The authors previously reported that male offspring of mother rats given alkaline ionized water (AKW) showed a significantly higher body weight by day 14 after birth than did offspring of mother rats given tap water (TPQ) ; furthermore, marked myocardial necrosis and fibrosis were observed particularly in the former male offspring at the age of 15 weeks. In the present experiment we looked for differences in bioparameters, namely the milk yeild of mothers and suckled milk volume of the offspring, between the AKW-and the TPW-treated groups in order to reveal the factors which cause the unusual body weight gain in the offspring. Even though we were able to repeat our previous observation (the body weight of the male offspring of the AKW group increased significantly more by day 14 and 20 after birth and of the female by day 20 after birth than did that of the TPW group (p<0.05), no significant difference was noted in any of the bioparameters, including those related to milk production and consumption. It is thus suspected that the water-hydrated cation, which was transferred either to the fetus through the placenta or to the offspring through the milk, might be the cause of the unusual body weight increase. Since calcium plays an important role in skeletal formation, it is tentatively concluded tht the higher calcium concentration of AKW enriched the mother, serum calcium which was transferred to the fetus through the placenta and to the offspring through the milk.

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In this study, we conducted a simultaneous analysis of sperm count and viability in rats by flow cytometry (FCM). Epididymal fluids were taken from the caudal epididymis of 12 to 13 week-old Sparague-Dawley rats. The fluids were weighed and mixed with Dulbecco's phosphate buffered saline (D-PBS). Propidium iodide, which can stain only dead sperm, was used to distinguish viable and dead sperm. The sperm count and viability analyzed by FCM were 1.28×10⁶/mg and 78.0%, respectively. These values were consistent with the corresponding values (1.39×10⁶/mg and 81.0%) that were directly determined microscopically in the fluids of the same sample. In addition, when the original mixture containing sperm was diluted two times and four times with D-PBS, or was diluted two times with D-PBS containing only killed sperm, the sperm count and viability determined by FCM also correlated well with the sperm count (r=0.96, P<0.01) and sperm motility (r=0.99, P<0.01) by direct microscopic observation, respectively. In conclusion, the present flow cytometric analysis would be practical for the simultaneous determination of sperm count and viability in rat epididymal fluids.

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Methylmercury (MeHg) specifically depolymerizes microtubules and inhibits cell proliferation in mouse glioma cells. The effect of microtubule depolymerization by MeHg on tubulin synthesis was studied. Tubulin synthesis analyzed by two-dimensional electrophoresis using [³⁵S] methionine as a tracer was markedly inhibited in mouse glioma cells exposed to 5×10^-6M MeHg for 3 hr, which completely depolymerized microtubules. Under this condition, density of the protein bands other than tubulin in autoradiogram remained unchanged on gradient urea-polyacrylamide gels. Furthermore, the decrease in tubulin mRNA level was relevant to that in tubulin synthesis, although action mRNA levels remaind unchanged. In addition, specific transcription rates of β-tubulin genes appeared to be unaffected under the same experimental condition as above. Thus, it is concluded tht the disruption of microtubules by MeHg resulted in the inhibition of the synthesis of tubulin itself through autoregulatory repression in post-transcriptional processes as in the case of colchicine treatment.

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Recently, we demonstrated that nitric oxide (NO) reduces ATP generation via oxidative phosphorylation coupled with the mitochondrial respiratory chain in PC12 cells resulting in induction of apoptotic cell death. To further study the correlation between NO-induced ATP depletion and neuronal death, we examined the effect of NO on glycolytic ATP generation in PC12 cells, a neuronal model. When the oxidative phosphorylation was maximally suppressed by DNP and oligomycin, which are inhibitors of the mitochondrial respiratory chain, the cellular ATP contents were reduced by sodium nitro-prusside (SNP). In addition, the cellular ATP contents were further decreased along with a decrease in the activity of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a key enzyme in the glycolytic pathway. Benzamide, an inhibitor of poly (ADP-ribose) synthetase, could protect the depletion of NAD but had no effedct on the depletion of ATP in PC12 cells induced by NO. These results suggest that the depletion of ATP in PC12 cells caused via the inhibition of G3PDH by NO is one of the mechanisms responsible for NO neurotoxicity.

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"Animal-Plant Warfare" is one of the hypotheses for the evolution of drug-metabolizing P450s. To address the validity of this hypothesis, we examined the induction of xenobiotic-metabolizing P450s by 12 plant toxins in rats, using hepatic activity for testosterone metabolism as the index. The compounds tested were aconitine, morphine, tubocurarine, physostigmine, pilocarpine, muscarine, cocaine, atropine, amygdalin, digitonin, nicotine and solanin. Drinking water containing a test compound was given to rats for 4 days, and the hepatic activity of testosterone metabolism was determined together with monitoring body weight gain and liver weight as the indices of toxicity. The results showed that while cocaine and nicotine have a minor ability to increase testosterone 16β-hydroxylase activity, a marker activity for the CYP2B1 and 2, all other compounds did not have any such effect. No correlation was observed between a change in 16β-hydroxylase and toxicity caused by toxins. Therefore, these results did not support the idea that the inducibility of the CYP2B subfamily in animals is acquired through "Animal-Plant Warfare". Several compounds examined here increased or decreased hepatic activities of testosterone 2α-, 6β-, 7α- and 16α-hydroxylation and 17-oxidation, indicating a possible effect on the CYP2A, 2C and 3A subfamily. Of these effects, a moderate correlation (r<0.49) was observed in the changes in the activities of 2α-/16α-hydroxylation and 17-oxidation vs. that in toxicity. It is therefore suggested that inhibition or suppression of the expression of CYP2C11 is one of the mechanisms in the toxicity of plant toxins for rats, although it comes from an examination using limited numbers of compounds.

