Yoshitaka Maeno

Induction of apoptotic cell death in rat thymus and spleen after a bolus injection of methamphetamine

We examined whether methamphetamine (MAP) induced apoptotic cell death in vivo. Male Wistar rats were injected intraperitoneally with 25 mg MAP/Kg body weight and were sacrificed at 4, 8 and 24 h. As early as 4 h after a single dose of MAP, DNA ladder bands representing DNA fragmentation into multiples of the internucleosomal DNA length of about 180 by were observed by gel electrophoresis in thymic and splenic DNA. DNA from control rats injected with 1 ml physiological saline/Kg body weight showed no ladder band patterns. The proportion of fragmented DNA from the thymus increased in a time-dependent manner up to 8 h and faint ladder band patterns were observed at 24 h, indicating that cell death via apoptosis occurred at an early stage and then apoptotic bodies were scavenged. DNA fragmentation in the thymus and spleen induced with MAP was also confirmed by the terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labeling (TUNEL) method in situ. In control thymus samples, stained cells were numerous in the cortex but sparse in the medulla. At the boundary area between the cortex and medulla, stained cells were seen as a layer. In the MAP-treated rats, stained cells were increased and dispersed equally in the cortex and medulla. In control spleen samples, stained cells were numerous in all areas excluding the germinal centers. Cells at the germinal centers were stained intensively in MAP-treated rat spleen. Light microscopical analyses allowed us to identify lymphocytes during the course of apoptotic cell death. Electron microscopic studies showed morphological landmarks for the process of cellular apoptosis in both organs e.g. lymphocytes with chromatin condensed into crescents at the periphery of the nuclei and apoptotic bodies. These results indicate that MAP induced cell death of the thymic and splenic lymphocytes via apoptosis.

Screening and determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry

Benzodiazepines are one of the most widely prescribed drugs for the treatment of a wide spectrum of clinical disorders. They are used as anticonvulsants, anxiolytics, hypnotics or muscle relaxants with different duration of action. In this paper, a simple and sensitive method for the determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry (GC/MS) is described. The drugs spiked in whole blood were extracted with an Oasis HLB solid-phase extraction cartridge (Waters), which contains a copolymer designed to have a hydrophilic-lipophilic balance. GC/MS analysis was performed using a Shimadzu QP-5000 equipped with a BPX5 capillary column (15 mx0.32 mm I.D., film thickness 0.25 microm, SGE). Nineteen benzodiazepines and two thienodiazepines were well separated from each other on their SIM chromatograms and also on the TIC with the exception of oxazolam to cloxazolam separation. The blank extract from whole blood gave no peaks that interfered with all benzodiazepines and thienodiazepines on the chromatogram. The calibration curves for selected benzodiazepines with fludiazepam as an internal standard showed excellent linearity over the concentration range 5-500 ng/ml blood with a correlation coefficients of >0.995. The detection limits ranged from 0.2 to 20 ng/ml blood. The method is simple and sensitive for the determination of benzodiazepines in whole blood and seems to be useful in the practice of forensic science.

Suppression of prostate cancer in a transgenic rat model via γ-tocopherol activation of caspase signaling

Epidemiological data indicate that intake of one form of vitamin E, γ‐tocopherol, may reduce prostate cancer risk, and several in vitro studies have demonstrated that γ‐tocopherol can inhibit prostate cancer cell growth. The purpose of the present study was to confirm effects of γ‐tocopherol on prostate cancer in the transgenic rat for adenocarcinoma of prostate (TRAP) model established in our laboratory.

Direct effects of methamphetamine on hypertrophy and microtubules in cultured adult rat ventricular myocytes.

Morphological alterations occasionally found in the myocardium of methamphetamine (MAP) abusers include hypertrophy, atrophy, disarrangement of myofibrils and fibrosis. These cardiac alterations have been thought to be due to an indirect action of MAP via catecholamines released by MAP administration. However, the direct effect of MAP on cardiomyocytes is not clear. In previous studies, we showed that cell size of isolated adult rat ventricular cardiomyocytes (ARCs) exposed to MAP was larger than that of untreated cells in culture supplemented with 10% fetal calf serum (FCS). In this study, to determine further the direct effect of MAP on cardiomyocytes, cultured ARCs were exposed to 0.05, 0.1 and 0.5 mM MAP for 7 days in culture medium without FCS following 6-day normal culture in medium containing FCS. Myocyte size was measured and microtubular (MT) structures which were associated with functional disorder of hearts were immunohistochemically observed using confocal microscopy. The size in treated ARCs significantly increased time- and dose-dependently as compared with untreated cells, but it decreased 7 days after exposure to 0.5 mM MAP. The increases in cell size, however, were lower than that in serum-supplemented cultures. MT structures in intact ARCs appeared as a filamentous network throughout the cytoplasm and around the nucleus. MAP exposure for 3 days promoted MT assembly, but in 7-day treated cells, MT and actin structures were injured. These results suggested that MAP directly induced cellular hypertrophy and might lead to cardiac functional disorder.

