Monitoring of glutamate-induced excitotoxicity by mitochondrial oxygen consumption.

Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na+ influx increases intracellular Ca2+ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na+ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na+ /K+ -ATPase exchanges intracellular Na+ with extracellular K+ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate-mediated excitotoxicity. Under a pyruvate-restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 µM. The OCR monitoring also revealed the contribution of the N-methyl-D-aspartate receptor and Na+ /K+ -ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte-dependent protection of neurons from glutamate-mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.

A mutant HCN4 channel in a family with bradycardia, left bundle branch block, and left ventricular noncompaction.

We found that a female infant presenting with left bundle branch block and left ventricular noncompaction carries uninvestigated gene mutations HCN4(G811E), SCN5A(L1988R), DMD(S2384Y), and EMD(R203H). Here, we explored the possible pathogenicity of HCN4(G811E), which results in a G811E substitution in hyperpolarization-activated cyclic nucleotide-gated channel 4, the main subunit of the cardiac pacemaker channel. Voltage-clamp measurements in a heterologous expression system of HEK293T cells showed that HCN4(G811E) slightly reduced whole-cell HCN4 channel conductance, whereas it did not affect the gating kinetics, unitary conductance, or cAMP-dependent modulation of voltage-dependence. Immunocytochemistry and immunoblot analysis showed that the G811E mutation did not impair the membrane trafficking of the channel subunit in the heterologous expression system. These findings indicate that HCN4(G811E) may not be a monogenic factor to cause the cardiac disorders.

Histopathology of a wavy medaka.

Wavy medakas are medakas that exhibit spinal curvature characterized by dorsoventrally curved vertebrae. We found a spontaneous wavy medaka in our experimental stock and subjected it to a histopathological examination. Macroscopically, the wavy medaka's spine formed an M shape, and its vertebrae displayed a dorsoventral curvature that started at the third vertebral bone. Microscopically, the vertebral cavities were filled with fibrous tissue, which was similar to that seen in the central parts of the intervertebral discs of a normal medaka. The vertebral joints were composed of vacuolated notochord cells without intervertebral disc formation. These changes were also observed in the caudal region, which exhibited less curvature. In the normal medaka, the intervertebral discs form via the regression of the notochord that plays a key role in the development of vertebrae and disc formation. We concluded that notochordal subinvolution had induced intervertebral disc dysplasia, leading to lordokyphosis, in the wavy medaka.

Pancreatic Ductal Adenocarcinoma in a Wistar Hannover GALAS Rat.

There are no reported spontaneous cases of pancreatic ductal adenocarcinoma (PDAC), and there are few reports about chemically-induced PDAC in rats. We encountered a PDAC in a Wistar Hannover GALAS rat that had been subjected to a medium-term multiorgan carcinogenicity bioassay. This article describes the histological and histochemical findings of the tumor. The tumor was located in the pancreatic tissue and had not invaded the liver parenchyma or the mucosal layer of the alimentary tract. The tumor cells were atypical and were mainly arranged in small tubules. In addition, abundant stroma and mucus production were observed in the tumor. In an immunohistochemical examination, the tumor cells were positive for cytokeratin, Sox9 and pancreas duodenum homeobox 1 and negative for amylase 2A and insulin. Therefore, the tumor was diagnosed as a PDAC based on its histological and histochemical findings. We considered that the tumor was caused by the carcinogens administered during the abovementioned bioassay.

Background data on developmental parameters during the gestation period in rats.

Background data during the gestation period were obtained from 128 Wistar Hannover GALAS rats and 26 Crl:CD(SD) pregnant rats in the control groups of our previous toxicity studies. The body weights of dams in the Wistar Hannover GALAS rats were significantly lower throughout the gestation period than those in the Crl:CD(SD) rats. In contrast, the time-dependent change in the body weight gain (%) of dams showed very similar trends in both strains. The mean number of live embryos/fetuses in the Wistar Hannover GALAS rats was 12.0, and was lower than that (14.5) in the Crl:CD(SD) rats. The placental weights gradually increased with pregnancy progression and reached a plateau on gestation day (GD) 19, although the embryo/fetal weights rapidly increased from GD 17 to GD 21. The embryo/fetal weights in the Wistar Hannover GALAS rats were significantly lower on only GD 21 than those in the Crl:CD(SD) rats. It is considered that this fetal weight difference between the strains develops during the fetal period, but not during the organogenesis period. In contrast, there were no differences in the placental weights between the two strains. Microscopically, the thickness of the labyrinth zone in the Wistar Hannover GALAS rats was thicker throughout the gestation period than that in the Crl:CD(SD) rats.