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The effect of a 14-day administration of FK-506 at 1 or 3 mg/kg on plasma lipids and myocardial energy and glucose metabolism in rats was investigated. FK-506 increased the level of blood glucose in rats when given daily at an oral dosage of 3 mg/kg for 14 days. The plasma level of total cholesterol was significantly increased by FK-506 at either 1 or 3 mg/kg, whereas that of triglyceride was not changed. There was no significant difference in energy charge potential and the lactate/pyruvate ratio of the myocardial tissue between FK-506-and solvent-treated groups. Because FK-506 administration for 14 consecutive days did not disturb myocardial energy and glucose metabolism, its cardiotoxic side effects were not recognized in the present experimental toxicological evaluation of rats.

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We have reported that a marked necrosis and subsequent fibrosis of myocardium occurred among male rats 15 weeks old given alkaline ionized water (AKW) during gestation and suckling periods, and after wearing. In this sstudy, it was examined whether similar lesions would occur in mother rats which were given AKW from day zero of gestation to day 20 of lactation. The myocardial lesion in the mother rats given AKW showed cell infiltration, vacuolation and fibrosis in the papillary muscle of the left ventricle, as were observed in male rats of 15 weeks old. Myocardial degeneration may cause a leakage of potassium into the blood that results in a higher concentration of potassium in the blood in the test group than in that of the control group given tap water.

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The effect of spine venom from the crown-of-thorns starfish (Acanthaster planci) on drug-metabolizing enxymes in rat liver was studied. The spine venom was prepared by saturation of spine homogenate with ammonium sulfate and the protein fracton precipitation 50% saturation was used as venom B. Venom A was the protein precipitated between 50 and 100% saturation. When venom B (100-200 mg/kg) was given to rats, liver microsomal GSH S-tramsferase and cytochrome P450 activities decreased while cytosolic GSH S-trasferase activity was not changed. The decrease in these microsomal enzyme activities was seen from 12 hr to 24 hr after giving 100 mg/kg of venom B. Rats give venom A died, suggesting an involvement of the lethal factor in venom A. The data showed that the spine venom B from A. planci depressed microsomal GSH S-transferse and cytochrome P450 activities in rat liver and that this venom was distinct from the lethal factor of the spine venom.

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In the present study, the correlation between mouse PLNA and those obtained from GP-PCA and from GP-ASA reactions fro sodium 2, 4, 6-trinitrobenzenesulfonate dihydrate (TNBS), penicillin G and cephalothin was investigated. Next, variou drugs were tested using the mouse PLNA to study whether PLN reactivity could be related to the potential of the compounds to induce allergic and autoimmune disorders in humans. The parameter of the PLNA was determined by the PLN cellularity index in BALB/c and A/J mice treated with a single subcutaneous injection of compounds. Hartley guinea pigs were immunized subcutaneously with the compounds without adjuvant, and then the GP-PCA and GP-ASA reactions were assessed. The examinations using mice and guinea pigs showed that mouse PLN responses to TNBS, penicillin G and cephalothin correlated with the allergenicity responses obtained in the GP-PCA reaction to three compounds. In the mouse PLNA, ten drugs considered to be well-known inducers of allergic side-effects in humans (i.e., penicillin G, cephalothin, ampicillin, carbenicillin, cefazolin, cefotaxime, streptomycin, 4-aminoantipyrine, chlorhexidine and sulfamethoxazole) caused increases in PLN cellularity indices as well. These results indicate that the PLNA may be useful as a short-term and simple test system for detecting low-molecular drugs exhibiting allergenicity potential.

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It has been established that progression of renal lesions in 5/6 nephrectomized rats, in which the total right kidney was removed and two poles of the left kidney were excised surgically, and are used as an animal model of renal failure in man, can be morphologically divided into three stages. In the present study, for establishing a renal toxicity study using this animal model on the physiological side, changes of biochemical parameters were sequentially investigated in 20 male 5/6 nephrectomized Wistar rats until 26 weeks after nephrectomy. Creatinine clearance (CLcre) and water as well as electrolyte reabsorption (FR water and FR Na, K, Cl) were reduced at weeks 2-4, then increased slightly from weeks 6 to 10, and reduced again thereafter. On the countrary, urinary protein was elevated throughout the experimental period, while albumin fraction was incereased after week 2 and low-molecular tubular protein increased after week 6 by electrophoresis. Urinary LDH also demonstrated high levels throughout the observation period, but ALP only increased after week 18. The present study thus confirmed that renal functon after 5/6 nephrectomy is indeed changed in three stages with clinical biochemical parameters, especially CLcre and FR Na, K, Cl being good indicators to distinguish the three stages of glomerular and tubular dysfunction, respectively. In addition, urinary protein-fractions by electrophoresis in this animal model were examined for the first time, proving useful approach to glomerular and tubular dysfunction.

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統計パッケージの普及に伴い, 毒性試験で用いられる統計手法も高度化しているが, わが国では, 生物統計学の専門家が不足しているため, 毒性試験の現場では統計学の利用について混乱が起きている。本稿では, 毒性試験において統計学を誤用しないために注意すべき点, 特によく用いられる検定と多重比較の意義, 統計パッケージと決定樹の利用の際の問題点, 論文における統計手法と結果の記載の注意点を総論的に述べる。

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内分泌攪乱化学物質の特徴について, その生体内作用, 障害性の考え方, それらに対する生物作用検出のための試験法の考え方, など, 主として生物学的側面からあらたな問題点を紹介し討論を加えた。それらをめぐっての, OECDやEPAなどの対応, 本邦における試験研究体制の動向などについても若干の解説をした。

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