Teruo Yamada

Prevention of halothane-induced hepatotoxicity by hemin pretreatment: Protective role of heme oxygenase-1 induction

Reductive metabolism of halothane in phenobarbital-pretreated rats is known to increase free radical formation that results in hepatotoxicity. It also is associated with a marked induction of microsomal heme oxygenase-1 (HO-1), suggesting that there is an alteration in heme metabolism. In this study, we examined heme metabolism in rats pretreated with phenobarbital, followed by exposure to halothane-hypoxia. In this model, there was a significant decrease in microsomal cytochrome P450 content in the liver, followed by a rapid increase in free heme concentration and a decrease in the level of mRNA for the nonspecific delta-aminolevulinate synthase. A transient but dramatic induction of HO-1 mRNA and a prolonged induction of heat shock protein 70 mRNA also occurred. The HO-1 protein was detected principally in the hepatocytes around the central vein. Serum alanine transaminase (ALT) activity, an indicator of hepatic dysfunction, increased continuously throughout the experiment. Hemin pretreatment induced hepatic HO-1 with abrogation of the halothane-induced hepatotoxicity in this model, as judged by ALT activity and normal histology. Our findings in this study thus indicate that halothane-induced hepatotoxicity is due not only to its reductive metabolite formation, but also to an increase in hepatic free heme concentration, which is a potent prooxidant; HO-1 induction is an important protective response against such changes. This is also the first study to demonstrate that hemin pretreatment, which induces HO-1 prior to exposure to halothane, effectively prevents halothane-induced hepatotoxicity.

Aggravation of ischemic neuronal damage in the rat hippocampus by impairment of histaminergic neurotransmission

Delayed damage to hippocampal CA1 pyramidal cells was observed in rats subjected to cerebral ischemia caused by 10 min of 4-vessel occlusion. Animals pretreated with alpha-fluoromethylhistidine, a suicide inhibitor of histidine decarboxylase, showed significantly more necrotic cells than did control animals. Mepyramine (H1-antagonist) and (R) alpha-methylhistamine (H3-agonist), but not zolantidine (H2-antagonist), significantly aggravated the delayed neuronal death. These results suggest that histaminergic neurons have a protective role, probably via H1-receptors, in the development of delayed neuronal death caused by cerebral ischemia.

Cathepsin-L, a Key Molecule in the Pathogenesis of Drug-Induced and I-Cell Disease-Mediated Gingival Overgrowth: A Study with Cathepsin-L-Deficient Mice

Drug-induced gingival overgrowth, the chronic side effect of calcium antagonists, is frequently seen due to the increase in patients with hypertension, although the etiology of the disease is largely unknown. I-cell disease, which accompanies gingival overgrowth, is characterized by a deficiency in UDP-N-acetyl-glucosamine and is classified as one of the lysosomal storage diseases. Here, we hypothesized that a common mechanism may underlie the etiology of gingival overgrowth seen in patients treated with calcium antagonist and in patients with I-cell disease. A calcium antagonist, nifedipine, specifically suppressed cathepsin-L activity and mRNA expression, but not that of cathepsin-B in cultured gingival fibroblasts. The activity of cathepsin-L was suppressed up to 50% at 24 hours after treatment of the cells with the reagent. The selective suppression of cathepsin-L activity appeared not to be dependent on Ca(2+), since treatment of the cells with thapsigargin suppressed both cathepsin-B and -L activity. Mice deficient in the cathepsin-L gene manifested enlarged gingivae. Histological observation of the gingivae demonstrated typical features of acanthosis, a phenotype very similar to that of experimentally induced gingival overgrowth. Since cathepsin-L deficiency was reported to be associated with thickening of the skin, impaired cathepsin-L activity may play a key role in the establishment of skin and gingival abnormalities seen in I-cell disease. In addition, reduced cathepsin-L activity may play an important role in inducing drug-induced gingival overgrowth.

The effects of perinatal anoxia or hypoxia on hippocampal kindling development in rats

The effects of anoxia and hypoxia (3% oxygen) at 10-12 post days of age on the development of ventral hippocampal kindling and its transfer to the contralateral ventral hippocampus were studied in adult male Sprague-Dawley rats. During oxygen deprivation, the heart rate decreased to 15% of the prehypoxic value in the animals exposed to anoxia and 40% in those exposed to hypoxia. As is observed in asphyxia of human newborns, our study included both ischemia and hypoxia. The susceptibility to kindling, which was measured by kindling rate, afterdischarge threshold, generalized seizure threshold, and total afterdischarge duration to stage 5, had a tendency to be enhanced in rats exposed to hypoxia compared with controls. The facilitating effects on primary site kindling were enhanced in the animals exposed to hypoxia compared with those exposed to anoxia. Transfer, which was indicated by kindling rate and afterdischarge threshold, was also slightly facilitated in the rats exposed to anoxia or hypoxia in the perinatal period. These results reveal that perinatal oxygen deficiency may not be sufficient to lead to the development of temporal lobe epilepsy. However, it is possible that perinatal hypoxia results in some pathophysiological change in the brain which leads to greater seizure susceptibility in adulthood.

