[Direct Positron Emission Imaging Using Ultrafast Timing Performance Detectors].

This is an explanatory paper on Sun Il Kwon et al., Nat. Photon. 15: 914-918, 2021 and some parts of this manuscript are translated from the paper. Medical imaging modalities such as X-ray computed tomography, Magnetic resonance imaging, positron emission tomography (PET), and single photon emission computed tomography, require image reconstruction processes, consequently constraining them to form cylindrical shapes. However, among them, only PET can use additional information, so called time of flight, on an event-by-event basis. If coincidence time resolution (CTR) of PET detectors improved to 30 ps, which corresponds to spatial resolution of 4.5 mm, directly localizing electron-positron annihilation point is possible, allowing us to circumvent image reconstruction processes and free us from the geometric constraint. We call this concept direct positron emission imaging (dPEI). We have developed ultrafast radiation detectors by focusing on Cherenkov photon detection. Furthermore, the CTR of 32 ps being equivalent to 4.8 mm spatial resolution is achieved by combining deep learning-based signal processing with the detectors. In this article, we explain how we developed the detectors and demonstrated the first dPEI using different types of phantoms, how we will tackle limitations to be addressed to make the dPEI more practical, and how dPEI will emerge as an imaging modality in nuclear medicine.

Ultrafast timing enables reconstruction-free positron emission imaging.

X-ray and gamma-ray photons are widely used for imaging but require a mathematical reconstruction step, known as tomography, to produce cross-sectional images from the measured data. Theoretically, the back-to-back annihilation photons produced by positron-electron annihilation can be directly localized in three-dimensional space using time-of-flight information without tomographic reconstruction. However, this has not yet been demonstrated due to the insufficient timing performance of available radiation detectors. Here, we develop techniques based on detecting prompt Cerenkov photons, which when combined with a convolutional neural network for timing estimation resulted in an average timing precision of 32 picoseconds, corresponding to a spatial precision of 4.8 mm. We show this is sufficient to produce cross-sectional images of a positron-emitting radionuclide directly from the detected coincident annihilation photons, without using any tomographic reconstruction algorithm. The reconstruction-free imaging demonstrated here directly localizes positron emission, and frees the design of an imaging system from the geometric and sampling constraints that normally present for tomographic reconstruction.

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.

Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: summary of the collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS).

The repeated-dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect hepatocarcinogens. We conducted a collaborative study to assess the performance of this assay and to evaluate the possibility of integrating it into general toxicological studies. Twenty-four testing laboratories belonging to the Mammalian Mutagenicity Study Group, a subgroup of the Japanese Environmental Mutagen Society, participated in this trial. Twenty-two model chemicals, including some hepatocarcinogens, were tested in 14- and/or 28-day RDLMN assays. As a result, 14 out of the 16 hepatocarcinogens were positive, including 9 genotoxic hepatocarcinogens, which were reported negative in the bone marrow/peripheral blood micronucleus (MN) assay by a single treatment. These outcomes show the high sensitivity of the RDLMN assay to hepatocarcinogens. Regarding the specificity, 4 out of the 6 non-liver targeted genotoxic carcinogens gave negative responses. This shows the high organ specificity of the RDLMN assay. In addition to the RDLMN assay, we simultaneously conducted gastrointestinal tract MN assays using 6 of the above carcinogens as an optional trial of the collaborative study. The MN assay using the glandular stomach, which is the first contact site of the test chemical when administered by oral gavage, was able to detect chromosomal aberrations with 3 test chemicals including a stomach-targeted carcinogen. The treatment regime was the 14- and/or 28-day repeated-dose, and the regime is sufficiently promising to incorporate these methods into repeated-dose toxicological studies. The outcomes of our collaborative study indicated that the new techniques to detect chromosomal aberrations in vivo in several tissues worked successfully.

Repeated dose liver micronucleus assay using adult mice with multiple genotoxicity assays concurrently performed as a combination test.

Recently, the liver micronucleus (MN) assay using young adult rats with repeated administrations has been investigated by employing a new method without partial hepatectomy or in situcollagenase perfusion as the repeated dose liver MN (RDLMN) assay by Narumi et al. (2012). In our study, in order to investigate the possibility of the RDLMN assay using young adult mice instead of rats and the feasibility of employing some genotoxicity assays along with the RDLMN assay as a combination test, two genotoxic carcinogens (N,N-diethylnitrosoamine (DEN) and cisplatin (CIS)) and a nongenotoxic carcinogen (phenobarbital sodium (PHE)) were administered to mice for 15 or 29 days. Then, the liver MN assay, peripheral blood (PB) MN assay and comet assay using the liver and kidney were concurrently performed as a combination test. DEN showed positive responses to all endpoints except MN induction in PB after 15 days of repeat administration. A cross-linking agent, CIS, showed MN induction in liver after 29 days of repeat administration, and in PB after 15 and 29 days of repeat administration, although the comet assay yielded negative responses for both organs at both sampling times. PHE yielded negative responses for all endpoints. In conclusion, it is suggested that the RDLMN assay using mice is a feasible method to be integrated into the general repeated toxicity test along with the combination assays, i.e., comet assay or PB MN assay, which would help in risk assessment for carcinogenicity by comparing the results of combination assays with each other.