G Protein-Coupled Glutamate and GABA Receptors Form Complexes and Mutually Modulate Their Signals.

Molecular networks containing various proteins mediate many types of cellular processes. Elucidation of how the proteins interact will improve our understanding of the molecular integration and physiological and pharmacological propensities of the network. One of the most complicated and unexplained interactions between proteins is the inter-G protein-coupled receptor (GPCR) interaction. Recently, many studies have suggested that an interaction between neurotransmitter GPCRs may mediate diverse modalities of neural responses. The B-type gamma-aminobutyric acid (GABA) receptor (GBR) and type-1 metabotropic glutamate receptor (mGluR1) are GPCRs for GABA and glutamate, respectively, and each plays distinct roles in controlling neurotransmission. We have previously reported the possibility of their functional interaction in central neurons. Here, we examined the interaction of these GPCRs using stable cell lines and rat cerebella. Cell-surface imaging and coimmunoprecipitation analysis revealed that these GPCRs interact on the cell surface. Furthermore, fluorometry revealed that these GPCRs mutually modulate signal transduction. These findings provide solid evidence that mGluR1 and GBR have intrinsic abilities to form complexes and to mutually modulate signaling. These findings indicate that synaptic plasticity relies on a network of proteins far more complex than previously assumed.

Possible carcinogenic risks of copper gluconate and their prevention by co-administered green tea catechins evaluated by a rat medium-term multi-organ carcinogenicity bioassay protocol.

Carcinogenic risks of copper gluconate, green tea catechins and their combined exposure were evaluated using a rat medium-term multi-organ carcinogenicity bioassay protocol. Male BrlHan:WIST@Jcl (GALAS) rats were given N-nitrosodiethylamine (DEN), N-methylnitrosourea (MNU), 1,2-dimethylhydrazine (DMH), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN) for a total multiple initiation period of 4 weeks (DMBDD treatment). Rats were then given a diet containing copper gluconate at a concentration of 0, 10, 300, 3000 or 6000 ppm with or without a co-administration of catechins starting 1 week later by admixing in the drinking water at a concentration of 5000 ppm. All survivors were sacrificed at the end of week 29. Number of putatively preneoplastic, glutathione S-transferase placental form-positive, liver lesions significantly increased by copper gluconate of 300 ppm or greater. In addition, both incidence and grade of hyperplasia in the forestomach significantly increased by copper gluconate of 6000 ppm. Catechins, exerting no effects by themselves, inhibited these effects of copper gluconate. The present results indicate that copper gluconate may possess carcinogenic risks for the liver and forestomach at its high dose level, and that co-administered green tea catechins may exert preventive effects.

Effect of 6-mercaptopurine on rat placenta.

In order to investigate the toxic effects of 6-mercaptopurine (6-MP) on placental development, we examined sequential morphology in the placentas from rats exposed to 6-MP. 6-MP was intraperitoneally administered at 60 mg/kg during gestation days (GDs) 11 to 12, and the placentas were sampled on GD 13, 15 or 21. In the 6-MP-treated group, maternal body weight suppression, increased death embryo/fetus ratio and some malformations were observed. The placenta weights were decreased on GDs 15 and 21. Macroscopically, placentas on GD 21 were small, brittle and thin with a white peripheral rim. Histopathologically, in the labyrinth zone, 6-MP treatment mainly evoked decreased mitosis on GDs 13 and 15, increased apoptotic cell on GDs 13, 15 and 21 and thinning on GDs 15 and 21. In the basal zone, 6-MP evoked decreased mitosis on GDs 13, and PAS-positive material in the spongiotrophoblasts was still detected on GD 15. Thickening of the basal zone was observed with cytolysis of glycogen cells, apoptosis and an increased number of composed cells on GD 21. In conclusion, 6-MP administration in pregnant rats induced growth arrest of the labyrinth zone and developmental delay in the basal zone, leading to small placentas. The fetotoxicity of 6-MP may be responsible for its direct anti-proliferative effects and resulting placental dysfunction.

Histopathological effect of ketoconazole on rat placenta.

