Unexpected Liver and Kidney Pathology in C57BL/6J Mice Fed a High-fat Diet and Given Azoxymethane to Induce Colon Cancer.

Multiple animal models have been developed to investigate the pathogenesis of colorectal cancer and to evaluate potential treatments. One model system uses azoxymethane, a metabolite of cycasin, alone and in conjunction with dextran sodium sulfate to induce colon cancer in rodents. Azoxymethane is metabolized by hepatic P450 enzymes and can also be eliminated through the kidneys. In this study, C57BL/6J mice were fed either standard or high-fat diet and then all mice received azoxymethane at 10 mg/kg body weight twice a week for 6 wk. Shortly after the end of treatment, high mortality occurred in mice in the high-fat diet group. Postmortem examination revealed hepatic and renal pathology in mice on both diets. Histologic changes in liver included hepatocytomegaly with nuclear pleomorphism and bile duct hyperplasia accompanied by mixed inflammatory-cell infiltrates. Changes in the kidneys ranged from basophilia of tubular epithelium to tubular atrophy. The results indicate that further optimization of this model is needed when feeding a high-fat diet and giving multiple azoxymethane doses to induce colon cancer in C57BL/6J mice.

Toxic phytochemicals and their potential risks for human cancer.

Consuming plants for their presumed health benefits has occurred since early civilizations. Phytochemicals are found in various plants that are frequently included in the human diet and are generally thought to be safe for consumption because they are produced naturally. However, this is not always the case and in fact many natural compounds found in several commonly consumed plants are potential carcinogens or tumor promoters and should be avoided.

Relative seed and fruit toxicity of the Australian cycads Macrozamia miquelii and Cycas ophiolitica: further evidence for a megafaunal seed dispersal syndrome in cycads, and its possible antiquity.

An apparent contradiction in the ecology of cycad plants is that their seeds are known to be highly poisonous, and yet they seem well adapted for seed dispersal by animals, as shown by their visually conspicuous seed cones and large seeds presented within a brightly colored fleshy "fruit" of sarcotesta. We tested if this sarcotesta could function as a reward for cycad seed dispersal fauna, by establishing if the toxic compound cycasin, known from the seeds, is absent from the sarcotesta. The Australian cycads Macrozamia miquelii and Cycas ophiolitica were tested (N = 10 individuals per species) using gas chromatography / mass spectrometry. Cycasin was detected at 0.34 % (fresh weight) in seed endosperm of M. miquelii and 0.28 % (fresh weight) in seed endosperm of C. ophiolitica. Cycasin was absent from the sarcotesta of the same propagules (none detected in the case of M. miquelii, and trace quantities detected in sarcotesta of only four of the ten C. ophiolitica propagules). This laboratory finding was supported by field observations of native animals eating the sarcotesta of these cycads but discarding the toxic seed intact. These results suggest cycads are adapted for dispersal fauna capable of swallowing the large, heavy propagules whole, digesting the non-toxic sarcotesta flesh internally, and then voiding the toxic seed intact. Megafauna species such as extant emus or cassowaries, or extinct Pleistocene megafauna such as Genyornis, are plausible candidates for such dispersal. Cycads are an ancient lineage, and the possible antiquity of their megafaunal seed dispersal adaptations are discussed.

Cycads and their association with certain neurodegenerative diseases.

INTRODUCTION: Cycads are ornamental plants that in some parts of the world are used as fresh food or raw material for producing flour with a high nutritional value. However, they also contain active compounds, including methylazoxymethanol, ß-methylamino-L-alanine, ß-alanine-L-oxalylamino and cycasin, which may produce neurotoxic effects. Some studies have associated consuming cycads and their derivatives with neurodegenerative diseases such as amyotrophic lateral sclerosis/Parkinsonism dementia complex, and other diseases characterised by motor impairment. Therefore, we must not forget that any product, no matter how natural, may present health risks or benefits depending on the chemical compounds it contains and the susceptibility of those who consume it. DEVELOPMENT: We completed a literature analysis to evaluate the neurotoxic properties of cycads and their association with neurological diseases in order to provide structured scientific information that may contribute to preventing health problems in people who use these plants. CONCLUSION: Cycads contain neurotoxic compounds that may contribute to the development of neurological diseases when ingested improperly. We must be mindful of the fact that while some plants have a high nutritional value and may fill the food gap for vulnerable populations, they can also be toxic and have a negative impact on health.