Effect of chlorpromazine on rat placenta development.

We examined the sequential histopathological changes in the placentas from rats exposed to chlorpromazine. Chlorpromazine was intraperitoneally administered on GD 14 at 50 and 100 mg/kg and the placentas were sampled on GDs 14.5, 15, 17 and 21. The incidence of dams with complete fetal resorption was increased from GD 17 up to 20% at 50 mg/kg and 44.4% at 100 mg/kg. The embryo/fetal weights reduced on GDs 15 and 17 at 50 mg/kg and during GDs 15-21 at 100 mg/kg. The placental weights reduced on GD 17 at 50 mg/kg and during GDs 14.5-21 at 100 mg/kg. Histopathologically, in the labyrinth zone, apoptotic cells were scattered in the trophoblastic septa without inhibition of cell proliferation on GDs 14.5 and 15 at 50 and 100 mg/kg in a dose-dependent manner. A decrease in trophoblasts led to labyrinth zone hypoplasia. In the basal zone, apoptotic cells were scattered on GDs 14.5 and 15 at 100 mg/kg, and most of them appeared to be glycogen cells. A decrease in glycogen cells induced the delayed development of glycogen cell islands and the subsequent remaining glycogen cell islands, and led to the cystic degeneration of glycogen cells. In addition, failure of development of the glycogen cell islands led to the impaired interstitial invasion of the glycogen cells, and then metrial gland hypoplasia occurred.

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.

Effect of estrogen on rat placental development depending on gestation stage.

We examined the sequential histopathological changes in the placenta from rats exposed to estrogen. 17 ß-estrogiol-3-benzoate was intraperitoneally administered at 100 µg/animal/day during GD 6 to GD 8 (GD6-8 treated group), GD 9 to GD 11 (GD9-11 treated group) and GD 12 to GD 14 (GD12-14 treated group), and the placentas were sampled on GDs 11, 13, 15, 17, and 21. Fetal mortality rates were increased up to approximately 50% in the GD6-8 and 9-11 treated groups, but there was no change of fetal weight on GD 21. An increase in placental weight and a reduction in fetal/placental weight ratio were detected during GD 17 to GD 21 in the GD6-8 treated group. Histopathologically, hypoplasia of metrial gland was detected with defective development of spiral arteries in the GD6-8 and GD9-11 treated groups. A decrease in the thickness of metrial gland was observed from GD 11 onwards in the GD6-8 treated group and from GD 13 onwards in the GD9-11 treated group. The endovascular trophoblasts invaded into the spiral arteries in the deep part of metrial gland in these treated groups. The number of phospho-histone H3 positive cells was decreased on GD 11 or GD 13 in these groups. In the decidua basalis, transitory necrosis was observed with hemorrhage on GD 13 in the GD6-8 and GD9-11 treated groups. In the labyrinth zone, cystic dilatation of the sinusoid was observed with congestion in the GD6-8 treated group, resulting in an increased placental weight. Therefore, we consider that estrogen inhibits the proliferation of decidualized endometrial stromal cells in the metrial gland, and leads to metrial gland hypoplasia with less development of the spiral arteries. The reduced utero-placental blood flow is supposed to be one of the important factors for poor reproductive performance.

Effect of cisplatin on rat placenta development.

