Biomedical subjects

Kanji Yamasaki

Publications and source records attributed to Kanji Yamasaki.

30 records · Page 2Linked to original sources

PubMed2002

Changes of serum alpha 2u-globulin in the subacute oral toxicity study of ethynyl estradiol and bisphenol A based on the draft protocol for the 'Enhanced OECD Test Guideline No. 407'.

To investigate the usefulness of serum alpha 2u-globulin changes as a new parameter for detecting endocrine-mediated effects, we performed a 28-day repeated-dose toxicity study using the administration of bisphenol A (BPA) or ethynyl estradiol (EE) in male rats, based on the draft protocol of the 'Enhanced OECD Test Guideline 407 (enhanced TG 407)'. BPA at doses of 0, 40, 200 and 1000 mg/kg per day or EE at doses of 0, 15, 75 and 375 microg/kg per day were orally administered to SD rats. The highest dose of BPA was reduced to 600 mg/kg per day from the second week of the study onwards because a male rat given 1000 mg/kg per day of BPA died within the first week, showing toxic clinical signs. In the assay using BPA, a reduction in the level of alpha 2u-globulin was detected in the group receiving a dose of 600 mg/kg per day. Reductions in the absolute and relative ventral prostate weights were only observed in the 600 mg/kg per day group. In the assay using EE, the alpha 2u-globulin level decreased significantly in the 375 microg/kg per day group. A reduction in the absolute and relative dorsolateral prostate weights was also observed in the 75 and 375 microg/kg per day groups, morphologically abnormal sperm were observed in the 375 microg/kg per day group. Furthermore, atrophic changes in the prostate and seminal vesicle and degenerative changes in the testis were observed in the 375 microg/kg per day group. Although the alpha 2u-globulin level was reduced in this assay using BPA and EE, further studies are necessary before this assay becomes a useful method for detecting endocrine-mediated effects.

Administration, Oral↗

PubMed2002

Comparison of toxicity studies based on the draft protocol for the 'Enhanced OECD Test Guideline no. 407' and the research protocol of 'Pubertal Development and Thyroid Function in Immature Male Rats' with 6-n-propyl-2-thiouracil.

Two repeated-dose studies of 6-n-propyl-2-thiouracil (PTU) in male rats based on the research protocol 'Pubertal Development and Thyroid Function in Immature Male Rats' (pubertal assay) proposed by the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) and the draft protocol of the 'Enhanced OECD Test Guideline 407' (enhanced TG 407) were performed to investigate the suitability of both assays as screening methods for the detection of endocrine-mediated effects and to compare their sensitivity for the endocrine-mediated effects. In the pubertal assay, PTU at doses of 0, 0.01, or 1 mg/kg per day was orally administered to male Sprague-Dawley rats for 30 days, starting at 23 days of age. In the enhanced TG 407 the same doses of PTU were orally administered to male Sprague-Dawley rats for 28 days, starting at 7 weeks of age. In the pubertal assay, decreased serum thyroxine (T4) and triiodothyronine (T3), increased thyroid and pituitary weights, hypertrophy of follicular epithelial cells in the thyroid, and increased basophilic cells in the pituitary were detected as endocrine-mediated effects of PTU in the 1 mg/kg group. In the enhanced TG 407, decreased T4 and T3 were detected in both the 0.01 and 1 mg/kg groups, together with increased thyroid-stimulating hormone in the 1 mg/kg group, increased thyroid and pituitary weights in the 1 mg/kg group, and hypertrophy of follicular epithelial cells in the thyroid and increased basophilic cells in the pituitary of the 1 mg/kg group. Thus, among the parameters tested, the thyroid hormone levels, organ weight changes, and the histopathological assessment allowed detection of the endocrine-related effects of PTU in both the pubertal assay and the enhanced TG 407, but the sensitivity of the hormone analysis was higher in the latter.

Animals↗

PubMed2002

Subacute oral toxicity study of ethynylestradiol and bisphenol A, based on the draft protocol for the "Enhanced OECD Test Guideline no. 407".