In order to investigate the morphological effects of ketoconazole on hypertrophied placentas, we examined the sequential histopathological changes in the placenta from rats exposed to ketoconazole. Ketoconazole was administered orally at 0 and 25 mg/kg/day during gestation days (GDs) 12 to 14, and the placentas were sampled on GDs 15, 17 and 21. All dams showed neither effect on body weight nor any abnormal clinical signs during the experimental period. In the treated group, the placentas appeared more hypertrophic with increases in the weight, diameter and thickness on GD 21. Histopathologically, increased thickness was noted in the labyrinth zone and basal zone on GDs 17 and 21, while on GD 15 the change had been already evident in the former zone. In the labyrinth zone, the mitotic figures of the trophoblasts were significantly elevated on GD 15. A multiple cystic dilatation of maternal sinusoids was observed in some placentas on GDs 15, 17 and 21. In the basal zone, an increase in spongiotrophoblasts and clusters of glycogen cells were detected on GDs 17 and 21. In the decidua basalis, there were no significant changes in either histology or thickness between the control and treated group during GDs 15 to 21. In conclusion, ketoconazole increased the population of composed cells in the labyrinth and basal zone, leading to placental hypertrophy in pregnant rats.

Lack of modulatory effect of the SCN5A R1193Q polymorphism on cardiac fast Na+ current at body temperature.

SCN5A encodes the main subunit of the NaV1.5 channel, which mediates the fast Na+ current responsible for generating cardiac action potentials. The single nucleotide polymorphism SCN5A(R1193Q), which results in an amino acid replacement in the subunit, is common in East Asia. SCN5A(R1193Q) is often identified in patients with type 3 long QT syndrome and Brugada syndrome. However, its linkage to arrhythmic disorders is under debate. Previous electrophysiological studies performed at room temperature inconsistently reported the gain- or loss-of-function effect of SCN5A(R1193Q) on the NaV1.5 channel. More recently, it was theoretically predicted that SCN5A(R1193Q) would exert a loss-of-function effect at body temperature. Here, we experimentally assessed whether SCN5A(R1193Q) modulates the NaV1.5 channel at various temperatures including normal and febrile body temperatures. We compared voltage-gated Na+ currents in SCN5A(R1193Q)-transfected and wild-type SCN5A-transfected HEK293T cells using a whole-cell voltage-clamp technique. First, we made comparisons at constant temperatures of 25°C, 36.5°C, and 38°C, and found no difference in the conductance density, voltage dependence of gating, or time dependence of gating. This suggested that SCN5A(R1193Q) does not modulate the NaV1.5 channel regardless of temperature. Second, we made comparisons while varying the temperature from 38°C to 26°C in 3 min, and again observed no difference in the time course of the amplitude or time dependence of gating during the temperature change. This also indicated that SCN5A(R1193Q) does not modulate the NaV1.5 channel in response to an acute body temperature change. Therefore, SCN5A(R1193Q) may not be a monogenic factor that triggers arrhythmic disorders.

Comparative study to elucidate the mechanism underlying the difference in airway hyperresponsiveness between two mouse strains.

The mechanism underlying airway hyperresponsiveness (AHR), a characteristic feature of asthma, remains obscure. We attempted to elucidate the mechanism responsible for the different degrees of AHR in two mouse strains, BALB/c and C57BL/6, following exposure to an anaphylactic trigger. When ovalbumin (OVA)-sensitized mice were challenged daily with OVA for up to three consecutive days, the BALB/c mice showed a higher degree of airway responsiveness to methacholine than did C57BL/6. Following the OVA challenge, eosinophils and macrophages in bronchoalveolar lavage fluid (BALF) from BALB/c increased significantly in number compared to those from C57BL/6. BALB/c mice also exhibited a higher serum IgE level than that of C57BL/6 after OVA challenge. The enhanced AHR and eosinophilic infiltration in BALF were significantly reduced by pretreatment with a selective cysteinyl-leukotriene type 1 receptor (cysLT(1)R) antagonist, montelukast. In the in vitro study, cysLT production was significantly lower in the dissected lung tissue from BALB/c than in tissue from C57BL/6 when both groups were stimulated with saline. The lungs from BALB/c generated significantly larger amounts of cysLTs on incubation with OVA rather than with saline, while the lungs from C57BL/6 did not show any significant increase in cysLTs with antigen stimulation. Significant upregulation of cysLT(1)R and cysLT(2)R mRNA expression was induced by OVA challenge in the lungs of BALB/c, but not in those of C57BL/6. It is suggested that, after an anaphylactic reaction, the degree of AHR is dependent on the genetic background and that cysLTs play an important role in the mechanism involved.