We examined the sequential histopathological changes in the placenta from rats exposed to cisplatin. Cisplatin was intraperitoneally administered at 2 mg/kg/day during GDs 11-12 (GD11,12-treated group), or GDs 13-14 (GD13,14-treated group), and the placentas were sampled on GDs 13, 15, 17 and 21. Fetal mortality rates were increased up to approximately 65% from GD 17 onward, and fetal weights were decreased on GD 21 in the GD11,12-treated group. A reduction in placental weights was detected from GD 15 onward, and the placentas on GD 21 were macroscopically small and thin in both treated groups. Histopathologically, in the GD13,14-treated group, an increase in apoptotic cells was detected on GDs 15 and 17 in the labyrinth zone, and on GD 21 in the basal zone, resulting in labyrinth zone hypoplasia. By contrast, in the GD11,12-treated group, an increase in apoptotic cells was detected on GDs 13, 15 and 17 in the labyrinth zone, and during the experimental period in the basal zone. A decrease in Phospho-Histone H3 positive cells was detected on GD 13 in the labyrinth zone and basal zone, resulting in hypoplasia of the labyrinth zone and basal zone. In addition, a marked decrease in glycogen cell-islands in the basal zone was also detected on GDs 15 and 17. There was a reduction in interstitial invasion of glycogen cell-like trophoblasts into the metrial gland on GD 15, resulting in metrial gland hypoplasia. Therefore, we consider that cisplatin administration in pregnant rats induces growth arrest of the labyrinth zone and basal zone, leading to small placenta. It is assumed that metrial gland hypoplasia is secondarily induced by the failure of glycogen cell island development associated with basal zone hypoplasia.

The impairment of metrial gland development in tamoxifen exposed rats.

We examined the sequential histopathological changes in the placenta from rats exposed to tamoxifen. Tamoxifen was administered intraperitoneally at doses of 0 and 2 mg/kg/day on gestation days (GDs) 8, 9 and 10, and the placentas were sampled on GDs 11, 13, 15, 17, and 21. The fetal mortality rates in the tamoxifen group were increased up to 56%. However, there were no effects on the weights of live embryos/fetuses and their placentas. Histopathologically, the size of metrial gland in the tamoxifen group was reduced on all sampling times. The spiral arteries appeared less well developed in the hypoplastic metrial gland. A decrease in uterine natural killer (uNK) cells and mitotic uNK cells around the spiral arteries in the metrial gland was detected from GD 13 onward and on GDs 11 and 13, respectively. There were no obvious changes in the labyrinth zone or basal zone. We consider that the anti-estrogen effect of tamoxifen inhibits the proliferation of decidualized endometrial stromal cells in the metrial gland and leads to inhibition of the proliferative activity of uNK cells, followed by defective development of spiral arteries, and metrial gland hypoplasia. It is assumed that the metrial gland hypoplasia might be involved in the tamoxifen-induced embryo/fetus-toxicity.

The relationship between fetal growth restriction and small placenta in 6-mercaptopurine exposed rat.

In order to investigate the effect of placental size on fetal intrauterine growth retardation (IURG), we examined the morphology and alterations in the expression of glucose transporter in the placentas of rats exposed to 6-mercaptopurine (6-MP). 6-MP was administered orally at 0 and 60 mg/kg/day on gestation day (GD) 9, 11, 13 or 15, and the placentas were sampled on GDs 17 and 21. The main findings in the treated groups were small placenta caused by mitotic inhibition and apoptosis, fetal resorption and IUGR with or without some malformations. The most sensitive period to 6-MP-induced fetal mortality was found to be in the GD9-treated group, and the small placenta and fetal abnormalities in the GD11-treated group, respectively. However, the litters in a quarter of the dams with the treatment on GD 11 had no fetotoxicity despite 25% decline in the placental weight. Histopathologically, the expression of glucose transporter GLUT3 was increased in the trophoblastic septa in all treated groups, particularly remarkable with proliferation of trophoblasts in the above litters, where the fetal-placental weight ratio was increased. Thus, we consider that the normal fetal growth and development can be maintained caused by adaptive change, even if the placental weight decreased by approximately 25% in 6-MP exposed rats.

Toxicological pathology in the rat placenta.

The placenta grows rapidly for a short period with high blood flow during pregnancy and has multifaceted functions, such as its barrier function, nutritional transport, drug metabolizing activity and endocrine action. Consequently, the placenta is a highly susceptible target organ for drug- or chemical-induced adverse effects, and many placenta-toxic agents have been reported. However, histopathological examination of the placenta is not generally performed, and the placental toxicity index is only the placental weight change in rat reproductive toxicity studies. The placental cells originate from the trophectoderm of the embryo and the endometrium of the dam, proliferate and differentiate into a variety of tissues with interaction each other according to the development sequence, resulting in formation of a placenta. Therefore, drug- or chemical-induced placental lesions show various histopathological features depending on the toxicants and the exposure period, and the pathogenesis of placental toxicity is complicated. Placental weight assessment appears not to be enough to evaluate placental toxicity, and reproductive toxicity studies should pay more attention to histopathological evaluation of placental tissue. The detailed histopathological approaches to investigation of the pathogenesis of placental toxicity are considered to provide an important tool for understanding the mechanism of teratogenicity and developmental toxicity with embryo lethality, and could benefit reproductive toxicity studies.