Methamphetamine induces an increase in cell size and reorganization of myofibrils in cultured adult rat cardiomyocytes

Abstract To investigate the direct effects of methamphetamine (MAP) on cardiac lesions seen in MAP abusers, isolated adult rat ventricular cardiomyocytes (ARCs) were exposed to MAP (0.05–1.0 mM) in medium 199 containing 10% fetal calf serum. Isolated ARCs attached to laminin-coated substrata and began to spread into polygonal shapes with pseudopodia at day 6 in normal culture. However, the cell attachment and spreading were inhibited by exposure to MAP (0.5 and 1.0 mM) for the first 7 days in culture. On the other hand, exposure to MAP (0.05 and 0.1 mM) for 7 days after a 6-day period of normal culture, led to a larger cross surface area of cells with more abundant actin bundles compared to control cells (p < 0.05). This development of spreading area resembled that of norepinephrine-treated ARCs. In addition, immunoreactive atrial natriuretic peptide (ANP) granules developed and accumulated around the nuclear region of ARCs exposed to MAP and the number of ANP positive cells tended to increase in a dose-dependent manner. These results suggest that chronic exposure to a high concentration of MAP may directly inhibit development of ARCs in culture and that a continuous exposure to a low concentration of MAP may facilitate the development of cellular hypertrophy. Therefore, hypertrophied cardiomyocytes in MAP abusers may be provoked by multifactorial incidents of direct and indirect actions of MAP.

Identification of fetal hemoglobin and simultaneous estimation of bloodstain age by high-performance liquid chromatography.

SummaryA method using reverse-phase high-performance liquid chromatography (HPLC) for the identification of fetal hemoglobin (Hb F) and the simultaneous estimation of bloodstain age is described. Umbilical cord and neonatal bloodstains can be differentiated from adult stains by the presence of γ-globin chains which are characteristic of Hb F. With this method, cord and neonatal blood could be distinguished from adult blood in stains up to 32 weeks old. The age of the stain was estimated from the ratio of the peak area of the α-globin chain to that of heme on the same chromatogram. The ratio decreased gradually with an increase in the age of the stain up to 20 weeks old. Studies performed at each time period revealed no significant difference in the ratios of cord and neonatal bloodstains or in the ratios of cord and adult stains. The regression equation calculated from the ratios (y) and the ages of stains in weeks (x) expressed logarithmically isy = 2.5758 − 0.2497 ln(x) and the coefficient of correlation is −0.7491 (n = 252,P < 0.001). The present method, having the advantages of simplicity, speed and sensitivity, should be of great value to forensic science.ZusammenfassungBeschrieben wird eine „reverse-phase” — HPLC-Methode für die Identifizierung von fetalem Hämoglobin (Hb F) und zur gleichzeitigen Bestimmung des Blutspuren-Alters. Blutspuren von Blut aus Nabelvenen und von Neugeborenen können von Blutspuren erwachsener Personen durch die Anwesenheit der γ-Globin-Ketten differenziert werden, welche für Hb F charakteristisch sind. Mit Hilfe dieser Methode gelang die Differenzierung zwischen bis zu 32 Wochen alten Blutspuren aus Nabelschnüren und Neugeborenen einerseits und von Erwachsenen andererseits. Das Alter der Spur wurde bestimmt aus dem Verhältnis der Peak-Fläche, der α-Globin-Kette zu jener des Häm auf demselben Chromatogramm. Das Verhältnis dieser beiden Flächen nahm mit zunehmendem Alter der Blutspur graduell ab, — dies bis zu einem Spurenalter von 20 Wochen. Studien, welche zu jenem Zeitpunkt durchgeführt wurden, deckten keine signifikanten Unterschiede in diesen Peak-Verhältnissen von Nabelvenen- und Neugeborenen-Blutspuren und auch nicht in den Verhältnissen von Nabelvenen und Erwachsenen-Spuren auf. Die Regressions-Gleichung, welche von den Verhältnissen (y) und dem Alter der Spuren in Wochen (x) errechnet und logarithmisch ausgedrückt wird, lautet:y = 2.5758 − 0.2497 ln(x) und der Korrelations-Koeffizient beträgt −0.7491 (n = 252,P < 0.001). Die vorliegende Methode, welche einfach, schnell und empfindlich ist, sollte für die forensischen Wissenschaften von großem Interesse sein.

