Fumio Chatani

Circadian and short-term variabilities in blood pressure and heart rate measured by telemetry in rabbits and rats.

Blood pressure (BP) and heart rate (HR) were measured by telemetry in conscious unrestrained rabbits to clarify the profile of their variabilities. The variabilities were assessed for two periods, 24 h (circadian rhythm) and 1 h (short-term variability), and compared with those in rats. BP and HR in rabbits were lower than those in rats but the circadian rhythms in rabbits showed nocturnal patterns as is the case in rats. In contrast, short-term variabilities in BP in rabbits were considerably larger than those in rats. The short-term variability in BP in rabbits was suppressed by alpha-adrenergic blocking without changes in basal values but not by beta-adrenergic blocking or angiotensin converting enzyme inhibition. These results demonstrate the need to take the unique characteristics of short-term variability in BP in rabbit into consideration when the circadian rhythm is focused on and that the short-term variability in BP in rabbits is caused mainly by activation of alpha-adrenergic receptors.

Advantages of Human Hepatocyte-Derived Transformants Expressing a Series of Human Cytochrome P450 Isoforms for Genotoxicity Examination

Metabolites of chemicals can often be ultimate genotoxic species; thus, in vitro routine testing requires the use of rat liver S9. However, there is a question as to whether this represents an appropriate surrogate for human metabolism. We have previously demonstrated the usefulness of HepG2 transformants expressing major human cytochrome P450 (CYP) isoforms to assess the genotoxicity of metabolites. We further assessed the advantages of these transformants from the following three aspects. First, the sensitivity of these transformants was confirmed with micronucleus (MN) induction by 7,12-dimethylbenz[a]anthracene or ifosfamide in transformants expressing the corresponding CYP1A1 or CYP2B6 and CYP2C9, respectively. Second, by using these transformants, beta-endosulfan, a chemical for which the CYP isoforms contributing to its genotoxicity are unknown, was found to induce MN through the CYP3A4-mediated pathway. This result was confirmed by the facts that the decreased CYP3A4 activity using a inhibitor or short interfering RNA (siRNA) repressed MN induction by beta-endosulfan and that endosulfan sulfate, one of the metabolites produced by CYP3A4, induced MN in the transformants harboring an empty vector. Third, the interaction between phase I and II drug-metabolizing enzymes was demonstrated by MN induction with inhibitors of uridine diphosphate (UDP)-glucuronosyltransferases in tamoxifen-treated transformants harboring the corresponding CYP3A4 or with inhibitors of glutathione S-transferase in safrole-treated transformants harboring the corresponding CYP2D6, whereas neither tamoxifen nor safrole alone induced MN in any transformant. These advantages provide the benefits of newly established transformants for in vitro genotoxicity testing that reflects comprehensive metabolic pathways including not only human CYP isoforms but also the phase II enzymes.

Possible mechanism for the anemia induced by candesartan cilexetil (TCV-116), an angiotensin II receptor antagonist, in rats

Candesartan cilexetil (TCV-116), an angiotensin II receptor antagonist, was administered orally to male F344/Jcl and Crj:CD (SD) rats at 1000 mg kg(-1) day(-1) for 1-28 days, and the possible mechanism for the anemia induced by TCV-116 was investigated. In the TCV-116 group, the erythrocyte count, hematocrit value and hemoglobin concentration were decreased by 7-8% as compared with the values in the control group after dosing for 28 days. The plasma and renal erythropoietin levels, the reticulocyte count in the peripheral blood and the erythroid cell count upon bone marrow examination were decreased on day 7, but there were no accompanying histopathological renal lesions. Renal blood flow was increased, and mean blood pressure was decreased after TCV-116. These results suggest that the primary cause of the anemia induced by TCV-116 treatment is the increase in renal blood flow followed by a decrease in erythropoietin production.

Reduced responsiveness of kisspeptin neurons to estrogenic positive feedback associated with age-related disappearance of LH surge in middle-age female rats