The use of an automated hematology analyzer to observe cell growth in the chromosome aberration test using human lymphocytes.

Human lymphocytes have been frequently used for in vitro chromosome aberration or micronucleus tests on whole-blood culture. However, it is difficult to observe or confirm the cell growth of lymphocytes just before chemical treatment compared with cultured cell lines, such as CHL or CHO cells. In order to overcome this drawback of using whole-blood culture, we investigated a possibility of using an automated hematology analyzer (AHA) (Sysmex XT-2000i, SYSMEX Corp. (Hyogo, Japan)) to measure the growth of lymphocytes applying a manual function of this apparatus. In this study, whole-blood samples were cultured for 4 days, and the growth of lymphocytes was measured once a day using a standard flow cytometer (FCM) with antibody CD3 and DNA staining solution, and by the AHA simultaneously. The results showed that growth curves produced employing the two methods coincided fairly well. Therefore, it can be concluded that the growth of lymphocytes in whole-blood culture can be measured using AHA in a straightforward and rapid way in in vitro chromosome aberration or micronucleus tests.

Evaluation of a liver micronucleus assay in young rats (III): a study using nine hepatotoxicants by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS).

We have been investigating a liver micronucleus assay to detect genotoxic chemicals using young rats for several years, and had established its advantages with respect to using autonomous proliferation of young rat hepatocytes. Nine chemicals known to induce hepatotoxic effects such as necrosis (2,6-dinitrotolune, bromobenzene, isoniazid, phenacetin, allyl alcohol and thioacetamide), cholestasis (chlorpromazine hydrochloride and alpha-naphthyl isothiocyanate) and oxidative stress (clofibrate) were selected for this study. A liver micronucleus assay was conducted in 4-week-old male F344 rats using two or three dose levels of test chemicals given orally by gavage to evaluate the compound's ability to induce micronucleated hepatocytes. Several of these test chemicals were additionally examined in a peripheral blood micronucleus assay conducted concurrently and in the same animals. The genotoxic rodent hepatocarcinogen, 2,6-dinitrotoluene showed a positive result in the liver micronucleus assay, but the nongenotoxic hepatocarcinogens, clofibrate and thioacetamide gave negative responses. Bromobenzene, known to produce DNA adducts but is noncarcinogenic in rodent liver, was judged equivocal in this assay. alpha-Naphthyl isothiocyanate is noncarcinogenic and showed negative response in the liver. The other four chemicals, known to be either noncarcinogenic or carcinogenic in other non-liver target organs, showed negative results in the liver micronucleus assay. Based on the results in the present study and previous report described above, it was concluded that this technique is able to effectively predict genotoxic rodent hepatocarcinogenicity, and does not give false positives due to hepatotoxicity.

Evaluation of a liver micronucleus assay in young rats (IV): a study using a double-dosing/single-sampling method by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS).

A collaborative study was conducted to evaluate whether a liver micronucleus assay using four-week-old male F344 rats can be used to detect genotoxic rat hepatocarcinogens using double-dosing with a single-sampling 4 days after the second dose. The assay methods were thoroughly validated by the seven laboratories involved in the study. Seven chemicals, 2,4-diaminotoluene, diethyl nitrosamine, p-dimethylaminoazobenzene, 1,2-dimethylhydrazine dihydrochloride, 2,4-dinitrotolunene, 2,6-dinitrotoluene and mitomycin C, known to produce positive responses in the single-dosing/triple-sampling method were selected for use in the present study, and each chemical was examined in two laboratories with the exception of 2,4-dinitrotolunene. Although several of the compounds were examined at lower doses for reasons of toxicity than in the single-dosing/triple-sampling method, all chemicals tested in the present study induced micronuclei in liver cells indicating a positive result. These findings suggest that the liver micronucleus assay can be used in young rats to detect genotoxic rat hepatocarcinogens using a double-dosing/single-sampling procedure. Further, the number of animals used in the liver micronucleus assay can be reduced by one-third to a half by using the double-dosing/single-sampling method. This reduction in animal numbers also has significant savings in time and resource for liver perfusion and hepatocyte isolation.

Multi-endpoint genotoxic assay using L5178Y (Tk(+/-) -3.7.2c) cells.

