Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid β-protein (Aβ) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10-to 15-nm spherical Aβ oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuronspecific Na + /K + -ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aβ-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn 879 and Trp 880 is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.NMR | computational modeling | abnormal protein-protein interaction in synapse | hyperexcitotoxicity | protein-protein interaction inhibitors

Our goal was to elucidate roles of Nrf2 in in vivo defense against pentachlorophenol (PCP), an environmental pollutant and hepatocarcinogen in mice. We examined oxidative stress and cell proliferation, along with other hepatotoxicological parameters, in the livers of nrf2-deficient (wild:+/+, heterozygous:+/-, homozygous:-/-) animals fed PCP in their diet at doses of 0, 150, 300, 600, or 1200 ppm for 4 weeks. For measurement of methoxyresorufin-O-demethylase (CYP 1A2), NAD(P):quinone oxidoreductase 1 (NQO1), and UDP-glucuronosyltransferase (UDP-GT), an additional study was performed with all but the 150-ppm dose. Significant elevation of 8-hydroxydeoxyguanosine (8-OH-dG) levels in the liver DNA was observed only in -/- mice treated with PCP at 1200 ppm. Levels of thiobarbituric-acid-reactive substances (TBARS) were also raised significantly compared to those of the relevant +/+ mice. Bromodeoxyuridine labeling indices (BrdU-LIs) of hepatocytes in -/- mice were significantly higher at all doses than those in the relevant +/+ mice. Relative liver weights were unchanged in mice lacking Nrf2, whereas liver weight in +/+ and +/- mice was increased. Significant elevations of serum ALP activity, but not ALT and AST activity, occurred at 600 ppm and above in -/- mice compared to the relevant +/+ mice. Histopathologically, centrilobular hepatocyte necrosis was severe in the -/- mice that received 600 ppm. Although CYP 1A2 activity was elevated in all treated mice, increases in NQO1 levels and UDP-GT activities did not occur only in -/- mice. These data suggest that Nrf2 plays a key role in prevention of PCP-induced oxidative stress and cell proliferation.

In order to cast light on carcinogen-specific molecular mechanisms underlying experimental hepatocarcinogenesis in rats, in vivo mutagenicity and mutation spectra of known genotoxic rat hepatocarcinogens N-nitrosopyrrolidine (NPYR), and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), as well as the nongenotoxic hepatocarcinogen di(2-ethylhexyl)phthalate (DEHP) and the noncarcinogen acetaminophen (AAP), were investigated in guanine phosphoribosyltransferase (gpt) delta transgenic rats, a recently developed animal model for genotoxicity analysis. After 13-wk treatment, glutathione S-transferase placental form (GST-P)-positive liver cell foci were significantly increased in NPYR-treated and IQ-treated rats. In the DEHP-treated rats, marked hepatomegaly with centrilobular hypertrophy of hepatocytes occurred, although GST-P staining was consistently negative. Positive mutagenicity was detected in IQ- and NPYR-treated rats. Mutant frequencies (MFs) in the liver DNA were 188.0 x 10(-6) and 56.5 x 10(-6), approximately 35-fold and 10-fold higher, respectively, than that of nontreatment control rats (5.5 x 10(-6)). There were no increases in MFs in the DEHP- or AAP-treated rats as compared to the nontreatment control value. IQ induced mainly base substitutions leading to G:C to T:A transversions (56.9%) and deletions of G:C base pairs. In contrast, NPYR primarily caused specific A:T to G:C transitions (49.3%), which are very rare in the other groups. These data provided support for the conclusion that IQ and NPYR hepatocarcinogenesis depends on genotoxic processes and specific DNA adduct formation while DEHP exerts its influence via a nongenotoxic promotional pathway. Our data also indicate that analysis of specific in vivo mutational responses with transgenic animal models can provide crucial information for understanding the molecular mechanisms underlying chemical carcinogenesis.