Age-related disappearance of the LH surge is one of major biomarkers of reproductive aging in female rats. Kisspeptin neurons in the hypothalamic anteroventral periventricular nucleus (AVPV) are proposed as the critical regulator of the preovulatory LH surge in response to estrogenic positive feedback. Here we investigated the possible involvement of the AVPV kisspeptin neurons in the disappearance of the LH surge in middle-age rats. Middle-age rats exhibiting persistent estrus (M-PE) did not show an LH surge although neither Kiss1 mRNA nor peptide in the AVPV was differentially expressed when compared to young rats exhibiting normal estrous cycles (YN). M-PE released LH in response to exogenous kisspeptin in a similar dose-dependent manner as YN, suggesting that their GnRH neurons still maintained responsiveness to kisspeptin. To investigate the estrogenic positive feedback effect on kisspeptin neurons in the AVPV, rats were ovariectomized and supplemented with estradiol (OVX+E2). We performed in situ hybridization and immunohistochemistry for Kiss1 mRNA and cFos, respectively, and found that M-PE exhibited a significantly lower percentage of Kiss1 mRNA positive neurons with cFos immunoreactivity, although the total number of kisspeptin neurons was not different from that in cyclic rats. Furthermore, OVX+E2 M-PE did not show the surge-like LH release under high estradiol administration while YN did. Thus our current study suggests that the reduced responsiveness of the AVPV kisspeptin neurons to estrogenic positive feedback presumably results in the decrease in kisspeptin secretion from neurons and eventually causes the age-related disappearance of the LH surge in middle age female rats.

Involvement of p53 function in different magnitude of genotoxic and cytotoxic responses in in vitro micronucleus assays

In in vitro micronucleus (MN) assays the sensitivity to MN induction or cytotoxicity can vary depending on the kind of cells employed. This study was conducted to examine the involvement of the p53 function in the different sensitivities between Chinese hamster lung (CHL) cells and human lymphoblastoid TK6 cells in MN assays. MN induction and cytotoxicity were compared using MN-inducing chemicals reported as DNA reactive clastogens, non-DNA reactive clastogens or aneugens. The study revealed that the maximum levels of MN induction in p53-compromised CHL cells were higher than those in p53-competent TK6 cells, but MN were significantly induced in TK6 cells at lower concentrations than in CHL cells. Most of the test chemicals produced a more severe cytotoxicity in TK6 cells, suggesting TK6 cells are more sensitive for cytotoxicity than CHL cells. An additional experiment with 9 MN inducers revealed that the magnitude of MN induction and cytotoxicity were comparable between p53-competent TK6 cells and its p53-null mutant NH32 cells at the same concentrations. Furthermore, the MN frequencies induced by methylmethane sulfonate, aphidicolin and hydroxyurea in NH32 cells were identical to those in TK6 cells at different recovery times. From these results, it is suggested that the p53 abrogation does not explain the difference in sensitivity to MN induction or cytotoxicity between CHL and TK6 cells. In this regard, p53 abrogated NH32 cells can be an option for the in vitro MN assay.

Characterization of Hydronephrosis in Neonatal Rats from Dams Receiving Candesartan Cilexetil (TCV-116), an Angiotensin II Type 1 Receptor Antagonist

The characteristics and mechanisms of hydronephrosis in neonatal rats induced by candesartan cilexetil (TCV-116), a potent angiotensin II (AngII) type 1 receptor antagonist, were examined. TCV-116 (300 mg/kg/day) was orally administered to dams for 4 weeks from gestation day 15 through lactation day 21. On lactation days 0, 4, 7, 14, and 22, the kidneys of the pups were examined. Hydronephrosis was observed starting on lactation day 14 accompanied by other histological changes, atrophy of the renal papillary tubules, dilatation of the renal tubules, and basophilic renal tubules in the cortex. These changes could also be observed at 10 weeks of age, 7 weeks after the last dose was administered. These renal structural abnormalities were consistent with that seen in other renin-angiotensin system antagonists. TCV-116 (300 mg/kg/day) was then administered to dams for four separate 1-week periods: gestation days 15 through 21, lactation days 0 to 6, lactation days 7 to 13, and lactation days 14 to 21. Pups were most susceptible to the induction of hydronephrosis when TCV-116 was administered from lactation days 0 to 6 and lactation days 7 to 13. The increased incidence of hydronephrosis and renal histological changes in the pups was prevented by administering mineralocorticoid, deoxycorticosterone acetate (10 mg/kg/day), subcutaneously to the pups from lactation days 7 to 13. Also, plasma aldosterone concentration in the pups was decreased after three daily treatments of TCV-116, accompanied by the increased plasma potassium concentration and urine Na/K ratio and the decreased urine osmolality. Therefore, we considered that the development of hydronephrosis in pups is closely related to the AngII blockade for the first 2 weeks after birth, and the reduction of aldosterone secretion by the inhibition of AngII leads to the disorder of the sodium and potassium homeostasis in neonates, and subsequent increase in urine volume may be involved in the mechanisms of hydronephrosis. We conclude that the hydronephrosis was caused by the sodium imbalance resulted from the pharmacological action of TCV-116 during the neonatal period.

