Differential effects of isoflurane and halothane on the induction of heat shock proteins.

Isoflurane is considered to be a less hepatotoxic volatile anesthetic than halothane since it not only undergoes quantitatively much less metabolism to form toxic reactive intermediates, but also preserves better hepatic blood flow. However, the biochemical basis for the reduced hepatotoxicity has not been elucidated. In this study, we examined the induction of two heat shock proteins, heat shock protein 70 (HSP70) and heme oxygenase-1 (HO-1), in the livers of rats pretreated with or without phenobarbital, followed by exposure to isoflurane or halothane under hypoxic conditions. In the phenobarbital-pretreated rats, the maximal induction of HSP70 was observed by halothane-hypoxia treatment, followed by a half-maximal induction by isoflurane-hypoxia treatment, and less than 30% induction by hypoxia treatment alone. Serum alanine aminotransferase (ALT) activity, an indicator of hepatic dysfunction, which correlated well with the extent of centrilobular necrosis, showed similar changes with increases in HSP70 mRNA. In contrast, HO-1 mRNA was induced only by treatment with halothane-hypoxia. In addition, changes in the expression of HSP70 and HO-1 mRNAs were correlated with their protein expression in the liver. In non-pretreated rats, neither isoflurane-hypoxia exposure nor halothane-hypoxia exposure caused apparent hepatic injury. There was also no induction of HSP70 or HO-1 mRNA by these treatments in non-pretreated animals. These findings demonstrate that there is a significant difference in hepatic injury, and in the induction of HO-1 and HSP70 between halothane-hypoxia and isoflurane-hypoxia treatments. Isoflurane is known to be safer than halothane, which may, in part, be accounted for by the generation of less oxidative stress in the presence of isoflurane, as assessed by reduced induction of heat shock proteins compared with halothane treatment.

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.

Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 2). Testicular toxicity in rats treated orally with ethinylestradiol for 2 weeks.

Parameters of ethinylestradiol-induced testicular toxicity were evaluated with organ weight determination, histopathological examination and quantitative morphometry. Male Sprague-Dawley rats were administered ethinylestradiol orally at 3 or 10 mg/kg/day for 2 weeks and 3 mg/kg/day for 4 weeks. Final body weights in all treated groups were lower than in the respective control group. Decreased absolute and/or relative organ weights of epididymides, prostate, seminal vesicles and testes were observed in all treated groups. In the testes, apoptosis of round spermatids, atrophy of seminiferous tubules, exfoliation of spermatids or spermatocytes, and vacuolar degeneration of Sertoli cells were only observed with 4 weeks treatment. Apoptosis of pachytene spermatocytes and atrophy of Leydig cells were also more marked in the 4 week treated group than after 2 weeks. Therefore, degenerative histopathological changes in testes were more remarkable after 4 weeks treatment than in the 2 weeks treatment groups. However, retention of spermatids was less after 4 weeks treatment and the TUNEL index, calculated as the number of TUNEL-positive spermatocytes or spermatids, was increased in all treated groups. These results suggest that ethinylestradiol-induced testicular toxicity can be detected in male rats administered the compound for 2 weeks and that the TUNEL method for in situ detection of apoptosis is effective for evaluation of testicular toxicity.

Inhibition by beraprost sodium of thrombin-induced increase in endothelial macromolecular permeability.

Effect of beraprost sodium (BPS), a long-acting and orally active stable analogue of PGI2, on the macromolecular permeability of cultured vascular endothelial cells (HUVEC) was detected by the transport of FITC-albumin. Thrombin treatment resulted in induction of FITC-albumin transport across the endothelial cell monolayer. The albumin transport induced by thrombin was not accompanied by any damage to the cells. BPS had no effect on the permeability of resting endothelial monolayers, while BPS inhibited the thrombin-induced increase in the albumin permeability in a dose-dependent manner (30-1000 nM). Treatment of the cells with PGI2 or dibutyryl cAMP caused a significant inhibition of the thrombin-induced increase in the albumin permeability. These results strongly suggested that BPS suppressed the thrombin-induced macromolecular permeability in HUVEC through the elevation of its intracellular cAMP, and that BPS was a suppressor against inflammatory vascular changes such as exudation.

Purification and properties of L-histidine decarboxylase from mouse stomach.

L-Histidine decarboxylase was purified to electrophoretic homogeneity from mouse stomach according to a procedure described previously [Ohmori E, Fukui T, Imanishi N, Yatsunami K and Ichikawa A, J Biochem (Tokyo) 107: 834-839, 1990]. The purified enzyme exhibited a specific activity of 750 nmol histamine formed per min per mg protein, which constituted a 37,500-fold purification compared to the crude extract, with a 1.6% yield. The molecular mass of the enzyme was estimated to be 54 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 100 kDa by gel filtration. The isoelectric point of the enzyme was determined to be pH 5.4. The Km value for L-histidine was estimated to be 0.29 mM. The single mRNA encoding the amino acid sequence of the mouse stomach enzyme was examined and its size was found to be 2.7 kb. These molecular and catalytic property values of the L-histidine decarboxylase of mouse stomach are quite similar to those of the enzyme from mouse mastocytoma P-815 cells.