Effect of methanolic extract of Vernonia amygdalina (common bitter leaf) on lipid peroxidation and antioxidant enzymes in rats exposed to cycasin.

This study investigated the effect of a methanolic extract of Vernonia amygdalina (VA) on lipid peroxidation and antioxidant status of the colon of rats maintained on a normal diet containing 5% Cycas revoluta (cycads). Fifty male Wistar albino rats were randomly assigned into five groups of ten experimental animals in a study that lasted for six weeks. One control group was maintained on a normal diet only while another group was fed a normal diet containing 5% cycads. The other three groups were maintained on the normal diet and 5% cycads and orally fed 200 mg VA/kg body weight for 1, 5 or 6 weeks. The results obtained revealed that the level of malondialdehyde (an index of lipid peroxidation) was significantly elevated (p < 0.05) in rats exposed to cycads only compared with the control. However, oral administration of VA in conjunction with exposure to cycads appeared to reduce the extent of lipid peroxidation to values that are not significantly (p > 0.05) different from those of the control. The activity of Superoxide Dismutase (SOD) was significantly reduced (p < 0.05) in the experimental animals fed cycads compared with the controls. Oral administration of VA seemed to counteract the effect of cycads on SOD in the colon as no significant difference (p > 0.05) was observed in rats fed VA compared with the controls. The results of this study suggest that methanolic extract of VA may mitigate the biochemical consequences of cycasin-induced lipid peroxidation in the colon of rats.

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.

Two genera of Aulacoscelinae beetles reflexively bleed azoxyglycosides found in their host cycads.

Aulacoscelinae beetles have an ancient relationship with cycads (Cycadophyta: Zamiaceae), which contain highly toxic azoxyglycoside (AZG) compounds. How these "primitive" leaf beetles deal with such host-derived compounds remains largely unknown. Collections were made of adult Aulacoscelis appendiculata from Zamia cf. elegantissima in Panama, A. vogti from Dioon edule in Mexico, and Janbechynea paradoxa from Zamia boliviana in Bolivia. Total AZG levels were quantified in both cycad leaves and adult beetles by high performance liquid chromatography (HPLC). On average, cycad leaves contained between 0.5-0.8% AZG (frozen weight, FW), while adult beetles feeding on the same leaves contained even higher levels of the compounds (average 0.9-1.5% FW). High AZG levels were isolated from reflex bleeding secreted at the leg joints when beetles were disturbed. Nuclear magnetic resonance and mass spectroscopy identified two AZGs, cycasin and macrozamin, in the reflex bleeding; this is the first account of potentially plant-derived compounds in secretions of the Aulacoscelinae. These data as well as the basal phylogenetic position of the Aulacoscelinae suggest that sequestration of plant secondary metabolites appeared early in leaf beetle evolution.

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)

[Studies on the chemical constituents of the roots of Anemone altaica].

OBJECTIVE: To investigate the chemical constituents of the roots of Anemone altaica Fisch. ex C. A. May. METHODS: The constituents of n-BuOH-soluble portion were isolated and purified by means of chromatography. Compounds were identified by their physical characteristics and spectral features. RESULTS: Six compounds were isolated and identified as cimigenol-3-O-beta-D-xylopyranoside (1), cimigenol-3-O-beta-D-xylopyranol (1 -->3)-beta-D-xylopyranoside (2), isolariciresinol-9-O-beta-D-glucopyranoside (3), adenosine (4), uridine (5) and methyl-beta-D-glucopyranoside (6). CONCLUSION: All compounds are isolated from this genus for the first time.