We performed a 28-day repeated-dose toxicity study of ethynylestradiol (EE) and bisphenol A (BPA) based on the draft protocol of the "Enhanced OECD Test Guideline no. 407", and assessed the sensitivity of a list of parameters for detecting endocrine-related effects of endocrine disruption. Doses of EE at 0, 10, 50 or 200 microg/kg per day, or BPA at 0, 40, 200 or 1000 mg/kg per day were orally administered to Sprague-Dawley rats. The highest dose of BPA was decreased to 600 mg/kg per day from the second week of administration because a male rat given 1000 mg/kg BPA had died within 1 week with toxic clinical signs. In the assay using EE, the decrease of prostate, seminal vesicle and pituitary weights, increase of the testis weight, atrophic changes of the prostate, seminal vesicle and mammary gland, and degenerative changes in the testes were detected in male rats in the 50 and/or 200 microg/kg groups. In females of the 200 microg/kg group, decrease of the ovary weight, increase of the uterine weight, atrophy of the ovary, hypertrophy or squamous metaplasia of the uterine epithelial cells and mucification in the vagina were observed. Furthermore, diestrous, estrous or the unknown stage was prolonged in the 50 and 200 microg/kg groups of rats. Endocrine-mediated effects of EE were not detected in general observations, hematology, serum biochemistry, or hormonal or spermatological examinations. In the assay using BPA, the diestrous stages were prolonged at the highest dose, but changes related to endocrine effects were not detected in other examinations. Thus, among the parameters tested, the weight of endocrine-linked organs and their histopathological assessment and estrous cycle stage allowed the detection of the endocrine-related effect of EE, whereas the estrous cycle stage was only a useful parameter to detect the effect of BPA.

Administration, Oral↗

PubMed2002

The efficacy of endocrine disruptor screening tests in detecting anti-estrogenic effects downstream of receptor-ligand interactions.

Several predictive test methods for endocrine disrupters have been evaluated by international organizations. In this study, we performed a series of predictive tests for endocrine disrupters, i.e. the receptor binding assay, reporter gene assay, and immature rat uterotrophic assay, on all-trans retinoic acid (tRA), which may cause antiestrogenic activity via their receptors, interfere with estrogenic action at estrogen responsive element level, and we examine the efficacy of endocrine disruptor screening tests in detecting anti-estrogenic effects downstream of receptor-ligand interactions. Despite showing complete lack of binding affinity to ER in the receptor binding assay, tRA exhibited clear antagonist activity without any agonist activity in the reporter gene assay. In the in vivo test, tRA was subcutaneously administered to immature Crj:CD (SD) IGS rats at doses of 5 and 25 mg/kg per day for 3 days, beginning at 20 days of age. Additional groups of rats given tRA at the above doses were also subcutaneously injected with ethinyl estradiol (EE) at a dose of 0.6 microg per rat per day. A vehicle control group given olive oil alone and a positive control group given EE alone were also established. Although no uterotrophic activity was detected in any of the rats given only tRA, co-treatment with 5 and 25 mg/kg tRA and EE reduced the EE-induced increases in uterine weight. We confirmed that the ER antagonist activity of tRA may be mediated by transcriptional interference after ER-ligand complex binding to an estrogen responsive element of the gene by the gel mobility shift analysis. These findings suggest the reporter gene assay and uterotrophic assay can detect anti-estrogenic effects downstream of receptor-ligand interactions, but the receptor binding assay can not detect this type of interference. In any case, a screening strategy for endocrine disrupters, especially the primary screening battery for prioritizing the chemicals to be tested in the higher screening stages, should be designed to detect various kinds of chemicals possessing endocrine modulating activity including a retinoid-like endocrine modulator. Accordingly, reporter gene assay or uterotrophic assay should be conducted in the early stage of screening process for endocrine disrupting chemicals, because they can detect antagonist activity caused by both inhibition of receptor-ligand interaction and transcriptional interference. Particularly, the reporter gene assay may be a promising prescreening procedure, because it can be adopted in the high throughput screening process for thousands of chemicals and it requires no use of experimental animals.