Myrosinase is a cytosolic plant enzyme present in daikon ( Raphanus sativus, Japanese white radish) roots that hydrolyzes 4-methylthio-3-butenyl glucosinolate (MTBGLS) into the natural pungent agent 4-methylthio-3-butenyl isothiocyanate (MTBITC), which possesses antimicrobial, antimutagenic, and anticarcinogenic properties. The concentration of MTBGLS, myrosinase activity, and production of MTBITC in seven daikon varieties (one conventional and six heirlooms) were determined to rank the activity of the glucosinolate-myrosinase system and identify critical factors influencing the production of MTBITC. The six heirloom varieties produced 2.0-11.5 times higher levels of MTBITC as compared to the conventional variety, Aokubi, which is consumed by the present Japanese population. The myrosinase was located exclusively in the outer epidermal layer in Aokubi, and MTBGLS was widely distributed throughout the root tissue. Although the skin is a potentially rich source of myrosinase in Aokubi, the skin is usually peeled off in the current practice of preparing daikon for cooking. New practices are therefore proposed for the preparation of daikon tubers that eliminate the peeling of the skin to avoid removing the enzyme needed to convert MTBGLS to the health-beneficial MTBITC. It is also concluded that the consumption of heirloom daikon varieties in addition to changes in food preparation will optimize the health benefits of daikon.

In order to explore a possibility that the custom of drinking green tea infusion is efficacious for reducing the carcinogenic risk of environmental exposure to pentachlorophenol (PCP), we examined the effects in a hepato- and cholangiocarcinogenesis model in mice exposed to diethylnitrosamine (DEN). In the first experiment, groups of 15 male mice were initially treated with DEN at a dose of 20 p.p.m. in the drinking water for the first 8 weeks followed by a 4 week recovery interval by PCP at concentrations of 0 (basal diet), 300 or 600 p.p.m. in the diet for 23 weeks. Further groups of animals were treated with DEN and PCP in the same manner and received 2% green tea infusion (GT) instead of the drinking water from week 10 until death. PCP exposure at the high dose promoted DEN-induced hepatocarcinogenesis, and also caused progression of cystic hyperplasias of the intrahepatic bile ducts to cholangiocellular tumors. Co-administration of GT was able to prevent the increases of incidences and multiplicities of DEN-induced hepatocellular tumors and also arrest the progression of cholangiocellular tumors. In the second experiment, co-treatment with GT in the drinking water from 1 week before 300 or 600 p.p.m. PCP treatment in the diet to the end of the experiment at week 3 in B6C3F1 male mice suppressed increases of serum ALT activities, 8-oxodeoxyguanosine levels in liver DNA and bromodeoxyuridine labeling indices of hepatocytes and intrahepatic biliary epithelial cells induced by PCP. These findings suggest that regular intake of green tea may reduce the carcinogenic risk posed by an environmental pollutant, PCP, presumably due to effects on oxidative stress.

