Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-ß assembly.

Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid ß-protein (Aß) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aß oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific 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-Aß-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.

Comparison of the glucosinolate-myrosinase systems among daikon (Raphanus sativus, Japanese white radish) varieties.

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.

A possible role of nrf2 in prevention of renal oxidative damage by ferric nitrilotriacetate.

To ascertain the possible roles of nuclear erythroid 2 p45-related factor 2 (Nrf2), a key transcription factor of phase 2 drug-metabolizing enzymes, in renal cellular defense against oxidative stress, wild-type and Nrf2-knockout -/- mice were treated with ferric nitrilotriacetate (Fe-NTA) at doses of 3 or 6 mg iron/kg body weight. After Fe-NTA treatment, Nrf2 -/- mice consistently showed lower levels of glutathione (GSH) in the kidney at the low dose and the liver at the high dose than the wild-type mice. Gamma-glutamylcysteine ligase (GCL) activity in the kidney and liver of Nrf2-/- mice was also consistently lower than in wild-type mice after the Fe-NTA treatment. Histopathological examination revealed that nephrotoxicity of Fe-NTA, reflected in necrosis of renal tubule epithelial cells following nuclear damage, was more severe in the Nrf2-/- mice than in their wild-type counterparts. Overall, the data suggest that Nrf2 -/- mice are unable to compensate for depletion of renal GSH because of oxidative stress, being more susceptible to Fe-NTA-induced nephrotoxicity. In conclusion, the present study showed that Nrf2 might play an important role in protecting cells from oxidative stress in the kidney through its regulation of antioxidant enzymes.

Increased susceptibility to hepatocarcinogenicity of Nrf2-deficient mice exposed to 2-amino-3-methylimidazo[4,5-f]quinoline.

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 IQ-treated 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(-/-) mice suggest the existence of different contributions of Nrf2 to IQ hepatocarcinogenesis between mice of the two sexes.

Lung as a new target in rats of 2-amino-3-methylimidazo[4,5-f]quinoline carcinogenesis: results of a two-stage model initiated with N-bis(2-hydroxypropyl)nitrosamine.

The effects of IQ on the promotion stage of DHPN-induced lung carcinogenesis and contributions of oxidative stress were investigated in rats. Groups of 20 male 6-week-old F344 rats were given 0.1% DHPN in their drinking water for 2 weeks for initiation. From the age of 9 weeks, they were treated with 0, 150 and 300 p.p.m. of IQ in the diet for 27 weeks. Control rats were similarly fed 300 p.p.m. IQ or basal diet alone without the preceding initiation. IQ clearly (P < 0.01) enhanced the multiplicity of lung tumors in a dose-dependent manner (DHPN alone, 3.63 +/- 1.80; DHPN +150 p.p.m. IQ, 11.50 +/- 5.04; DHPN +300 p.p.m. IQ, 18.83 +/- 4.58 [no./rat]). In addition, the incidence of lung tumors in the 300 p.p.m. IQ alone group (25%) was significantly (P < 0.05) higher than that in the non-treatment group (0%). In a second experiment, male rats were given IQ at doses of 0 and 300 p.p.m. in the diet for one week in order to analyze 8-OHdG formation, levels of TBARS and BrdU-LI in the lungs. There were no changes in 8-OHdG or TBARS levels, but significant elevation of BrdU-LI occurred in the IQ administration group. The overall data clearly indicate that IQ is a potent lung carcinogen in rats, in which oxidative stress may not be involved in lung carcinogenesis.

Dose-dependent promotion of rat forestomach carcinogenesis by combined treatment with sodium nitrite and ascorbic acid after initiation with N-methyl-N'-nitro-N-nitrosoguanidine: possible contribution of nitric oxide-associated oxidative DNA damage.

