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.

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.

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.

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.

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.

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.

A practical procedure for testing DNA damage in vivo, proposed for a pre-screening of chemical carcinogens.

The alkaline elution method was adapted to the evaluation of DNA damage induced in vivo through a practical and reliable microfluorometric procedure, without any need for tissue pre-labeling. The DNA damage induced in vivo by treatment with a single dose of N-nitrosodimethylamine (DMNA), N-methyl-N-nitroso-urea (MNU), 1,2-dimethylhydrazine (DMH) or cycasin has been detected in different organs of mice or rats. The results obtained are rather consistent with the organotropism of these carcinogens, and show a satisfactory dose dependent of DNA damage. DMH and cycasin, both negative in the Ames' Salmonella/microsome mutagenicity test, are clearly positive with in vivo DNA damage/alkaline elution assay. This latter method, complemented with other short-term tests, may play a useful role in the pre-screening of chemical carcinogens.

Naturally occuring mutagens.

Naturally occurring mutagens have usually been discovered as a result of outbreaks of disease in agricultural livestock, or as a result of epidemiological studies of cancer of the liver in man. Subsequent work has then shown that the toxic agents responsible often have mutagenic properties. Examples are the pyrrolizidine alkaloids, cyasin, a range of mycotoxins produced by various fungi, and at least two unidentified toxic agents in bracken. Commonly the toxic agent itself does not show high biological activity, but after ingestion it is converted by metabolic processes into the active mutagen or carcinogen. Some of these toxic substances have been responsible for considerable losses of agricultural livestock and therefore are of economic significance. From the view-point of genetic hazards to man, the most significant compounds are probably the mycotoxins, e.g. aflatoxin, because of the common risk of fungal contamination of food, especially in tropical regions. No information is yet available on the effects of these mutagens on natural populations of animals. Plants containing the pyrrolizidine alkaloids are found in areas of Africa and the Middle East where plagues of the migratory locust occur. Although it is known that some of the alkaloids can induce chromosomal damage in grasshoppers, whether such damage ever becomes a significant factor under ecological conditions is not known. In some cases, insects have not only evolved resistance towards mutagenic alkaloids but have become dependent on them for certain purposes. The males of certain Danaid butterflies feed on plants containing pyrrolizidine alkaloids. After ingestion, the alkaloids are metabolised to dihydropyrrolizine derivatives, which are then secreted on special organs (hair pencils) and, following dissemination into the atmosphere, act as pheromones for the stimulation of mating behaviour.