Is cycasin in eumaeus minyas (lepidoptera: lycaenidae): a predator deterrent?

El secuestro de compuestos de defensa de las plantas hospederas puede requerir adaptaciones fisiológicas particulares para que las larvas los ingieran, acumulen y almacenen. Los compuestos de defensa son usados para varios propósitos, particularmente contra depredadores, donde la defensa química reduce el riesgo de depredación. La cicasina es un metabolito secundario presente en las cícadas (Cycadales), que son hospederas de mariposas del género Eumaeus (Ly caenidae). La cicasina es secuestrada por la mariposa aposemática E.minyas de su planta hospedera Zamia loddigesii (Zamiaceae) y es usada como defensa química. En experimentos de campo y laboratorio se evaluó la presencia de cicasina en todos los estadios de E.minyas. Mediante experimentos de palatabilidad en el laboratorio, se estableció que la cicasina pura repele a Solenopsis geminata (Hymenoptera: Formicidae), un depredador potencial de E.minyas y, al utilizar extractos de huevos, larvas y adultos de esta mariposa se obtiene el mismo efecto de manera experimental. Para evaluar la eficiencia de esta defensa química en huevos y larvas en condiciones naturales, se realizó un experimento de exclusión de depredadores. Usando un análisis de sobrevivencia se demostró que el número de huevos y larvas sobrevivientes fue significativamente más alto en las exclusiones, sugiriendo que la función protectiva de la cicasina en condiciones naturales no es totalmente efectiva contra los depredadores naturales. Este es el primer estudio que incluye manipulación experimental de la función protectiva de la cicasina en condiciones naturales. Sugerimos que la cicasina en la mariposa aposemática E.minyas tiene una función defensiva contra los depredadores; sin embargo, sus poblaciones pueden estar reguladas por el efecto negativo de sus depredadores, los cuales aparentemente saltan la barrera química defensiva y por el canibalismo

Cycad neurotoxins, consumption of flying foxes, and ALS-PDC disease in Guam.

The Chamorro people of Guam have been afflicted with a complex of neurodegenerative diseases (now known as ALS-PDC) with similarities to ALS, AD, and PD at a far higher rate than other populations throughout the world. Chamorro consumption of flying foxes may have generated sufficiently high cumulative doses of plant neurotoxins to result in ALS-PDC neuropathologies, since the flying foxes forage on neurotoxic cycad seeds.

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.

Substrate specificity and transglucosylation catalyzed by cycad beta-glucosidase.

Initial rates of transglucosylation with diglucosides and diglucose-azoxyglycosides as acceptor by cycad beta-glucosidase were tentatively obtained. The formation of beta-1,3 glucosidic linkage was predominant, except for neocycasin A (beta-laminaribioside of methylazoxymethanol, MAM) as an acceptor. With neocycasin A as an acceptor, beta-1,4 and beta-1,6 glucosidic linkages were formed but beta-1,3 linkage was not. Whereas with laminaribiose as acceptor, laminaritriose and triose with beta-1,6 linkage were formed, but triose with beta-1,4 linkage was not. On the other hand, with other diglucoses and neocycasin B (beta-gentiobioside of MAM) as acceptor, all the linkages formed were beta-1,3 glucosidic. The aglycone of azoxyglycosides, MAM, affected the kind of linkages formed in the trisaccharides. When initial rates of the linkage formation of the transglucosylation at 100 mM acceptor were compared with the hydrolysis rates obtained by Lineweaver-Burk plot, the order of formation rates of the di- and tri-glucosides by transglucosylation was the same as obtained for the hydrolysis parameter, kcat/Km. Km values for various substrates could be grouped according to the kind of the linkages (beta-1,3, beta-1,4, and beta-1,6) first split by the enzyme.

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.

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.