Indole-3-acetic acid induces microencephaly in mouse fetuses.

To determine the effect of indole-3-acetic acid (IAA), known as natural auxin, on developing fetus, pregnant mice were injected with 500 or 1000 mg/kg on various gestation days (Days). With the repeated treatment during Days 7-15, the fetal brains exhibited a reduction in size and weight in a dose-dependent manner on Day 18. Histopathologically, hypoplasia of the cortical plate, piriform cortex, hippocampus and thalamus were observed. From the single treatment on 1 day during Days 9-14, the sensitive period of IAA-induced microencephaly was found to be during Days 10-13 and the most significant response in the fetuses was seen on Day 11 or 12. With the repeated treatment during Days 11-13, apoptotic cells mainly increased in the medial and dorsal layer of the neuroepithelium and prepalate with a reduction in cell density in the telencephalon, diencephalon, mesencephalon and metencephalon on Day 12.5. p53-positve cells were detected associated with apoptotic cells in neuroepithelium. Therefore, IAA administration to pregnant mice induces apoptosis mediated by p53 in the embryo's neuroepithelium, decreases formation of neurons and leads to microencephaly in the fetuses.

Busulfan-induced apoptosis in rat placenta.

In order to investigate the effects of busulfan on the placenta, we examined the sequential histopathological changes in the placenta from rats exposed to busulfan during gestation days (Days) 12-14. Busulfan was intraperitoneally administered at 10 mg/kg on Days 12, 13 and 14, and the placentas were sampled on Day 13.5, 14.5, 15, 16 or 21. Macroscopically, small placenta was seen on Day 21 with scattered white spots and white peripheral rim. Histopathologically, in the treated group, there were increased apoptosis and decreased mitotic activities in the trophoblasts of the labyrinth zone on Days 13.5, 14.5, 15 and 16. In the basal zone, slightly increased apoptosis was seen on Day 13.5 and slightly decreased mitotic activity on Day 14.5. On Day 21, the labyrinth zone in the treated group was reduced in diameter. Degeneration and necrosis of trophoblasts, a diminution in thickness of the trophoblastic septa with a deposition of calcium and an irregular dilation of the maternal blood space were scattered in the labyrinth zone, although there were no conspicuous changes in the basal zone. The anti-proliferative effects of busulfan could have inhibited the development of the labyrinth zone, and led to small placentas. The fetotoxicity and teratogenicity of busulfan might be also responsible for these placental changes.

[Study for an allergic inflammation model using human lungs and its pharmacological application].

The complement system, which plays an important role in inmate immunity, is considered to be important in the pathophysiology of allergic asthma. A patient with allergic asthma shows the reversible characteristic system of bronchoconstriction, increased mucus secretion, and complicated airway inflammation. Various cytokines secreted from Th2 cells contribute to the system. Cysteinyl-leukotrienes (CysLTs) are also considered to be one of the important mediators involved in asthmatic pathophysiology. However, the effects of a drug on humans may not be the same as those on animals due to species differences in complement-related molecules. In this series of experiments, we tried to establish a model in which the effects of a drug on the production of CysLTs from human lung preparations were evaluated following an anaphylactic reaction. CysLT production increased when the passively sensitized lung tissues were stimulated with anti-IgE antibody. The coaddition of anaphylatoxin, C5a, with the anti-IgE antibody potentiated CysLT production. The response to C3a was weaker when compared with that to C5a. In addition, increased production of CysLTs by adding serum at a specific ratio was dose dependently inhibited by nonpeptide C5a receptor antagonist, W-54011, or a novel complementary peptide inhibitor of C5a, acetyl peptide A. From these results, it is suggested that C5a potentiates cysLT production from human lung tissues and contributes to allergic inflammation like asthma, and thus acetylated peptide A and W-54011 are useful for suppressing allergic inflammation in the lungs.

Effect of dibutyltin on placental and fetal toxicity in rat.