Quantification of In Situ Hybridization Signals in Rat Testes

We performed basic research into quantifying in situ hybridization (ISH) signals in rat testis, a suitable organ for the quantification because germ cells undergo synchronized development and show stage-specific gene expression. In this model experiment, rRNA was selected as the hybridizable RNA in paraffin sections. Specimens fixed with Bouins fixative and hybridized with digoxygenin-labeled probes could easily be analyzed quantitatively through “posterization” of the images. The amount of rRNA hybridized with the probe was greatest in early primary spermatocytes, followed by pachytene primary spermatocytes, then diplotene spermatocytes, and finally by secondary spermatocytes and spermatids. The amounts reached low levels in metaphase, anaphase, and telophase of meiotic division and early step 1 spermatids, and then slightly increased during spermiogenesis. ISH rRNA staining was a useful parameter for evaluation of the quantitative analysis of mRNA and the levels of hybridizable RNA in tissue sections. (J Histochem Cytochem 52:813–820, 2004)

Quantification of PERF 15 mRNA in tissue sections from rat testes

We previously conducted basic research to quantify in situ hybridization (ISH) signals in rat testes. In this experimental model, we selected ribosomal RNA (rRNA) as the hybridizable RNA in paraffin sections, since it allowed us to easily analyze ISH signals expressed with digoxygenin (DIG)-labeled probes quantitatively through “posterization” of the images. We applied this method to analyze the quantification of transcript, PERF 15 mRNA. PERF 15 is expressed specifically in the testes and localized in the rigid cytoskeletal structure of the sperm head, and has been considered to be involved in the apoptotic process of spermatogenic cells. Quantification of the signals may help to clarify the detailed function of PERF 15. We further analyzed the signals concomitant with a confocal laser scanning microscope. The peak of PERF 15 mRNA expression was found in diplotene spermatocytes, and the amount of PERF 15 mRNA was greatest in late pachytene and diplotene spermatocytes and early spermatids, followed by early pachytene spermatocytes, and then late spermatids. PERF 15 may be involved in the events leading to meiotic division, in which apoptosis is also involved. The present study may help to determine the concentration of mRNA in tissue sections.

Histochemical and immunohistochemical changes in rat hepatocytes after halothane exposure

AbstractPurpose. Histochemical and immunohistochemical changes were observed in hepatocytes to study the developing and recovery processes of halothane-induced hepatic injury from 0 to 7 days after halothane exposure. Methods. A total of 330 7-week-old male Sprague-Dawley rats, with or without phenobarbital preteatment, were exposed to halothane in 100%, 21%, 10% oxygen or oxygen alone for 2 h. Results. In the phenobarbital group, degenerated hepatocytes were observed immediately after exposure to 10% oxygen, both with and without halothane: glycogen and ribosomal ribonucleic acid (rRNA) disappeared immediately and 6 h after exposure, respectively, and necrosis developed in zones 3 to 2 at 6 h after exposure. From 12 h to day 1, the necrosis was more marked and more widely observed in the cells with halothane than those without halothane. However, all tissues returned to normal by day 7. Conclusion. Disappearance of glycogen at 0 h and rRNA at 6 h after exposure to halothane under 10% oxygen is considered to be one of the factors inducing necrosis around the central vein. Recovery of the hepatolobular structure was attributed to the rearrangement of the remaining hepatocytes in the portal vein area.

Guanidino Compounds in Patients with Acute Renal Failure

Alterations in urea cycle activity in acute renal failure (ARF) may have important metabolic consequences. There are many reports which study the concentration of guanidino compounds (GC) in the sera of patients with chronic renal failure (CRF) and the relationship of these substances to the degree of renal failure1. In a recent publication2, we pointed out that there were very few reports on GC in ARF and that the relative importance of the plasma level of GC in the pathogenesis of ARF was uncertain. To predict the clinical course of ARF, it might be useful clinically to know of changes in GC, many of which are potential uremic toxins. As reported at the fifth Symposium on the Analysis of GC in Japan (Osaka, 1982), it is of interest to trace variations in GC for determining the course and prognosis of ARF. The purpose of this study is to trace the clinical meaning of GC in ARF, and to observe changes in GC by making circle diagrams called guanidinograms. We describe our cumulative studies on GC in 50 patients with ARF.

Guanidino Compounds and Hemodialysis

Despite a large number of reports that have appeared recently in regard to guanidino compounds and their role in causing uremia and aggravating renal failure, many points are still unclear. There are very few reports on guanidino compounds in acute renal failure, and those that are available have deficiencies related to the clinical assessment or etiological research. Furthermore, many of the patients admitted to the Intensive Care Unit have renal failure in a high risk situation of multiple organ failure, thus contributing to the difficulty of treatment by dialysis. The clinical picture varies, and can include those with infective foci, decreased immunological capacity, and unstable circulatory and respiratory system, abnormality of the central nervous system, and hepatic failure or disseminated intravascular coagulation. The pathophysiology of acute renal failure was investigated to elucidate the miscellaneous complicating factors by analyzing guanidino compounds in patients requiring hemodialysis.

Effect of anesthetics on the self-sustained oscillation in an artificial membrane induced by repetitive conformational change of DOPH molecules between hydrophilic and hydrophobic phases

AbstractPurpose. The mechanism of anesthesia was approached from a study of an artificial excitable membrane that well reproduced the active electrical properties of the nerve membrane. Methods. Self-sustained oscillations of the membrane potential in a model membrane in which dioleyl phosphate (DOPH) was infiltrated into the pores of a millipore filter were utilized to investigate the effect of volatile anesthetic agents on the repetitive conformational change of DOPH molecules between hydrophilic multibilayers and hydrophobic oil droplets, while this process was coupled with diffusion of K+ across the membrane placed between KCl aqueous solutions. Results. The period of the self-sustained oscillations increased due to the addition of volatile anesthetics to the aqueous solutions, and there were critical values of concentrations of volatile anesthetics above which the self-sustained oscillations disappeared. Conclusion. The volatile anesthetic agents affected the hydrophobic oil droplets of the DOPH molecules and impeded their repetitive conformational change between the hydrophilic and hydrophobic phases, just as local anesthetics had been reported to do.