Dose-dependent promotion effects of combined treatment with sodium nitrite (NaNO2) and ascorbic acid (AsA) on gastric carcinogenesis were examined in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Groups of 15 6-week-old F344 male rats were given 0.01% MNNG in their drinking water for 10 weeks to initiate carcinogenesis in the glandular stomach and a single intragastric administration of 100 mg/kg/bodyweight of MNNG by stomach tube at week 9 to initiate carcinogenesis in the forestomach. From week 11, they received either drinking water containing 0.05, 0.1 or 0.2% NaNO2 and a diet supplemented with 0.1 or 0.2% AsA in combination, each individual chemical alone or a basal diet until the end of week 42. In the forestomach, the incidence of hyperplasia was increased dose dependently by the treatment with NaNO2 alone. Incidences of neoplastic lesions were dramatically increased by the combined treatment with NaNO2 and AsA in a dose-dependent manner, but AsA itself had no effect. In the glandular stomach, only toxicity and regenerative changes were increased by the high-dose combination. In a second short-term experiment conducted for sequential observation, necrosis and strong inflammation were found in the forestomach epithelium shortly after commencing combined treatment with 1.0% AsA and 0.2% NaNO2, followed by hyperplasia, whereas there were no obvious effects in the glandular stomach. In addition, after a 4 h treatment with 1.0% AsA and 0.2% NaNO2, a slight increase in the 8-hydroxy-deoxyguanosine levels in the forestomach epithelium was observed by high-performance liquid chromatography and an electrochemical detection system, albeit without statistical significance. In vitro, electron spin resonance demonstrated nitric oxide formation during incubation with NaNO2 and AsA under acidic conditions. Thus, NaNO2 was demonstrated to exert promoter action in the forestomach, with AsA acting as a strong copromoter through cytotoxicity and regenerative cell proliferation, possibly mediated by oxidative DNA damage, but the combined treatment with NaNO2 and AsA had little influence on glandular stomach carcinogenesis.

Protective effects of benzyl isothiocyanate and sulforaphane but not resveratrol against initiation of pancreatic carcinogenesis in hamsters.

Potential chemopreventive effects of naturally occurring agents were investigated using a new 16-week medium-term pancreatic carcinogenesis models in hamsters. Male 6-week-old Syrian hamsters were subcutaneously injected with 10mg/kg body weight N-nitrosobis(2-oxopropyl)amine (BOP) four times within a week, and fed a diet supplemented with 80ppm benzyl isothiocyanate (BITC), 80ppm sulforaphane (SFN) or 10ppm resveratrol (RES) during the initiation or post-initiation stages. For the initiation stage, each chemical was given for 3 weeks including 1 week before and after the BOP injections. With post-initiation exposure, the groups were changed from basal diet 1 week after the last BOP injection, and then fed each chemical for 14 weeks. All the animals were sacrificed after 16 weeks. The multiplicities of combined pancreatic lesions including atypical hyperplasias and adenocarcinomas were significantly decreased by BITC and SFN given in the initiation but not the post-initiation stage. On the other hand, RES, a naturally occurring inhibitor of cyclooxygenase-2 (COX-2) reported chemopreventive effects, failed to show significant effects on pancreatic carcinogenesis in either the initiation or post-initiation stages. Our data suggest that the naturally occurring isothiocyanates BITC and SFN can block BOP-initiation of hamster pancreatic carcinogenesis.

A crucial role of Nrf2 in in vivo defense against oxidative damage by an environmental pollutant, pentachlorophenol.

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.

Enhancing effects of simultaneous treatment with sodium nitrite on 2-amino-3-methylimidazo[4,5-f]quinoline-induced rat liver, colon and Zymbal's gland carcinogenesis after initiation with diethylnitrosamine and 1,2-dimethylhydrazine.

Combined effects of sodium nitrite (NaNO2) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) on liver, colon and Zymbal's gland carcinogenesis were assessed using a rat two-stage carcinogenesis model, with a focus on involvement of oxidative stress. Male 6-week-old F344 rats were given a single intraperitoneal injection of 200 mg/kg of diethylnitrosamine and 4 subcutaneous injections of 40 mg/kg of 1,2-dimethylhydrazine for initiation. Then, they were administered 0 or 300 ppm IQ in the diet or 0, 0.1 or 0.2% NaNO2 in their drinking water for 27 weeks. The treatment with NaNO2+IQ significantly enhanced colon and Zymbal's gland carcinogenesis and tended to enhance hepatocarcinogenesis. The incidence of lung tumors in the IQ-treated groups was significantly increased as compared with the initiation alone group. In a second experiment, male rats were given IQ or NaNO2 under the same conditions as before for 1 week, and at sacrifice, their liver and colon tissue or mucosa were collected for analysis of 8-hydroxydeoxyguanosine (8-OHdG), thiobarbituric acid reactive substances (TBARS), acrolein-modified protein and the bromodeoxyuridine-labeling index (BrdU-LI) (in the colon). In the colon, 8-OHdG, acrolein-modified protein levels and BrdU-LI were significantly increased by the combined treatment. These results indicate that the treatment with NaNO2 enhances IQ-induced colon and Zymbal's gland carcinogenesis in rats and that oxidative DNA damage and lipid peroxidation may partly be involved, especially in the colon. In addition, this experiment showed that IQ can act as a potent lung carcinogen in rats.