Species identification of blood and bloodstains by high-performance liquid chromatography.

SummaryA reverse-phase high-performance liquid chromatographic method for species identification of blood and bloodstains is described. The method employs a 300 Å pore SynChropak RP-4 column and ternary solvents (acetonitrile-trifluoroacetic acid-water) and can not only identify a species by its characteristic chromatogram, but also simultaneously demonstrates that it is of blood origin by the existence of the heme peak. Deformations in chromatographic profiles obtained with older bloodstains were observed, but the retention times of heme and the major peaks showed only minor changes. The species could be identified from bloodstains at least 3 months old and the present method has the advantage of simplicity, speed and sensitivity in the practice of forensic science.ZusammenfassungBeschrieben wird eine „reversedphase” HPLC-Methode zur Identifizierung und Spezifizierung von Blut und Blutspuren. Die Analysenmethode besteht aus einer SynChropak RP-4-Säule und einem ternären Lösungsmittelgradientensystem (Acetonitril/Trifluoressigsäure/Wasser). Mit dieser Methode wird durch die Detektion des Häm-Peaks die Probe als Blut identifiziert und die Herkunft (human/tierisch) gleichzeitig durch charakteristische Chromatogramme UV-spektrometrisch spezifiziert. Deformationen der chromatographischen Profile wurden bei älteren Blutspuren beobachtet, wobei sich aber die Retentionzeiten der Hauptkomponenten nur geringfügig änderten. Der Speziesnachweis gelang bei bis zu drei Monate alten Blutspuren. Die vorgestellte Methode hat den Vorteil der Einfachheit, der Schnelligkeit und der Empfindlichkeit für die forensische Praxis.

Mitochondrial sequence haplotype in the Japanese population

Sequence polymorphysms of the mitochondrial DNA (mtDNA) control region, hypervariable regions I and II, from 50 unrelated Japanese were determined by PCR amplification and cycle sequencing.

Detection of cardiomyocyte apoptosis in forensic autopsy cases

Abstract The purpose of the present study was to determine reliable parameters for the detection of apoptotic cells for use as a diagnostic marker during the early stage of acute myocardial infarction (AMI) in forensic autopsy cases. Myocardial tissues taken from forensic autopsy cases were examined by immunohistochemical and molecular-biological methods using the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labelling (TUNEL) and the DNA laddering methods. In cases of AMI with a time period between 2 h from onset to death and 20 h post-mortem time, the nuclei of cardiomyocytes were stained positive with the TUNEL method and DNA fragmentation of myocardial cells was detected by agarose gel electrophoresis. Similar findings were obtained in cases of carbon monoxide (CO) intoxication. However, no apoptotic cells were found in other cases such as methamphetamine (MAP) intoxication, tetrodotoxin intoxication, alcohol intoxication, asphyxia, head injury, heart injury or myocarditis. These findings suggested that it would be possible to apply TUNEL-positive cells as a diagnostic marker during the early stages of AMI.

An autopsy case of poisoning by massive absorption of cresol a short time before death

A 65-year-old male patient who was hospitalized with schizophrenia died about 15 min later after ingestion of a large volume of saponated cresol solution in a mental hospital. Fatal levels of free p- and m-cresol in the heart blood were detected at 458.8 and 957.3 microg/ml, respectively, which far exceeded the fatal levels determined previously. The levels in the heart muscle, liver and spleen tissues were also extremely high, and there was 250 ml of cresol-odor-emitting fluid in the stomach. The levels of glucuronic-acid-conjugated p- and m-cresols in the heart blood were 38.2 and 85.6 microg/ml, respectively. Although the high levels of cresols in the heart blood may be due to diffusion from the stomach contents, it is surmised that the essential levels of free and conjugated forms in blood were at least 99 and 240 microg/ml, respectively, considering the results of postmortem examinations and some case reports. It was concluded that about 340 microg/ml of the total cresols was absorbed in a very short period following oral ingestion of saponated cresol solution in this case.