Animals↗

PubMed2002

Comparison of reporter gene assay and immature rat uterotrophic assay of twenty-three chemicals.

We performed a reporter gene assay for ERalpha-mediated transcriptional activation and an immature rat uterotrophic assay of 23 chemicals, to study the relationship between these two assays and to examine the usefulness of the reporter gene assay. The chemicals analyzed in the study were as follows: benzophenone, bisphenol A, bisphenol B, bisphenol F, p-cumyl phenol, dibutyl phthalate, dicyclohexylphthalate, dihydrotestosterone, equilin, 17alpha-estradiol, estrone, ethynyl estradiol, genistein, hematoxylin, nonylphenol mixture, 4-n-nonylphenol, norethindrone, norgestrel, octachlorostyrene, 4-n-octylphenol, 4-tert-octylphenol, tributyltin-chloride and zearalenone. To perform the reporter gene assay, HeLa cells were transfected with a rat ERalpha expression construct and an estrogen-regulated luciferase reporter construct. The transcriptional activities of each chemical were tested over concentrations ranging from 10 pM to 10 microM and the EC50, PC50 and PC10 values were calculated. In the immature rat uterotrophic assay, the doses of 21 chemicals, with the exception of dibutyl phthalate and ethynyl estradiol, were 0, 2, 20 and 200 mg/kg; each group consisted of six rats. The doses of dibutyl phthalate and ethynyl estradiol were 0, 40, 200 and 1000 mg/kg per day and 0, 0.2, 2 and 20 microg/kg per day, respectively. In the reporter gene assay, the PC10 values were calculated for 15 chemicals: bisphenol A, bisphenol B, bisphenol F, p-cumyl phenol, dihydrotestosterone, equilin, 17alpha-estradiol, estrone, ethynyl estradiol, genistein, nonylphenol mixture, norethindrone, norgestrel, 4-tert-octylphenol and zearalenone. These chemicals corresponded to the chemicals that tested positive in the uterotrophic assay. The other chemicals were negative in the reporter and uterotrophic assays. Although the EC50 and PC50 values could only be calculated for five and six chemicals, respectively, the PC10 values were shown to be well correlated with the EC50 values by a correlation analysis (R(2)=0.9202). These findings demonstrate that PC10 values are preferable to EC50 and PC50 values for predicting the estrogenic activities of chemicals.

Animals↗

PubMed2002

Uterotrophic and Hershberger assays for n-butylbenzene in rats.

We performed the uterotrophic and Hershberger assays proposed by the OECD to investigate the estrogenic and androgenic effects of n-butylbenzene (nBB). For the uterotrophic assay, nBB was injected subcutaneously at doses of 0, 40, 200, 1000 and 2000 mg/kg to 19-day-old rats for 3 days. In some rats, ethynylestradiol (EE) was also injected subcutaneously at a dose of 0.6 microg/kg after the administration of nBB. There were essentially no differences in the uterine wet or blotted weights between the control and any of the nBB-treated groups, or between the control and any of the nBB plus EE-treated groups. For the Hershberger assay, nBB was administered orally at doses of 0, 200 and 600 mg/kg to 56-day-old castrated rats for 10 days. In some rats, 0.4 mg/kg testosterone propionate (TP) was also administered by subcutaneous injection after the administration of nBB. Doses of 0, 200 and 600 mg/kg nBB were also administered orally to non-castrated rats. The weights of the accessory sex organs of the castrated rats showed no significant differences between the control and any of the nBB-treated groups or between the control and the nBB plus TP-treated groups. No significant differences in the weights of the accessory sex organs of the non-castrated rats were observed between the control and the nBB-treated groups. These findings suggest that nBB does not have any endocrine-disrupting properties on in vivo screening tests.

Administration, Oral↗

PubMed2002

Age-related changes of genital systems in the female Crj:CD (SD) IGS rats during sexual maturation.