Lack of enhancing effects of sodium nitrite on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in female Sprague-Dawley rats.

A number of heterocyclic amines (HCAs) have been shown to exert enhanced carcinogenic and mutagenic potential when given simultaneously with sodium nitrite (NaNO(2)). In the present experiment, effects of combined treatment with NaNO(2) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), one of the most prevalent carcinogenic HCAs in the human environment, were assessed with regard to mammary tumor induction in female Sprague-Dawley (SD) rats. Animals at 6 weeks of age were given intragastric doses of 100mg/kg body weight of PhIP twice a week for 4 weeks, during which period 0 or 0.2% NaNO(2) was administered in the drinking water. Control rats received 0.2% NaNO(2) alone for the 4 weeks or non-supplemented water during the entire 48 week experimental period, without carcinogen treatment. The first tumor in the PhIP+NaNO(2) group appeared significantly later than with PhIP alone, and during the experimental period, the incidence, multiplicity and volume of mammary tumors in this group tended towards decreased, although values did not significantly differ at the terminal sacrifice. These results indicate that NaNO(2) does not enhance PhIP-induced rat mammary carcinogenesis, rather possessing some potential for inhibition.

Effects of N-acetylcysteine, quercetin, and phytic acid on spontaneous hepatic and renal lesions in LEC rats.

The effects of anti-oxidants were examined in Long-Evans Cinnamon (LEC) rats, which develop acute hepatic injury, and subsequent hepatic and renal tumors due to accumulation of excess Cu. The rats, at the age of 15 weeks, were supplied a diet containing either 1% of N-acetylcysteine (NAC), quercetin (QC), or phytic acid (PA), or basal diet alone. At weeks 2 and 6 posttreatment, animals were sacrificed for collection of blood and tissue samples. In the NAC-treated group, the development of hepatic and renal lesions was dramatically reduced. In addition, accumulation of Cu and Fe in the liver was suppressed. Acrolein-modified protein, a new marker for lipid peroxidation, was not detected in the liver or kidney of NAC treated rats, even though deposition was evident in control. Neither QC nor PA affected the development of spontaneous hepatic lesions. These results indicate that oxidative stress was reduced by NAC in the liver and kidney, and suggest that Cu and Fe may be involved in the generation of oxidative stress in the liver. In addition, it was suggested that the different effects of the anti-oxidants on lesion development in LEC rats might be related to different mechanisms of action with regard to oxidative stress.

Induction of colon tumors in C57BL/6J mice fed MeIQx, IQ, or PhIP followed by dextran sulfate sodium treatment.

Heterocyclic amines (HCAs) have been shown to induce tumors in several organs of rodents, but except for MeIQ and PhIP, other HCAs such as MeIQx and IQ consistently failed to induce colon tumors in mice, whereas MeIQ, IQ, and PhIP exerted colon tumorigenicity in rats. Recently, we found that dietary MeIQx induces genotoxicity in the colon as well as the liver of two different types of reporter gene transgenic mice at subcarcinogenic doses such as 300 ppm. However, in the present study, dietary MeIQx did not significantly induce any tumors in C57BL/6J mice or gpt delta mice even when fed at 300 ppm for 78 weeks, suggesting that the treatment of MeIQx alone was not sufficient to promote colon tumors. In order to clarify a possibility whether such HCAs can induce colon tumors, C57BL/6J mice were fed MeIQx, IQ, or PhIP at a dose of 300 ppm for 12 weeks and, thereafter, twice received 1-week treatment with dextran sulfate sodium (DSS), 2 weeks apart. After 20 weeks, colon tumors including adenocarcinomas were found at incidences of 22%, 24%, and 45% in the groups receiving MeIQx, IQ, and PhIP, respectively, which were significantly (p < 0.05 or 0.01) different from the DSS alone value (0%). Thus our results clearly indicate that, in addition to PhIP, MeIQx and IQ can induce colon tumors in mice under an experimental condition promoting colon tumors.

In vivo mutational analysis of liver DNA in gpt delta transgenic rats treated with the hepatocarcinogens N-nitrosopyrrolidine, 2-amino-3-methylimidazo[4,5-f]quinoline, and di(2-ethylhexyl)phthalate.

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.

Specificity of co-promoting effects of caffeine on thyroid carcinogenesis in rats pretreated with N-bis(2-hydroxypropyl)nitrosamine.

