Hajime Watanabe

Low dose effect of in utero exposure to bisphenol A and diethylstilbestrol on female mouse reproduction

In utero exposure to bisphenol-A (BPA) at doses relevant to human consumption has been reported to accelerate weight gain and puberty in female mice, but the effect of low dose BPA on female reproduction has not been described. In this study, we investigated low dose effects of BPA on sexual maturation and reproduction in female ICR/Jcl mice. Pregnant ICR mice (F0) were injected (s.c.) with BPA (2 and 20 microg/kg), diethylstilbestrol (DES; 0.02, 0.2, and 2 microg/kg) or oil vehicle once per day from gestational days 11-17. For both female and male offspring (F1), body weights were measured on postnatal day (PND) 0 (the day of birth), 11, 22, and 60, and anogenital distance (AGD) was measured on PNDs 22 and 60. Pups were weaned at PND 22 and males were caged separately from females. Vaginal smears were taken daily beginning the day of vaginal opening for 30 days. The age at vaginal opening was significantly earlier in all exposed females except for 2 microg/kg BPA females compared to oil controls. Body weight at vaginal opening was lower than controls in all exposed females. The first vaginal estrus was earlier in all exposed females except for the 2 microg/kg BPA group females compared to controls. From PND 90 to 120, gestationally exposed F1 female mice were mated with unexposed males. Total numbers of pups and sex ratio in F1 mice exposed to BPA or DES, and those of their offspring (F2) were not different from controls in any treatment group. The present results indicate that prenatal exposure to low doses of BPA and DES induces early vaginal opening, but does not affect reproductive functioning at the first breeding.

Developmental effects of perinatal exposure to bisphenol-A and diethylstilbestrol on reproductive organs in female mice

Reproductive tract development is influenced by estrogen. The aim of this study was to determine the effects of an environmental estrogenic chemical bisphenol-A (BPA) on prenatal and postnatal development of female mouse reproductive organs. In the prenatal treatment group, BPA or the synthetic estrogen diethylstilbestrol (DES) were given by subcutaneous (s.c.) injections to pregnant mice during gestational days 10-18. Some offspring treated prenatally with 10 and 100 mg/kg bw BPA or 0.67 and 67 microg/kg bw DES were ovariectomized at 30 days and sacrificed at 40 days of age. Vaginal smears were examined in the remaining offspring, then these offspring were mated with normal males. Prenatal exposure to 10 mg/kg BPA reduced the number of mice with corpora lutea compared to sesame oil controls at 30 days, but more than 80% of mice from either prenatally exposed BPA group were fertile at 90 days. Mice exposed prenatally to maternal doses of 67 microg/kg DES were sterile and showed ovary-independent vaginal and uterine epithelial stratification; however, mice exposed prenatally to BPA did not show ovary-independent vaginal and uterine changes. The number of offspring and litter sex ratio from mice exposed prenatally to BPA (10 or 100 mg/kg) or 0.67 microg/kg DES were not different compared to controls. In postnatal treatment group, female mice were given s.c. injections of BPA (15 or 150 microg/pup) or DES (0.3 or 3 microg/pup) for 5 days from the day of birth, then some mice were ovariectomized at 30 days and examined at 40 and 90 days. In the remaining mice, vaginal smears were examined from 61 to 90 days and ovarian histology was evaluated at 90 days. Mice exposed postnatally to 150 microg BPA exhibited ovary-independent vaginal epithelial stratification. Postnatal DES (0.3 and 3 microg) treatment also induced ovary-independent vaginal stratification. Polyovular follicles having more than one oocyte in a follicle were induced by postnatal injections of BPA (150 microg) or DES (0.3 or 3 microg) at 30 days. These findings indicate for the first time that a large dose of BPA can induce ovary-independent vaginal epithelial changes when given postnatally but not prenatally.

Developmental effects of estrogenic agents on mice, fish, and frogs: a mini-review.

Laboratory experiments have demonstrated that exposure of rodents to sex hormones during prenatal or early postnatal life can cause permanent and irreversible alterations of the endocrine and reproductive organs, such as ovary, fallopian tube, uterus, cervix, vagina, and mammary gland in females; and testis, epididymis, prostate, and seminal vesicle in males; as well as non reproductive organs including bones and muscle and immune and nervous systems in both sexes. Early development of Xenopus laevis into the tadpole and Fundulus heteroclitus goes through a rapid cell division, gastrulation, neurulation, and organogenesis within 1 week after fertilization. The developing embryo is very fragile and sensitive to estrogenic agents. Thus, these animals can be used as a suitable model for examining the effect of endocrine disruptors (hormonally active agents) on the development of aquatic living beings, which are most likely to be exposed to the compounds.