The age-related changes of vaginal opening, body weight, the weights of the uterus and ovary, together with histological examination, serum 17beta-estradiol (E2) and progesterone levels were examined in intact female Crj:CD (SD) IGS rats between 21 and 36 days of age to understand the basic biological profile of changes of the female genital system during sexual maturation in the rat for female pubertal assays. With the beginning of the elevation of serum E2 level from 28 days of age, all parameters except body weight started to show drastic change until 31 days of age. The highest incidence of vaginal opening was recorded at 34 days of age. On macroscopic examinations, a number of rats showed uterine imbibition but vaginal opening. Immediately after the confirmation of the vaginal opening, the genital systems of three rats were observed microscopically. Both ovaries already had multiple corpora lutea, and degeneration of endometrial epithelial cells was observed. In conclusion, we obtained essential data on genital tract development of female Crj:CD (SD) IGS rats for in vivo screening assays that will contribute to detect potential endocrine active chemicals. In addition, it is assumed that the first ovulation precedes or occurs simultaneously with vaginal opening.

Age Factors↗

PubMedDate not supplied

Changes in serum alpha2u-globulin levels in castrated male rats treated with testosterone propionate in a Hershberger assay.

The Hershberger assay has been proposed as a candidate screening test method for the detection of androgenic and anti-androgenic chemicals and is being validated presently under the test guideline programme of the Organization for Economic Cooperation and Development (OECD). Rat alpha2u-globulin is male rat-specific protein appearing in their serum and urine, and the protein is known to be induced by androgens. We investigated the usefulness of measuring serum alpha2u-globulin levels as a parameter for the androgenic activity of chemicals tested in the Hershberger assay. The serum alpha2u-globulin level was measured after the administration of testosterone propionate at dosages of 0, 20, 100 or 500 microg kg(-1) day(-1) for ten consecutive days in the castrated male rats. The ventral prostate, balbocavernosus/levator ani muscles, glans penis and Cowper's gland were collected and weighed. Although all the androgen-responsive organ weights were increased significantly at dosages of 100 and 500 microg kg(-1) day(-1), the serum alpha2u-globulin level was increased significantly only at a dosage of 500 microg kg(-1) day(-1). These results show that the serum alpha2u-globulin level may be a useful biomarker for detecting androgenic activity caused by test chemicals, but it is less sensitive than the organ weights of androgen-responsive tissues in the Hershberger assay.

Alpha-Globulins↗

PubMedDate not supplied

Advantage of using CBA/N strain mice in a non-radioisotopic modification of the local lymph node assay.

The murine local lymph node assay (LLNA) is currently recognized as a stand-alone test method for determining the skin sensitizing potential of chemicals. It has been incorporated into the official test guidelines published by some authorities, including the OECD. To avoid the use of radioisotopes, efforts have been made recently to develop non-radioisotopic modifications of the LLNA. A non-radioisotopic modification of the LLNA was developed previously using 5-bromo-2'-deoxyuridine (BrdU) incorporation (non-RI LLNA). However, the non-RI LLNA was found to be somewhat less sensitive than the standard assay. This study reports the advantage of using mice of the CBA/N strain in the non-RI LLNA to improve the sensitivity of this method. The non-RI LLNA was performed using CBA/JN and CBA/N mice exposed to one of four confirmed skin sensitizers, 2,4-dinitrochlorobenzene (DNCB), eugenol (EG), isoeugenol (IEG) or alpha-hexylcinnamic aldehyde (HCA), and to one non-sensitizer, propylene glycol (PG). The EC3 values for DNCB, IEG, EG, HCA and PG were calculated to be 0.1%, 9.6%, 40.6%, 45.5% and >50% in CBA/JN mice and 0.08%, 1.9%, 10.7%, 20.3% and >50% in CBA/N mice, respectively. The EC3 values for DNCB, IEG, EG, HCA and PG in the standard LLNA using CBA/Ca mice and radioisotopes were reported elsewhere as being 0.08%, 1.3%, 13.0%, 8.0% and >50%, respectively. The EC3 values derived from the CBA/N mice in the non-RI LLNA were nearly equivalent to the EC3 values obtained using the standard radioisotopic LLNA with CBA/Ca mice. These data suggest that the use of CBA/N mice may provide a realistic opportunity to develop a version of the LLNA that does not have a requirement for the use of radioisotopes, but which nevertheless has sensitivity approaching, or comparable to, the standard method.

