Is neurodegenerative disease a long-latency response to early-life genotoxin exposure?

Western Pacific amyotrophic lateral sclerosis and parkinsonism-dementia complex, a disappearing neurodegenerative disease linked to use of the neurotoxic cycad plant for food and/or medicine, is intensively studied because the neuropathology (tauopathy) is similar to that of Alzheimer's disease. Cycads contain neurotoxic and genotoxic principles, notably cycasin and methylazoxymethanol, the latter sharing chemical relations with nitrosamines, which are derived from nitrates and nitrites in preserved meats and fertilizers, and also used in the rubber and leather industries. This review includes new data that influence understanding of the neurobiological actions of cycad and related genotoxins and the putative mechanisms by which they might trigger neurodegenerative disease.

El tratamiento con progesterona previene las alteraciones motoras inducidas por la intoxicación con semillas de cícada (Dioon spinulosum) en la rata macho / Treatment with progesterone prevented the motor impairment induced by poisoning with seeds of Cicada (Dioon spinulosum) in the male rat

El consumo crónico de semillas de cícadas ha sido asociado con enfermedades neurodegenerativas, las cuales predominan en el género masculino. En México, las semillas decícada (Dioon spinulosum) son usadas como sustituto de maíz y a nivel experimental producen un déficit motor; probablemente causado por sus componentes neurotóxicos. En este sentido, la progesterona ejerce efectos neuroprotectores contra traumatismo cerebral, hipoxia, así como la muerte neuronal inducida por colchicina en el SNC; por lo que podría prevenir los efectos neurotóxicos producidos por el consumo crónico de semillas de Dioon spinulosum en ratas, una posibilidad explorada en el presente estudio. Se emplearon 32 ratas macho de la cepa Wistar distribuidas en cuatro grupos: a) un grupo control recibió 1 ml de agua purificada (v.o.) y 0,2 ml de aceite de maíz (s.c.); b) un grupo cícada recibió 5g/kg de la semilla de cícada (v.o.) y 0,2 ml de aceite de maíz (s.c.); c) un grupo más recibió 1 ml de agua purificada (v.o.) y 3 mg/kg de progesterona (s.c.); y d) el último grupo recibió semilla de cícada y progesterona. Los tratamientos fueron administrados diariamente durante 40 días y el efecto fue evaluado en las pruebas de actividad locomotora y nado forzado a los 20, 30 y 40 días de tratamiento. El grupo cícada redujo el número de cuadros cruzados en la prueba de actividad locomotora. En la prueba de nado forzado, los animales tratados con la semilla de cícada fueron los únicos que presentaron la conducta de giro, un efecto que fue prevenido por el tratamiento previo con progesterona. La administración de vehículo y progesterona no produjo per se la conducta de giro. En conclusión, la progesterona previene las alteraciones motoras inducidas por el consumo crónico de semillas de cícada en ratas forzadas a nadar (AU)

The chronic consumption of cycad seeds has been associated with neurodegenerative diseases, which are predominant in masculine gender. In Mexico, the cycad seeds (Dioon spinulosum) are used as a maize substitute and at experimental level produce a motor deficit; probably caused by its neurotoxic compounds. Progesterone could prevent the neurotoxic effects against traumatic brain injury, hypoxia and neuronal death induced by colchicine in the CNS. In addition, progesterone could prevent these neurotoxic effects produced by the chronic administration of cycad seeds (Dioon spinulosum) in rats, a possibility explored in the present study. Male wistar rats were randomly divided in 4 groups: a) a control group received 1 ml purified water (PO) and oil (0.2 ml, s.c.);b) a cycad group received a cycad seed 5g/kg (PO) and oil (0.2 ml,s.c.); c) another group received 1 ml of purified water (PO) and progesterone (3mg/kg, s.c.) and d) the last group received cycad seed and progesterone. All treatments were administered daily during 40 days and the effect was evaluated in behavioral tests (open filed and forced swim) were conducted on 20, 30 and 40 days of treatment. The cycad group decreased the number of squares crossed in the open field whereas, in the forced swim test, rotational behavior was only observed in the group administered with cycad seeds, this effect was prevented by previous progesterone treatment. The vehicle and progesterone administration did not produce the rotational behavior per se. In conclusion, progesterone prevents the motor deficit induced by the chronic consumption of cycad seeds in forced swim rats (AU)

Cycad toxin-induced damage of rodent and human pancreatic beta-cells.