Changes in the plasma erythropoietin level in rats following fasting, ageing, and anaemia

Changes in the plasma erythropoietin (Epo) level in fasted and aged rats, and in male F344 rats following phenylhydrazine-and captopril-induced anaemia were investigated using an enzyme immunoassay. In male F344 rats, the plasma Epo level was decreased upon fasting, and was higher at night than in the day. In both sexes, the level was decreased from 5 to 10 weeks of age and remained almost constant from 10 to 19 weeks of age. The level in male Sprague-Dawley rats was higher than that in male Wistar and F344 rats at 6 and 10 weeks of age. After phenylhydrazine was administered to male F344 rats, anaemia was observed, and the plasma Epo level increased rapidly, followed by a delayed increase in the reticulocyte count. The plasma Epo level then decreased gradually to the initial level 2 weeks after dosing. Upon repeated administration of captopril, a decreased plasma Epo level on day 7 and anaemia on day 28 were observed in male F344 rats. These results suggest that monitoring the plasma Epo level would be useful in investigating the mechanism by which a chemical induces anaemia. It is also necessary to consider the feeding conditions, sampling time, age, sex, and strain of the rats when interpreting the Epo level.

Difference in susceptibility to morphological changes in the nucleus to aneugens between p53-competent and p53-abrogated lymphoblastoid cell lines (TK6 and NH32 cells) in the in vitro micronucleus assay

We previously reported that the proportion of large-size micronuclei (MN) can be a reliable parameter to discriminate aneugens from clastogens in the in vitro MN assay using Chinese hamster lung cells. The frequencies of polynuclear (PN) and mitotic (M) cells are also supposed to be useful parameters for the same purpose since they are known to be increased by aneugens. In the present study, we investigated whether morphological observations of the cell nucleus can be applied for the in vitro MN assay using the p53-competent human lymphoblastoid cell line, TK6 cells. Our present MN assay with six clastogens and six aneugens revealed that the frequencies of large-size MN or PN cells cannot distinguish aneugens from clastogens, while the frequencies of M cells can distinguish them, suggesting that the M-cell frequency is a recommended parameter to determine a mode of action for MN induction in the in vitro MN assay using TK6 cells. Our further investigation using p53-null mutant NH32 cells showed that the frequencies of large-size MN or PN cells induced by aneugen treatments were higher than those in TK6 cells but not by clastogen treatments. These findings suggest that p53 abrogation promotes the susceptibility for morphological changes in the nucleus to aneugens and that morphological observation of the cell nucleus including size-classifying MN counting could distinguish aneugens from clastogens in the MN assay using NH32 cells.

Role of Angiotensin II in Reflex Tachycardia During Hypotension Caused by a Calcium Channel Blocker

Blood pressure and heart rate were measured by telemetry in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) to investigate the contribution of angiotensin II to the reflex tachycardia resulting from exaggerated hypotension caused by a high dose of a calcium channel blocker. Pre-treatment with TCV-116, an angiotensin II AT1 receptor antagonist, or enalapril partially attenuated the reflex tachycardia induced by manidipine, but TCV-116 had almost no effect on the sinus tachycardia induced by isoproterenol. The suppressive effects of TCV-116 against the reflex tachycardia tended to be more obvious in WKY than in SHR, though the difference was not statistically significant. Concurrent administration of propranolol almost completely inhibited both the reflex tachycardia and the sinus tachycardia in SHR and WKY, indicating that the sympathetic nervous system contributes to both types of tachycardia. We demonstrated that angiotensin II may be involved in the reflex tachycardia induced by calcium channel blockers probably via activation of some component of the sympathetic nervous system other than postsynaptic factors at the sinus node.

Restraint‐induced maternal stress and alteration of ossification in mouse fetuses

Jcl:ICR pregnant mice were immobilized for 120 minutes from days 8–12 of gestation, and their fetuses were examined for skeletal features on day 18 of gestation. In the stressed group, decreased maternal bodyweight gain and lower fetal weight were noted. In this group, the incidences of segmentation defects, fused ribs, absent lumbar vertebrae and full supernumerary ribs were increased in fetuses. In addition, fusion of the basi‐ and ex‐occipital bones was frequently observed in this group (12.9%). This finding was seen at an incidence of 1.4% in the control group, usually in newborns during the ossification process of the occipital bone. Therefore, the fused basi‐ and ex‐occipital bones were considered to be due to altered ossification, but not to be an abnormality. In summary, immobilization of Jcl:ICR mice during the period of fetal organogenesis induced altered ossification of the occipital bones as well as some abnormalities and supernumerary ribs.