Acrolein↗

PubMedDate not supplied

Assessment of the skin sensitization potency of eugenol and its dimers using a non-radioisotopic modification of the local lymph node assay.

Allergic contact dermatitis is a serious health problem. There is a need to identify and characterize skin sensitization hazards, particularly with respect to relative potency, so that accurate risk assessments can be developed. For these purposes the murine local lymph node assay (LLNA) was developed. Here, we have investigated further a modi fi cation of this assay, non-radioisotopic LLNA, which in place of tritiated thymidine to measure lymph node cell proliferation employs incorporation of 5-bromo-2'-deoxyuridine. Using this method we have examined the skin sensitizing activity of eugenol, a known human contact allergen, and its dimers 2,2'-dihydroxyl-3,3'-dimethoxy-5,5'-diallyl-biphenyl (DHEA) and 4,5'-diallyl-2'-hydroxy-2,3'-dimethoxy phenyl ether (DHEB). Activity in the guinea pig maximization test (GPMT) also measured. On the basis of GPMT assays, eugenol was classified as a mild skin sensitizer, DHEA as a weak skin sensitizer and DHEB as an extreme skin sensitizer. In the non-radioisotopic LLNA all chemicals were found to give positive responses insofar as each was able to provoke a stimulation index (SI) of >or=3 at one or more test concentrations. The relative skin sensitizing potency of these chemicals was evaluated in the non-radioisotopic LLNA by derivation of an ec(3) value (the concentration of chemical required to provoke an SI of 3). The ec(3) values calculated were 25.1% for eugenol, >30% for DHEA and 2.3% for DHEB. Collectively these data suggest that assessments of relative potency deriving from non-radioisotopic LLNA responses correlate well with evaluations based on GPMT results. These investigations provide support for the proposal that the non-radioisotopic LLNA may serve as an effective alternative to the GPMT where there is a need to avoid the use of radioisotopes.

Allergens↗

PubMedDate not supplied

Effect of gonadotropin-releasing hormone antagonist on ovarian and uterine weights in immature female rats.

The immature rat uterotrophic assay has been proposed as a screening test method for detecting estrogenic and antiestrogenic chemicals. Although the immature rat uterotrophic assay is advantageous because the test animals are not traumatized by the ovariectomizing process, the effect of endogenous estrogen on ovarian and uterine weight in the immature animals that are used in immature rat uterotrophic assay has not received much attention. In this study, 19-day-old rats were treated with antide, a gonadotropin-releasing hormone antagonist, antide, to block gonadal production of endogenous estrogen. Uterine and ovarian weights of the antide-treated animals were markedly lower than those of control animals. This finding suggests that endogenous gonadal estrogen may already be acting on the uterus and ovaries in immature rats. Blocking endogenous estrogen with a gonadotropin-releasing hormone antagonist may enhance the sensitivity of the immature rat uterotrophic assay; however, the possibility that this protocol may interfere with the ability of the immature rat uterotrophic assay to detect centrally-mediated effects can not be discounted.

Animals↗

PubMedDate not supplied

Evaluation of an in utero through lactational exposure protocol for detection of estrogenic effects of ethinyl estradiol on the offspring of rats: preliminary trial.

As a preliminary trial of an in utero through lactational exposure protocol, ethinyl estradiol, 0, 0.5, 5, or 50 micro g/kg/day, was administered by gavage to pregnant Crj: CD (SD) IGS BR rats from gestational day (GD) 7 to day 18 after delivery to evaluate the efficacy of this protocol and to estimate optimal endpoints. The dams showed no abnormalities. Cleft phallus was observed in female offspring at 50 micro g/kg. Other than a retardation of body weight gain in both sexes at 50 micro g/kg/day, no other abnormal findings were detected. The fact that cleft phallus was the only change induced suggests that the protocol is applicable to the detection of effects of estrogenic chemicals given to pregnant rats on offspring development and that the morphology of the female external genitalia may be a useful endpoint.

Abnormalities, Drug-Induced↗