Kazutoshi Sayama

Characterization of the estrogenic activities of zearalenone and zeranol in vivo and in vitro.

In the present study, we compared the estrogenic activity of zearalenone (ZEN) and zeranol (ZOL) by determining their relative receptor binding affinities for human ERalpha and ERbeta and also by determining their uterotropic activity in ovariectomized female mice. ZOL displayed a much higher binding affinity for human ERalpha and ERbeta than ZEN did. The IC(50) values of ZEN and ZOL for binding to human ERalpha were 240.4 and 21.79nM, respectively, and the IC(50) values for binding to ERbeta were 165.7 and 42.76nM, respectively. In ovariectomized female ICR mice, s.c. administration of ZEN at doses >or=2mg/kg/day for 3 consecutive days significantly increased uterine wet weight compared with the control group, and administration of ZOL increased the uterine wet weight at lower doses (>or=0.5mg/kg/day for 3 days). Based on available X-ray crystal structures of human ERalpha and ERbeta, we have also conducted molecular modeling studies to probe the binding characteristics of ZEN and ZOL for human ERalpha and ERbeta. Our data revealed that ZEN and ZOL were able to occupy the active site of the human ERalpha and ERbeta in a strikingly similar manner as 17beta-estradiol, such that the phenolic rings of ZEN and ZOL occupied the same receptor region as occupied by the A-ring of 17beta-estradiol. The primary reason that ZOL and ZEN is less potent than 17beta-estradiol is likely because 17beta-estradiol could bind to the receptor pocket without significantly changing its conformation, while ZOL or ZEN would require considerable conformational alterations upon binding to the estrogen receptors (ERs).

Biology of mouse mammary tumor virus (MMTV)

Mouse mammary tumor viruses (MMTV) replicate in the mammary gland, appear as infectious particles in mothers milk and invade the sucking pups from the intestinal tract. The immune system is essential for MMTV in the gut to reach the mammary gland. These properties make the life cycle of MMTV unique. We review the oncologically and immunologically intriguing events caused by MMTV in relation to the life cycle of the virus.

Green Tea Extract-induced Acute Hepatotoxicity in Rats

Although green tea is considered to be a healthy beverage, hepatotoxicity associated with the consumption of green tea extract has been reported. In the present study, we characterized the hepatotoxicity of green tea extract in rats and explored the responsible mechanism. Six-week-old IGS rats received a single intraperitoneal (ip) injection of 200 mg/kg green tea extract (THEA-FLAN 90S). At 8, 24, 48 and 72 hrs and 1 and 3 months after exposure, liver damage was assessed by using blood-chemistry, histopathology, and immunohistochemistry to detect cell death (TUNEL and caspase-3) and proliferative activity (PCNA). Analyses of malondialdehyde (MDA) in serum and the liver and of MDA and thymidine glycol (TG) by immunohistochemistry, as oxidative stress markers, were performed. Placental glutathione S-transferase (GST-P), which is a marker of hepatocarcinogenesis, was also immunohistochemically stained. To examine toxicity at older ages, 200 mg/kg green tea extract was administered to 18-wk-old female rats. In 6-wk-old rats, 12% of males and 50% of females died within 72 hrs. In 18-wk-old rats, 88% died within 72 hrs. The serum levels of aspartate aminotransferase, alanine aminotransferase and/or total bilirubin increased in both males and females. Single-cell necrosis with positive signs of TUNEL and caspase-3 was seen in perilobular hepatocytes from 8 hrs onward in all lobular areas. PCNA-positive hepatocytes increased at 48 hrs. MDA levels in the serum and liver tended to increase, and MDA- and TG-positive hepatocytes were seen immunohistochemically. GST-P–positive hepatocellular altered foci were detected in one female rat at the 3-month time point. In conclusion, a single injection of green tea extract induced acute and severe hepatotoxicity, which might be associated with lipid peroxidation and DNA oxidative stress in hepatocytes.

Protection of brain and pancreas from high-fat diet: effects of catechin and caffeine.