When the mouse lymphoma Tk assay (MLA) provides a positive result, its cause can be roughly estimated by examining colony sizes. An increase in the number of large colonies means that the compound tested has point mutational potential, while an increase in small colonies indicates the potential for chromosome aberration. However, it was found to be difficult to clearly judge this in the case of caffeine known as a clastogen lacking the potential of point mutation. In our study, caffeine significantly increased the thymidine kinase (Tk) mutation frequencies derived from large colonies as well as those from small colonies in the standard protocol, although the frequencies derived from a small colony were higher than those from large colonies at higher doses. Therefore, we prolonged the expression period from 2 days, a standard period, to 6 days after treatment and then examined the Tk and Hprt mutations simultaneously. The result showed that caffeine gave a completely negative result on a mutation test for both Tk and Hprt. On the other hand, ethyl methanesulfonate (EMS), a genotoxic carcinogen, showed a positive result for both. Moreover, caffeine and EMS significantly increased the frequencies of micronucleated cells. In conclusion, when MLA gives a positive result and the cause is ambiguous, in order to identify the exact cause of the positive response, it is helpful to perform a confirmatory test investigating the potential of Tk and Hprt gene mutation simultaneously after 6-day expression and to perform an in vitro micronucleus assay during the expression period.

Evaluation of a liver micronucleus assay with 12 chemicals using young rats (II): a study by the Collaborative Study Group for the Micronucleus Test/Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group.

The partial hepatectomy method, co-treatment method with mitogens and an in vivo/in vitro assay method have been reported as in vivo liver micronucleus (MN) assays. These methods have disadvantages with respect to widespread use as an in vivo assay, i.e. they are time consuming, labour intensive and there is the possibility of interaction with the mitogens used. Therefore, we have attempted to develop a new method to overcome these disadvantages. The assay as described herein utilises the autonomous proliferation of hepatocytes of young rats. Nine chemicals have been evaluated using this method thus far. We have also assessed the sensitivity and detectability according to the following methods. A liver MN assay was performed in two strains of young rats using one or two doses of 12 chemicals to investigate the inducibility of micronucleated hepatocytes. For some of the chemicals, a peripheral blood MN assay was performed concurrently in the same animals. The following chemicals were used: diethylnitrosamine (DEN), 2-acetylaminofluorene (2AAF), 2,4-diaminotoluene (2,4-DAT), quinoline, p-dimethylaminoazobenzene (DAB), dimethylnitrosamine (DMN), ethylmethanesulphonate, 5-fluorouracil, mitomycin C (MMC), 1,2-dimethylhydrazine.2HCl, cyclophosphamide and 2,4-dinitrotoluene (2,4-DNT). The rodent hepatocarcinogens, quinoline, DAB and DMN showed positive responses in previous assays. The results of the present assay revealed new positive responses for single doses of 2AAF, 2,4-DAT, MMC, 1,2-dimethylhydrazine.2HCl and 2,4-DNT. These chemicals are known rodent hepatocarcinogens, whose clastogenicity is believed to be related to the formation of reactive metabolites generated through enzymatic activation, or the chemicals act directly. Two doses of 2AAF and DMN appeared to be more effective than a single dose in terms of MN induction. Although there were quantitative differences in the incidences of MNs, both strains of rat (F344 and SD) responded positively after treatment with DEN, DMN, 2,4-DAT, DAB, quinoline and 2AAF, suggesting that both strains are appropriate for the assay. Based on these results, it is concluded that this technique could be effective for detecting chemical clastogenicity in hepatocytes in vivo.

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.

Regioselective ring opening of benzylidene acetal protecting group(s) of hexopyranoside derivatives by DIBAL-H.

The reductive ring-opening reaction of benzylidene-protected glucosides and mannosides such as methyl 2,3-di-O-benzyl-4,6-O-benzylidene-alpha-d-glucoside (1) and methyl 2,3-di-O-benzyl-4,6-O-benzylidene-alpha-d-mannoside (4) by using a toluene stock solution of DIBAL-H and a dichloromethane stock solution of DIBAL-H gives mainly or selectively the corresponding 2,3,4-tri-O-benzyl derivatives (2, 5) and 2,3,6-tri-O-benzyl derivatives (3, 6), respectively, although that of methyl 2,3-di-O-benzyl-4,6-O-benzylidene-alpha-d-galactoside (7) gives methyl 2,3,6-tri-O-benzyl-alpha-d-galactoside (9) selectively irrespective of the solvent of the stock solution of DIBAL-H. Similarly, the reaction of the exo-isomer of methyl 2,3:4,6-di-O-benzylidene-alpha-d-mannopyranosides (exo-10) with a toluene stock solution and dichloromethane stock solutions of DIBAL-H selectively gives methyl 3-O-benzyl-4,6-O-benzylidene-alpha-d-mannoside (12) and methyl 2-O-benzyl-4,6-O-benzylidene-alpha-d-mannoside (11), respectively, although that of endo-10 selectively affords 11, irrespective of the solvent of the stock solution of DIBAL-H.

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.