In order to elucidate the effect of chorioallantoic and yolk sac placenta on the embryonic/fetal toxicity in dibutyltin dichloride (DBTCl)-exposed rats, we examined the histopathological changes and the tissue distribution of dibutyltin in the placentas and embryos. DBTCl was orally administered to the groups at doses of 0 mg/kg during gestation days (GD)s 7-9 (control group) and 20 mg/kg during GDs 7-9 (GD7-9 treated group), and GDs 10-12 (GD10-12 treated group). The total fetal mortality was increased, and malformations characterized by craniofacial dysmorphism were detected in the GD7-9 treated group. The embryonic/fetal weight and placental weight showed a decrease in both DBTCl-treated groups. Histologically, some embryos on GD 9.5 in the GD7-9 treated group underwent apoptosis without any changes of yolk sac. In the laser ablation-inductively coupled plasma-mass spectrometry analysis (LA-ICP-MS), tin was detected in the embryo, allantois, yolk sac, ectoplacental cone and decidual mass surrounding the conceptus on GD 9.5 in the GD7-9 treated group. Thus, it is considered that the embryo in this period is specifically sensitive to DBTCl-induced apoptosis, compared with other parts. The chorioallantoic placentas in both DBTCl-treated groups showed the developmental delay and hypoplasia in the fetal parts of placenta, resulting from apoptosis and mitotic inhibition. Thus, it was speculated that the DBTCl-induced malformations and fetal resorption resulted from the apoptosis in the embryo caused by the direct effect of DBTCl. The DBTCl-induced lesions in the chorioallantoic placenta were a non-specific transient developmental retardation in the fetal parts of placenta, leading to intrauterine growth retardation.

Effects of inducible nitric oxide synthase inhibitors on asthma depending on administration schedule.

The effectiveness of two inducible nitric oxide synthase (iNOS) inhibitors on allergic airway inflammation was investigated under different administration schedules. Rats sensitized to ovalbumin (OVA) were exposed to OVA for 3 consecutive days. Both iNOS inhibitors showed markedly different effects between two pretreatment schedules: pretreatment before each of three OVA exposures S1 and before the third exposure alone S2. S1 pretreatment resulted in higher pulmonary resistance than triple OVA alone. This potentiation was associated with increased eosinophil infiltration and malondialdehyde levels in the lungs, which were suppressed by superoxide dismutases (SODs) but not by methylprednisolone. However, the S2 administration of both iNOS inhibitors completely suppressed the airway response. Administration by schedule S1 completely suppressed plasma nitrite and nitrate levels, but that by S2 caused only a slight suppression. The triple OVA exposures resulted in the upregulation of iNOS in alveolar macrophages and arginase activity, Mn- and Cu/Zn-SOD expression, and nitrotyrosine and lipid peroxide deposition in the airway. However, inhibitors administered by schedule S1 suppressed this upregulation, but further potentiated nitrotyrosine, which in turn was inhibited by SOD. Although iNOS inhibitors may be beneficial for asthma, repeated administration may be detrimental because of extensive reduction of NO and downregulation of SOD.

[Complement activation and inflammation].

The complement system not only plays an important role in the defense system, but also contributes to the amplification of inflammation if activated in excess or inappropriately controlled. Complement activation through one of three pathways is tightly controlled by various regulators of complement activation (RCA) which are constitutively expressed on various cells in order to restrict excessive activation. Complement activation may generate polypeptides, so-called anaphylatoxins, and membrane attack complex (MAC) with large molecular mass. Anaphylatoxins (C3a, C4a, and C5a) produced by activation of the complement is considered to bridge innate and acquired immunity. Considering that C5a is more potent than C3a, but the serum concentration of C3 is 10 times higher than that of C5, the overall effects of C3a may be comparative with those of C5a. Since both anaphylatoxins are considered to exert their actions through rhodopsin-typed receptors, their receptor antagonists are targets for the discovery of anti-inflammatory and immune-modulating drugs. Complement activation may be related to the pathophysiology of various refractory disorders including ARDS, asthma, septic syndrome, SLE, rheumatoid arthritis, ischemia-reperfusion injury, and psoriasis etc. Pharmacological manipulation of the complement system may consist of various strategies including (1) inhibitors of complement activation at various levels, (2) receptor antagonists of anaphylatoxins, C3a and C5a, and (3) inhibitors of C5a including monoclonal antibody. Candidate agents concerning the above-mentioned manipulations have being produced and some are now in progress toward clinical trials in patients with certain diseases.

Effect of indole-3-acetic acid derivatives on neuroepithelium in rat embryos.