Environmental toxins may be risk factors for some forms of diabetes mellitus and neurodegenerative diseases. The medicinal and food use of seed from the cycad plant (Cycas spp.), which contains the genotoxin cycasin, is a proposed etiological factor for amyotrophic lateral sclerosis/Parkinsonism-dementia complex (ALS/PDC), a prototypical neurodegenerative disease found in the western Pacific. Patients with ALS/PDC have a very high prevalence of glucose intolerance and diabetes mellitus (in the range of 50-80%). We investigated whether the cycad plant toxin cycasin (methylazoxymethanol (MAM) beta-D-glucoside) or the aglycone MAM are toxic in vitro to mouse or human pancreatic islets of Langerhans. Mouse pancreatic islets treated for 6 days with cycasin impaired the beta-cell insulin response to glucose, but this effect was reversible after a further 4 days in culture without the toxin. When mouse islets were exposed for 24 hr to MAM/MAM acetate (MAMOAc; 0.1-1.0 mM), there was a dose-dependent impairment in insulin release and glucose metabolism, and a significant decrease in islet insulin and DNA content. At higher MAM/MAMOAc concentrations (1.0 mM), widespread islet cell destruction was observed. Glucose-induced insulin release remained impaired even after removal of MAM and a further culturing for 4 days without the toxin. MAM damages islets by two possible mechanisms: (a) nitric oxide generation, as judged by increased medium nitrite accumulation; and (b) DNA alkylation, as judged by increased levels of O6-methyldeoxyguanosine in cellular DNA. Incubation of mouse islets with hemin (10 or 100 microM), a nitric oxide scavenger, or nicotinamide (5-20 mM) protected beta-cells from a decrease in glucose oxidation by MAM. In separate studies, a 24 hr treatment of human beta-islet cells with MAMOAc (1.0 mM) produced a significant decrease in both insulin content and release in response to glucose. In conclusion, the present data indicate that cycasin and its aglycone MAM impair both rodent and human beta-cell function which may lead to the death of pancreatic islet cells. These data suggest that a "slow toxin" may be a common aetiological factor for both diabetes mellitus and neurodegenerative disease.

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.

Statistical analysis of a carcinogen mixture experiment. I. Liver carcinogens.

This paper describes factorial experiments designed to determine whether 2 liver carcinogens act synergistically to produce liver cancers in Fischer 344 rats. Four hepatocarcinogens, cycad flour, lasiocarpine (CAS: 303-34-4), aflatoxin B1 (CAS: 1162-65-8), and dipentylnitrosamine (CAS: 13256-06-9), were studied in pairwise combinations. Each of the 6 possible pairs was studied by means of 4 X 4 factorial experiment, each agent being fed at zero and at 3 non-zero doses. Methods of analysis designed explicitly for this study were derived to study interaction. These methods were supplemented by standard statistical methods appropriate for one-at-a-time studies. Antagonism was not discovered in any chemical mixture. Some chemical mixtures did interact synergistically. Findings for male and female animals were generally, but not always, in agreement.

Review: putative mutagens and carcinogens in foods. V. Cycad azoxyglycosides.

Cycasin is a member of a family of azoxyglycosides produced by cycads. It is mutagenic and carcinogenic only when deglucosylated to release its principal metabolite, methylazoxymethanol (MAM). Methylazoxymethanol is also the aglycone of other cycad azoxyglycosides and is responsible for their toxicologic properties. The way in which people can be exposed to cycad azoxyglycosides is through the consumption of foods prepared from cycads. MAM induces genetic alterations in various test systems in bacteria, yeast, plants, Drosophila, and mammalian cells. An important aspect of the biological activities of cycasin and MAM is the intimate connection between their metabolism and their toxicologic effects. In adult mammals, the deglucosylation of cycasin is catalyzed only by enzymes of the microflora of the gut. Cycasin is therefore active when administered orally but not when administered parenterally. In contrast, MAM is active regardless of the route of exposure. Major uncertainties remain regarding the intermediates generated from MAM spontaneously and metabolically. More knowledge of these intermediates is required for a better understanding of the molecular mechanisms underlying the toxicity of cycasin, MAM, and related compounds.

Comparison of the carcinogenicity of methylazoxymethanol-beta-D-glucosiduronic acid in conventional and germfree Sprague-Dawley rats.

Methylazoxymethanol-beta-D-glucosiduronic acid (MAM-GlcUA) was administered to young adult male Sprague-Dawley rats by oral and ip routes. Most neoplasms developed in rats that had received the compound orally. The most prevalent site for the neoplasms was the intestinal tract, predominantly the colon. Comparatively fewer tumors occurred in the liver and kidneys. Germfree rats did not develop tumors when MAM-GlcUA was administered either orally or ip.

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.

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.

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.