To investigate the effect of a high-fat diet on brain and pancreas functions, we used SAMP10 mice that have characteristics of brain atrophy and cognitive dysfunction with aging. Simultaneously, we investigated the effect of green tea catechin consumption on high-fat diet feeding, because green tea catechin has been reported to improve brain atrophy, brain dysfunction and obesity. The body weight of mice fed a high-fat diet from 2 to 12 months was higher than that of the control, although the calorie intake was not. The high-fat diet also increased insulin secretion; however, the hypersecretion of insulin and obesity were suppressed when mice were fed a high-fat diet with green tea catechin and caffeine. Furthermore, brain atrophy was suppressed and the working memory, tested using Y-maze, improved in mice fed a high-fat diet containing green tea catechin and caffeine. The secretion of insulin might affect both obesity and brain function. A strong correlation was found between working memory and insulin release in mice fed a high-fat diet with green tea catechin and/or caffeine. The results indicate the protective effect of green tea catechin and caffeine on the functions of brain and pancreas in mice fed a high-fat diet.

Immunological characterization of C3H mice congenic for Faslprcg, C3H/HeJ-Faslprcg/Faslprcg

Faslpr (lpr) and Faslprcg (lprcg ) are allelic mutations of the Fas gene that is involved in apoptosis or programmed cell death. Lpr greatly reduces the expression of functional Fas and lprcg expresses the death domain-disabled, non-functional Fas on the cell surface. C3H/HeJ mice congenic for lprcg (C3H-lprcg ) were established and compared with C3H/HeJ-lpr/lpr (C3H-lpr) mice for their immunological and pathological features. Lymphadenopathy, splenomegaly, development of CD4-CD8-B220+ or double-negative (DN) T cells, renal pathology, and lymphoid cell infiltration in the lung and liver were not significantly different between C3H-lprcg and C3H-lpr mice. Noticeably, however, the production of serum immunoglobulin, autoantibodies against double-strand DNA and serum immune complexes were significantly lower in C3H-lprcg than in C3H-lpr mice. The results indicate that the death signal through the death domain of Fas is responsible for lymphoproliferation due to the accumulation of DN T cells and suggest that the region of Fas outside the death domain may be involved in autoantibody production. The newly-developed congenic C3H-lprcg mice will provide a powerful tool for research into the function of Fas apart from apoptosis.

Effects of an antibiotic protease inhibitor, actinonin on the growth within collagen gels of non-metastatic and metastatic mouse mammary tumors of the same origin

Of five autonomous sublines established independently from the transplantable hormone-dependent mouse mammary tumor, TPDMT-4, three but not two acquired metastatic potential. In in vitro culture using collagen gels, actinonin, an antibiotic protease inhibitor exerted a stronger growth-inhibiting effect on the metastatic than on the parent and non-metastatic tumors. Zymographic analysis demonstrated the active forms of gelatinases in the metastatic but not in the non-metastatic sublines and the complete inhibition of the enzyme activities by actinonin. Gelatinases/type IV collagenases might play an important role in tumor progression towards metastatic phenotype and actinonin may suppress tumor growth through inhibiting collagenase.

Maternal prolactin during late pregnancy is important in generating nurturing behavior in the offspring