Indole-3-acetic acid (IAA), a natural auxin, induces microencephaly in rats exposed to IAA during gestation days (Days) 12-14, corresponding to the early stage of cerebral cortex development. The purpose of this study was to examine the effects of 5 IAA derivatives administration in pregnant rats on neuroepithelial cells in the embryos. N-Methylindole-3-acetic acid (1Me-IAA), 2-Methylindole-3-acetic acid (2Me-IAA), 2-Methyl-5-methoxyindole-3-acetic acid (2Me-5MeO-IAA), 5-Methoxyindole-3-acetic acid (5MeO-IAA), Indole butyric acid (IBA), and IAA were administered at 1,000 mg/kg except for 2Me-IAA at 500 mg/kg on Days 12, 13 and 14, and then embryos/fetuses were harvested on Day 14.5 or 21. The dams in the 1Me-IAA and 2Me-IAA groups exhibited rigidity and a decrease in locomotor activity. Although a decrease in the absolute brain weight was observed in the 1Me-IAA, 5MeO-IAA, IBA and IAA groups, a decrease in the relative brain weight was observed in only the IAA group. Histopathologically, apoptotic cells were observed mainly in the medial and dorsal layer of the neuroepithelium in the 5MeO-IAA and IAA groups on Day 14.5. The degree of induced neuroepithelial cell apoptosis was less in the 5MeO-IAA group than in the IAA group. However, it was confirmed that the histopathological changes induced by 5MeO-IAA were quite similar to the lesions induced by IAA and may have resulted from the same mechanisms.

Evaluation of repeated dose micronucleus assays of the liver using N-nitrosopyrrolidine: a report of the collaborative study by CSGMT/JEMS.MMS.

The repeated dose liver micronucleus (RDLMN) assay has the potential to detect liver carcinogens, and can be integrated into a general toxicological study. To assess the performance of the assay, N-nitrosopyrrolidine (NPYR), a genotoxic hepatocarcinogen, was tested in 14- or 28-day RDLMN assays. NPYR was orally administered to rats at a daily dose of 25, 50 or 100 mg/kg. One day after the last administration, a portion of the liver was removed and hepatocyte micronucleus (MN) specimens were prepared by the new method recently established by Narumi et al. In addition, a bone marrow MN assay and a histopathological examination of the liver were conducted. The detection of Phospho-Histone H3 was performed by immunohistochemistry to evaluate the proliferation rate of hepatocytes. The results showed significant increase in the number of micronucleated hepatocytes and Phospho-Histone H3-positive cells from the lowest dose in both 14- and 28-day RDLMN assays. On the other hand, the bone marrow MN assay yielded a negative result, which was in accordance with the existing report of the bone marrow MN assay using mice. Upon histopathological examination, inflammatory lesions and hypertrophy were noted, which may explain the increase in the hepatocyte proliferation and the enhancement of MN induction by NPYR. Our findings indicate that the RDLMN assay could be a useful tool for comprehensive risk assessment of carcinogenicity by providing information on both genotoxicity and histopathology when integrated into a general repeat dosing toxicity assay.

Histomorphological comparison of rat placentas by different timing of chlorpromazine-administration.

The effects of chlorpromazine-treatment timing on the development of the placenta in pregnant rats were examined. Chlorpromazine was administered intraperitoneally at 100mg/kg on gestation day (GD) 11 (GD11-treated group), GD 13 (GD13-treated group) or GD 15 (GD15-treated group) into pregnant rats. All treated dams exhibited decreased body weight, prone position, hypothermia, loss or decrease of locomotor activity, etc. The fetal mortality rates were increased up to 42.9% in the GD11- and GD13-treated groups and up to 16.7% in the GD15-treated group. The embryo/fetal weight was on a declining trend from GD 16 onward, and the intrauterine growth retardation (IUGR) rates on GD 21 were increased in all treated groups. The placental weight showed a declining trend from GD 15 onward in all treated groups. Histopathologically, apoptosis was detected 1 or 2 days after treatment, and led to hypoplasia in the labyrinth zone and metrial gland, and cystic degeneration in the basal zone on GD 21 in all treated groups. There was no difference in the histopathological lesions on GD 21 among the treated groups. Thus, it is considered that chlorpromazine-induced placental toxicity is characterized in that there is no obvious specific sensitive period from GD 11 to GD 15. Chlorpromazine induced a non-specific transient development retardation of the placenta by apoptosis independently of the cell proliferation period in each part/zone.