No abstract

To elucidate the roles of the transcription factor NF-E2-related factor (Nrf2) in hepatocarcinogenesis induced by 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ), a mutagenic and carcinogenic heterocyclic amine, Nrf2-deficient mice were treated with 300 p.p.m. IQ in their diet for 1, 4 or 52 weeks. In the long-term experiment, the multiplicity and incidence of liver tumors in male and female IQtreated Nrf2 deficient (-/-) mice were significantly higher than those in their counterpart wild-type (+/+) mice exposed to IQ. In the short-term experiment, although IQ exposure to Nrf2(+/+) mice of both sexes did not modify UDP-glucuronosyltransferase values, glutathione S-transferase values were significantly increased due to IQ treatment, in contrast to no alteration in male and female Nrf2(-/-) mice. Levels of oxidative stress markers such as 8-hydroxydeoxyguanosine and thiobarbituric acid reactive substances in the livers of all treated mice were not changed by IQ treatment. IQ-specific DNA adduct levels were elevated only in female Nrf2(-/-) mice, although the increase was not significant. IQ treatment caused an increase in proliferating cell nuclear antigen labeling indices only in male Nrf2(-/-) mice. The present data clearly show that Nrf2(-/-) mice of both sexes are susceptible to IQ hepatocarcinogenicity, which might result from IQ accumulation due to failure of metabolizing enzyme induction. In addition, inconsistent results concerning IQ-specific adducts and proliferating cell nuclear antigen labeling indices in male and female Nrf2(-/ (1) and also in cigarette smoke condensate (2) . IQ is carcinogenic to the liver, colon, small intestine, Zymbal's gland, clitoral gland and skin in F344 rats, (3) to the liver, forestomach and lung in CDF 1 mice, (4) and to the liver in B6C3F 1 mice (5) in long-term experiments. Recently, we have found that rat lung is also a target of IQ carcinogenicity.-(6) Its carcinogenesis is considered to involve mainly IQ bioactivation, that is phase I hepatic CYP1A1/2-mediated N-hydroxylation followed by phase II (mainly N-acetyltransferase and sulfotransferase) esterification of N-hydroxylamines to reactive ester derivatives that bind to DNA covalently. (7,8) In addition, it was suggested recently that oxidative stress may also participate in HCA carcinogenesis, based on the findings that induction of preneoplastic or neoplastic lesions following HCA exposure was significantly inhibited when given simultaneously with antioxidants in rats, (9,10) and that superoxide anion radicals were generated during metabolism of HCA by NADPH/cytochrome P-450 reductase in vitro using a spin-trapping method.(11) We have demonstrated enhancement of IQ-induced colon tumor development due to cotreatment with NaNO 2 and the participation of oxidative stress such as oxidative DNA damage and lipid peroxidation in this phenomenon. However, IQ-induced liver tumors were only slightly increased by the addition of NaNO 2 , the relation to oxidative stress being equivocal.(12) Thus, IQ causes carcinogenesis in a b...

In association with the international validation project to establish an OECD Enhanced Test Guideline 407, we performed a 28-day repeated-dose toxicity study of methoxychlor, a chlorinated hydrocarbon pesticide with pro-estrogenic and anti-androgenic activities. Attention was paid to the sensitivity of certain additional parameters for detecting endocrine related effects of endocrine disrupting chemicals based on the existing TG 407. Seven-week-old Crj:CD(SD)IGS rats were allocated to one of four groups, each consisting often males and ten females, and methoxychlor was administered once daily by gavage at doses of 0 (control), 20, 100 or 500 mg/kg body weight per day. Male rats were killed on the day after the 28th administration. Female rats were killed on the day of the diestrus stage during 4 days after the 28th administration. Male rats receiving methoxychlor showed mainly atrophy of mammary acinus in the 20 mg/ kg and higher groups, together with decreases in prostate and seminal vesicle weights, and atrophy of epididymis, prostate, seminal vesicle and coagulating gland in the 100 and 500 mg/kg groups. In addition, decrease in serum testosterone level, increase in follicle-stimulating hormone level, decrease in testis and epididymis weights, atrophy of semiferous tubules and Leydig cells, decrease in the number of sperm in the caudal epididymis and their motility were observed in the 500 mg/kg group. Female rats receiving methoxychlor showed mainly abnormal estrous cycles, decrease in serum luteinizing hormone level, decrease in ovary weight, proliferation of mammary acinus, atrophy of ovary due to decrease in follicles and corpus luteum in histopathology, hypertrophy of endometrial epithelium of uterus and vagina epithelium in the 100 and 500 mg/kg groups. Among the parameters tested in the present experimental system, effects of methoxychlor on endocrine-related organs were detected with regard to serum hormone, organ weights, histopathological examination in both sexes, estrus cycle in females and sperm examination in males. Based on these results, a no-observed-adverse-effect level (NOAEL) in the present study was estimated to be below 20 mg/kg per day. In particular, the adverse effects were effectively detected in organ weights of accessory sex organs and histopathological examination.