chamber were separated by a membrane filter from rat cecal organisms and test chemical contained in the other chamber. The genetic activities of cycasin breakdown products for mutation, gene conversion, and mitotic crossing-over in samples incubated aerobically are reported. Samples containing cycasin alone had a small but clearly increased frequency of genetic damage. Samples containing rat cecal organisms without cycasin showed no increase in genetic activity. Anaerobic incubation resulted in no increase in genetic activity in any of the samples.
Aerobiosis↗
Induction of tumors with cycasin in newborn and preweanling rats.
Comprehensive studies of carcinogenesis in newborn or preweanling SD rats were conducted under various dose schedules of cycasin (CAS: 14901-08-7) administration. When cycasin was given sc to newborn rats at day 0, tumors were detected in more than 80% of rats of both sexes; kidney tumors were by far the most common. The incidences of tumors declined in the older groups, namely, over 60% in both sexes in the 7-day group, 55% in males and 8.3% in females in 14-day rats, and 0% in 21-day groups. By multiple administration, tumor incidences elevated considerably. Administration ip of cycasin also gave rise to tumor induction in newborn rats. A total of 435 kidney tumors found in the experiments were studied pathologically. Most of them were classified as mesenchymal tumor; some of them metastasized. A few other tumors were found in the liver and colon.
Adenocarcinoma↗
A partially purified beta-glucosidase from Bifidobacterium adolescentis converts cycasin to a mutagenic compound.
beta-Glucosidase was extracted from sonicated Bifidobacterium adolescentis Int-57 and partially purified by Sepharose CL-6B gel-filtration and DEAE-cellulose ion-exchange chromatography. The partially purified enzyme was confirmed to convert cycasin to a mutagen in the Ames and SOS chromotests. beta-Glucosidase negative strains were unable to activate cycasin mutagenically.
Bifidobacterium↗
CYCASIN: RADIOMIMETIC EFFECT.
Cycasin, methylazoxy-methanol-beta-glucoside, a carcinogenic compound that occurs naturally in cycad plants, induces chromosome aberrations in the root-tip cells of Allium cepa. Germination and growth in an aqueous solution of cycasin at a concentration of only 3 percent of that found in Cycas circinalis induced as many aberrations in chromosomes as did about 200 roentgens of gamma irradiation.
Carcinogens↗
Intestinal tumors of rats by gastric or intestinal administration of cycad extract and cycasin.
Sprague-Dawley rats were given gastric intubation of cycad extract (group 1), rectal infusion of cycasin (group 2), or rectal indusion of cycad extract after external colostomy at 1/3 proximal portion of the large intestine (group 3). In group 1, intestinal tumors developed in any portion of the intestinal tract ranging from the duodenum to the rectum. In group 2, tumors developed in mucosa of the large intestine. In group 3, however, tumors arose from both sites of intestinal mucosa which were in contact and not in contact directly with the cycad extract infused. Possible hypothesis for intestinal tumor development by cycad extract and cycasin was presented.
Adenocarcinoma↗