The specificity of copromotion effects of caffeine with known goitrogenic factors on thyroid carcinogenesis was examined in rats pretreated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). Male F344 rats were divided into 8 groups, each consisting of 10 animals, and received a single sc injection of 2,800 mg/kg DHPN. From one week after the DHPN initiation, they were given basal diet, iodine deficiency (ID) diet, 500 ppm phenobarbital (PB) solution or 1,000 ppm sulfadimethoxine (SDM) solution with or without 1,500 ppm caffeine feeding for 12 weeks. The caffeine, PB, SDM, and ID treatments significantly (p < 0.05 or 0.01) increased the relative thyroid weights, and the increases with PB or ID were further (p < 0.05 or 0.01) enhanced in combination with caffeine. SDM drastically promoted thyroid carcinogenesis in association with increased serum TSH levels regardless of the caffeine treatment. Thyroid follicular carcinomas and adenomas were more frequently observed in the additional caffeine groups than in the ID alone groups. The incidence and multiplicity of focal thyroid follicular hyperplasias in the ID-treated groups were significantly (p < 0.05 and 0.01) elevated in the case of combination with caffeine. Increases in serum TSH levels with PB or ID were also further enhanced in combination with caffeine. Serum thyroid hormone levels were significantly (p < 0.01) decreased by SDM but significantly (p < 0.05 or 0.01) increased by caffeine, PB or ID. Our results clearly indicate that dietary caffeine at a high dose of 1,500 ppm interacts with ID, but neither SDM nor PB, to promote rat thyroid carcinogenesis although the combined caffeine + PB treatment somewhat affected thyroid weights as well as thyroid hormone levels.

Dose-related changes of oxidative stress and cell proliferation in kidneys of male and female F344 rats exposed to potassium bromate.

It is still of importance to investigate renal carcinogenesis by potassium bromate (KBrO3), a by-product of water disinfection by ozonation, for assessment of the risk to man. Five female F344 rats in each group were given KBrO3 at a dose of 300 mg/kg by single i.g. intubation or at a dose of 80 mg/kg by single i.p. injection, and were killed 48 h after the administration for measurements of thiobarbituric acid-reactive substances (TBARS) and 8-oxodeoxyguanosine (8-oxodG) levels in the kidney. Both levels in the treated animals were significantly elevated as compared with the control values. In a second experiment, 5 male and female F344 rats in each group were administered KBrO3 at concentrations of 0, 15, 30, 60, 125, 250 and 500 ppm in the drinking water for 4 weeks. KBrO3 in the drinking water did not elevate TBARS in either sex at any of the doses examined, but 8-oxodG formation in both sexes at 250 ppm and above was significantly higher than in the controls. Additionally, the bromodeoxyuridine-labeling index for proximal convoluted tubules was significantly increased at 30 ppm and above in the males, and at 250 ppm and above in the females. Alpha2u-globulin accumulation in the kidneys of male rats was increased with statistical significance at 125 ppm and above. These findings suggest that DNA oxidation induced by KBrO3 may occur independently of lipid peroxidation and more than 250 ppm KBrO3 in the drinking water can exert a carcinogenic effect by way of oxidative stress.

Lack of significant inhibitory effects of a plant lignan tracheloside on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in female Sprague-Dawley rats.

Tracheloside, one of the plant lignans which can be extracted from the debris after safflower oil is produced from the seeds of Carthamus tinctorious, is an analogue of another plant lignan, arctiin, the side-chain C-2 of the five-membered ring being changed from a hydrogen to a hydroxyl group. We have already demonstrated that arctiin has chemopreventive effect on mammary carcinogenesis. Therefore, chemopreventive effects of tracheloside on the initiation or post-initiation period of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in female rats were examined. For initiation, female Sprague-Dawley (SD) rats at the 6 weeks of age were given intragastric administrations of 100 mg/kg body weight of PhIP once a week for 8 weeks. The animals were treated with 0.2 or 0.02% tracheloside during or after this carcinogen exposure. Control rats were fed basal diet with PhIP initiation or 0.2% tracheloside or basal diet alone without initiation throughout the experimental period. All surviving animals were necropsied at the week 52 of administration. There were no clear treatment-related changes with statistical significance in all parameters for mammary carcinomas measured in this experiment. These results indicate that tracheloside may not exert significant effects on PhIP-induced mammary carcinogenesis at least under the present experiment condition.

A 13-week subchronic toxicity study of paprika color in F344 rats.