Roles of Disulfide Linkage and Calcium Ion-Mediated Interactions in Assembly and Disassembly of Virus-Like Particles Composed of Simian Virus 40 VP1 Capsid Protein

ABSTRACT The simian virus 40 capsid is composed of 72 pentamers of VP1 protein. Although the capsid is known to dissociate to pentamers in vitro following simultaneous treatment with reducing and chelating agents, the functional roles of disulfide linkage and calcium ion-mediated interactions are not clear. To elucidate the roles of these interactions, we introduced amino acid substitutions in VP1 at cysteine residues and at residues involved in calcium binding. We expressed the mutant proteins in a baculovirus system and analyzed both their assembly into virus-like particles (VLPs) in insect cells and the disassembly of those VLPs in vitro. We found that disulfide linkages at both Cys-9 and Cys-104 conferred resistance to proteinase K digestion on VLPs, although neither linkage was essential for the formation of VLPs in insect cells. In particular, reduction of the disulfide linkage at Cys-9 was found to be critical for VLP dissociation to VP1 pentamers in the absence of calcium ions, indicating that disulfide linkage at Cys-9 prevents VLP dissociation, probably by increasing the stability of calcium ion binding. We found that amino acid substitutions at carboxy-terminal calcium ion binding sites (Glu-329, Glu-330, and Asp-345) resulted in the frequent formation of unusual tubular particles as well as VLPs in insect cells, indicating that these residues affect the accuracy of capsid assembly. In addition, unexpectedly, amino acid substitutions at any of the calcium ion binding sites tested, especially at Glu-157, resulted in increased stability of VLPs in the absence of calcium ions in vitro. These results suggest that appropriate affinities of calcium ion binding are responsible for both assembly and disassembly of the capsid.

Developmental toxicity of estrogenic chemicals on rodents and other species

ABSTRACTu2002 Antenatal sex‐hormone exposure induces lesions in mouse reproductive organs, which are similar to those in humans exposed in utero to a synthetic estrogen, diethylstilbestrol. The developing organisms including rodents, fish and amphibians are particularly sensitive to exposure to estrogenic chemicals during a critical window. Exposure to estrogens during the critical period induces long‐term changes in reproductive as well as non‐reproductive organs, including persistent molecular alterations. The antenatal mouse model can be utilized as an indicator of possible long‐term consequences of exposure to exogenous estrogenic compounds including possible environmental endocrine disrupters. Many chemicals released into the environment potentially disrupt the endocrine system in wildlife and humans, some of which exhibit estrogenic activity by binding to the estrogen receptors. Estrogen responsive genes, therefore, need to be identified to understand the molecular basis of estrogenic actions. In order to understand molecular mechanisms of estrogenic chemicals on developing organisms, we are identifying estrogen responsive genes using cDNA microarray, quantitative RT‐PCR, and differential display methods, and genes related to the estrogen‐independent vaginal changes in mice induced by estrogens during the critical window. In this review, discussion of our own findings related to endocrine distuptor issue will be provided.

A secure code for recipient watermarking against conspiracy attacks by all users

To protect copyrights of data from illegal copying, a scheme called recipient watermarking which can specify dishonest users who got the data legally and copied it illegally has been studied. A recipient watermarking consists of two processes, called coding process and embedding process. Even though there exists a secure embedding process on which it is impossible to erase the codeword by conspiracy of users, there are some cases that they can make the data by which the server cannot specify even one of them as a illegal user because of the defect of the coding process of the information about users.

A Tracing Algorithm for Short 2-Secure Probabilistic Fingerprinting Codes Strongly Protecting Innocent Users

We give a tracing algorithm for 2-secure probabilis- tic fingerprinting codes with the property that it never accuses innocent users when there are up to 2 attackers. Moreover, by using our code and tracing algorithm, innocent users are also unlikely to be accused even if either the number of attackers or attackers abilities exceed our assumption. Our code is the first example of collusion-secure fingerprinting codes with both of these two properties. Furthermore, our code has shorter length among the preceding 2-secure codes, and possesses further properties desirable in a practical use.

A group testing based deterministic tracing algorithm for a short random fingerprint code

Digital watermarking techniques have been used for preventing an illegal copying and re-distributing of contents. A collusion attack is a strong attack against digital watermarking schemes. To prevent such attacks, a collusion secure fingerprint code can be used. Collusion secure fingerprint code have been studied widely and many codes have been proposed. However, a code length of them are too long to embed it into a content using digital watermarking.

A cellular automata based HB # -like low complexity authentication technique

This paper addresses the problem of developing low-complexity authentication challenge-response protocols based on employment of pseudorandom sequences generated by cellular automata (CA). The proposed protocol is an improved variant of the recently reported HB# protocol. Oppositely from the reported HB-protocols the proposed one is based in the following: (i) The response vector consists of the effective bits and dummy bits embedded in a pseudorandom manner: The effective bits are corrupted parity-check bits, and the dummy ones are pure random bits; (ii) The states of secret key controlled CA are employed for generation of the effective response bits and for the pseudorandom embedding of the effective and the dummy bits. The proposed protocol provides a framework for increasing the security and reducing the implementation complexity and communications overhead in comparison with the previously reported HB# one.

Large-scale gene expression analysis for evaluation of endocrine disruptors

The capacity of numerous chemicals released into the environment to disrupt the development and function of the endocrine system of wildlife and humans is drawing public attention. Early evidence that estrogenic chemicals could pose a threat to human health during development came from studies of diethylstilbestrol (DES) used to prevent premature birth and spontaneous abortion. Laboratory experiments have demonstrated that exposure of animals to sex-hormones during perinatal life cause permanent and irreversible alterations to the endocrine and reproductive systems as well as the immune system, nervous system, bone, muscle and liver in both sexes.