Significance Maternal child neglect is an increasingly prevalent public health issue, but the underlying biologic processes causing this phenomenon are still largely unknown. This study examines the prenatal environment of mice that later exhibit neglectful behavior. We have found that, in mice, factors determining the neglect phenotype in a mother are present during her own fetal period. We present evidence that maternal prolactin (PRL) could be a key factor for generating nurturing behavior in offspring by activating neural circuits required for the expression of nurturing behaviors. Although PRL is known as a trigger for maternal behavior in dams, this is a function of PRL that has a transgenerational impact. Although maternal nurturing behavior is extremely important for the preservation of a species, our knowledge of the biological underpinnings of these behaviors is insufficient. Here we show that the degree of a mother’s nurturing behavior is regulated by factors present during her own fetal development. We found that Cin85-deficient (Cin85−/−) mother mice had reduced pituitary hormone prolactin (PRL) secretion as a result of excessive dopamine signaling in the brain. Their offspring matured normally and produced their own pups; however, nurturing behaviors such as pup retrieval and nursing were strongly inhibited. Surprisingly, when WT embryos were transplanted into the fallopian tubes of Cin85−/− mice, they also exhibited inhibited nurturing behavior as adults. Conversely, when Cin85−/− embryos were transplanted into the fallopian tubes of WT mice, the resultant pups exhibited normal nurturing behaviors as adults. When PRL was administered to Cin85−/− mice during late pregnancy, a higher proportion of the resultant pups exhibited nurturing behaviors as adults. This correlates with our findings that neural circuitry associated with nurturing behaviors was less active in pups born to Cin85−/− mothers, but PRL administration to mothers restored neural activity to normal levels. These results suggest that the prenatal period is extremely important in determining the expression of nurturing behaviors in the subsequent generation, and that maternal PRL is one of the critical factors for expression. In conclusion, perinatally secreted maternal PRL affects the expression of nurturing behaviors not only in a mother, but also in her pups when they have reached adulthood.

Ustalic acid as a toxin and related compounds from the mushroom Tricholoma ustale

A toxin and related compounds were isolated from a poisonous mushroom Tricholoma ustale. Their structures were determined by analyses of the spectral data and synthesis.

Effects of catechins and caffeine on the development of atherosclerosis in mice

Atherosclerosis is one of the diseases related to metabolic syndrome which is caused by obesity. Previous reports have shown that green tea and its components have anti-obesity effect. We examined whether catechins and caffeine can prevent the development of atherosclerosis by oral administration, singly or in combination to the atherosclerosis model mice. Results demonstrated that the number of atherosclerotic regions in the aorta was significantly reduced by the combined treatment, and the atherosclerotic area was also improved. Serum HDL-C increased by caffeine single treatment, but no effect on the TG and TC by any treatments. Moreover, ECG illuviated to atheromatous lesions in aorta and the illuviation was enhanced by caffeine. The mRNA expression levels of LOX-1 and TNF-α showed a tendency to suppress by the combined treatment. These results indicated that the combined administration of catechins and caffeine has the inhibitory effect on the development of atherosclerosis in mice. Our research clearly demonstrated that the combined treatment of catechins and caffeine showed the inhibitory effect on the development of atherosclerosis in mice.

Significant role of Fas ligand-binding but defective Fas receptor (CD95) in lymph node hyperplasia composed of abnormal double-negative T cells.

The functional differences between two mutations of the Fas (CD95) locus, Faslpr (lpr) and Faslprcg (lprcg), were investigated using bone marrow (BM) transplantation on the C3H mouse background. Both lpr/lpr and lprcg/lprcg BM transferred caused lymph node (LN) hyperplasia in lpr/+ and lprcg/+ recipients, although it was clearly smaller than that in lpr/lpr and lprcg/lprcg recipients of lpr/lpr and lprcg/lprcg BM. In addition, both BM induced significantly larger LN hyperplasia in lprcg/+ than lpr/+ recipients. Appearance of CD4− CD8−[double negative (DN)] T cells in the periphery is the most consistent phenotype of Fas mutations. Importantly, the proportion of DN T cells was higher in larger LN hyperplasia in the order of lpr/+, lprcg/+ and lpr/lpr or lprcg/lprcg recipients. On the other hand, both lpr/lpr and lprcg/lprcg BM transferred into wild‐type (+/+) mice caused marked LN atrophy. The former, but not the latter, induced wasting syndrome. Faslg1d (gld)‐homozygous lpr/lpr BM transferred into +/+ mice elicited LN hyperplasia of the same extent as that in lpr/lpr mice transferred with lpr/lpr BM, but not wasting syndrome. Taken together with the fact that DN T cells massively express Fas ligand (FasL), this study implied that FasL overexpressed on DN cells may be involved in the accumulation of DN T cells in LN, LN atrophy and wasting syndrome, and that lprcg Fas, which can bind to Fas ligand but not transduce apoptosis signal into cells, may modulate these pathological conditions by interfering with the binding of FasL to Fas.