The fruit of the paprika (Capsicum annuum) has been widely used in various countries as a spice and food-coloring additive. As a part of the safety assessment of paprika color (Paprika oleoresin), a 13-week subchronic toxicity study was performed in F344 rats. To establish a no-observed-adverse-effect level (NOAEL) for application in subsequent long-term studies, rats were fed powder diet containing paprika color at dose levels of 0 (basal diet), 0.62, 1.25, 2.5 and 5% (maximum) for 13 weeks. During the experiment, there were no remarkable changes in general appearance and no deaths occurred in any experimental group. Although serum total cholesterol was dose-dependently increased in both sexes, no related histopathological changes were observed in the liver. Slight inflammatory cell infiltration in the myocardium and vacuolation of hepatocytes were noted in both control and paprika color-treated animals, but there were no clear differences between groups. In conclusion, paprika color even at 5% in the diet (0.67 g/rat/day or 2948.4 mg/kg bw/day for male rats and 0.43 g/rat/day or 3197.4 mg/kg bw/day for female rats) did not cause remarkable adverse effects in F344 rats. Thus, the NOAEL and the maximum dose level for carcinogenicity testing of paprika color were concluded to be 5% in the diet.

Synergistic interaction between excess caffeine and deficient iodine on the promotion of thyroid carcinogenesis in rats pretreated with N-bis(2-hydroxypropyl)nitrosamine.

The combined effects of caffeine (1,3,7-trimethylxanthine) with iodine deficiency (ID) were examined in a rat two-stage thyroid carcinogenesis model using N-bis(2-hydroxypropyl)nitrosamine (DHPN). Male F344 rats were divided into 6 groups each consisting of 10 animals, and received a single s.c. injection of 2800 mg/kg DHPN. From 1 week after the DHPN initiation, the rats were respectively fed a basal diet in which the protein was exchanged for 20% gluten, containing 1500 ppm caffeine + ID, 300 ppm caffeine + ID, 60 ppm caffeine + ID, 1500 ppm caffeine or ID or a basal diet alone for 12 weeks. Relative thyroid weights were significantly (P < 0.05) increased due to the development of proliferative lesions induced by the ID diet as compared to the DHPN-alone group value, which was enhanced by caffeine, albeit without statistical significance. Relative pituitary weights were significantly (P < 0.05) increased with 300 or 1500 ppm caffeine + ID as compared to the DHPN-alone group value. Serum thyroid stimulating hormone (TSH) levels were slightly increased by ID, an effect which was further enhanced by 300 or 1500 ppm caffeine. Serum thyroxine (T(4)) levels were slightly increased by caffeine or ID alone, but decreased by caffeine with ID. Histopathologically, thyroid follicular carcinomas were found only in the 1500 ppm caffeine + ID group, although thyroid follicular adenomas were detected in all the ID-treated groups. The multiplicity of focal thyroid follicular hyperplasias was significantly (P < 0.05) increased by 1500 ppm caffeine. These results indicate that caffeine may synergistically promote thyroid carcinogenesis with ID partially through a pituitary-dependent pathway in rats, implying the possible implication of routine caffeine intake in the promotion of thyroid carcinogenesis.

Prevention of dual promoting effects of pentachlorophenol, an environmental pollutant, on diethylnitrosamine-induced hepato- and cholangiocarcinogenesis in mice by green tea infusion.

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.

A repeated 28-day oral dose toxicity study of genistein in rats, based on the 'Enhanced OECD Test Guideline 407' for screening endocrine-disrupting chemicals.

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 genistein, which is known as a phytoestrogen. Attention was paid to the sensitivity of certain additional parameters, such as histopathology observations and organ weights of endocrine related organs, sperm characteristics, serum hormone levels and estrous cycle, for detecting endocrine-related effects of endocrine-disrupting chemicals based on the existing TG 407. Seven-week-old Crj:CD(SD)IGS rats were assigned to one of four groups, each consisting of ten males and ten females, and genistein was administered once daily by gavage at doses of 0 (control), 120, 400 or 1000 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 the 4 days after the 28th administration. Endocrine-disrupting effects of genistein were detected in females by histopathology. The changes included vacuolation and mucinification of the vaginal epithelium in the 400 and 1000 mg/kg groups; however, the incidences of the lesion were very low. Although increased serum prolactin levels were recorded in the males of the 1000 mg/kg group, we could not determine whether this was indeed induced by genistein. General toxicological effects of genistein were detected in blood chemistry, such as increased triglycerides and total protein and a decreased albumin/globulin ratio, as well as increased liver weight and glycogen deposition in the periportal hepatocytes. Based on these results, the no-observed-adverse-effect level (NOAEL) in the present study was estimated to be 120 mg/kg per day. In particular, endocrine-related effects were most sensitively detected by histopathology examination of sexual organs. However, the findings indicate that chemicals with weak endocrine-disrupting potential like genistein must be evaluated taking into consideration the results of other test systems.