Synergistic effects of 12-O-tetradecanoylphorbol-13-acetate and dexamethasone on de novo synthesis of histidine decarboxylase in mouse mastocytoma P-815 cells.

12-O-Tetradecanoylphorbol-13-acetate (TPA) markedly enhanced the increase in L-histidine decarboxylase (HDC) activity induced by dexamethasone in mouse mastocytoma P-815 cells, even with a concentration of the latter that had the maximal effect, whereas it induced a rapid and transient increase in HDC activity, which peaked after 3 h in the absence of dexamethasone. The synergistic effect of TPA on HDC activity induced by dexamethasone was detected after 4 h, a plateau level being reached by 6 h, which was similar to the time course with dexamethasone alone. TPA enhanced the induction of HDC activity by various glucocorticoids, but had no effect on the induction by dibutyryl cAMP, prostaglandin E2 or sodium butyrate. Both 1-oleoyl-2-acetylglycerol, a protein kinase C activator, and okadaic acid, a protein phosphatase inhibitor, enhanced the increase in HDC activity induced by dexamethasone, but 4 alpha-phorbol-12,13-didecanoate, an inactive derivative of TPA, did not. Protein kinase C inhibitors, such as staurosporin, H-7 and K255a, suppressed the increase in HDC activity induced by TPA with or without dexamethasone. The enhancement of HDC activity by dexamethasone was completely suppressed by cycloheximide or actinomycin D. Furthermore, TPA markedly enhanced the accumulation of HDC mRNA due to dexamethasone (5 to 10-fold, from 6 to 12 h after). TPA did not cause a significant increase in the level of either [3H]dexamethasone binding capacity or preformed HDC activity in cells. These results taken together suggest that dexamethasone-induced de novo synthesis of HDC in mastocytoma P-815 cells is up-regulated by TPA-activated protein kinase C through the mechanism involving an increased rate of transcription.

Synergistic effects of cyclic AMP and Ca2+ ionophore A23187 on de novo synthesis of histidine decarboxylase in mastocytoma P-815 cells.

In the preceding paper (Kawai, H. et al. (1992) Biochim. Biophys. Acta 1133, 172-178), we reported that in mastocytoma P-815 cells dexamethasone and 12-O-tetradecanoylphorbol-13-acetate (TPA) synergistically enhanced the de novo synthesis of L-histidine decarboxylase (HDC). Here we found that Ca2+ acted synergistically with cAMP in the induction of HDC mRNA and HDC activity in mastocytoma P-815 cells, and that the mechanism underlying the enzyme induction by Ca2+ plus cAMP was distinguishable from that by dexamethasone plus TPA. Ca2+ ionophore A23187, itself having no significant activity, markedly enhanced the induction of HDC activity by N6,O2'-dibutyryl cAMP (db cAMP) or cAMP-inducible prostaglandins such as PGE1, PGE2 and PGI2 in the presence of the phosphodiesterase inhibitor, Ro201724. However, A23187 had little effect on increases in HDC activity induced by other known stimulants, such as TPA, dexamethasone and sodium butyrate. These results suggest that A23187 has a specific effect on the induction of HDC activity due to an increased level of cAMP. The finding that both A23187 and cAMP enhanced HDC activity suggests that both Ca2+/calmodulin and cyclic nucleotide dependent protein kinase play essential roles in the process of enhancement of HDC activity. To examine this possibility, we studied the effects of W-7, an inhibitor of calmodulin, removal of extracellular Ca2+, and H-8, an inhibitor of cAMP-dependent protein kinase, on the enhancing activity of A23187 plus db cAMP. The enhancement of HDC activity by A23187 plus db cAMP was inhibited by W-7, removal of extracellular Ca2+, and H-8. The increase in HDC activity was due to the de novo synthesis of the enzyme, since it was suppressed by the addition of cycloheximide or actinomycin D, and was well correlated with the marked accumulation of a 2.7 kilobase HDC mRNA. Furthermore, the mechanism underlying the induction of HDC by db cAMP plus A23187 is distinguishable from that in the case of dexamethasone plus TPA, since preexposure to dexamethasone plus TPA for 12 h, for a plateau level to be reached, did not affect the subsequent increase in HDC activity due to db cAMP plus A23187.

Fluctuation of fetal rat hepatic histidine decarboxylase activity through the glucocorticoid-ACTH system.

Our studies suggest that the fluctuation of HDC activity in fetal liver in late gestation is regulated by the plasma glucocorticoid level through the pituitary-adrenal system. Taken together, these results support the conclusion that glucocorticoid promotes a rapid increase in HDC synthesis in fetal liver histamine-forming cells, as well as in mouse mastocytoma P-815 cells and rat glandular stomachs.

cDNA-derived amino acid sequence of L-histidine decarboxylase from mouse mastocytoma P-815 cells.

The primary structure of L-histidine decarboxylase (HDC: L-histidine carboxy-lyase, EC 4.1.1.22) from mouse mastocytoma P-815 cells has been determined by parallel analysis of the amino acid sequence of the protein and the nucleotide sequence of the corresponding cDNA. HDC contains 662 amino acid residues with a molecular mass of 74017, which is larger by about 21,000 Da than that of the previously purified HDC subunit (53 kDa), suggesting that HDC might be posttranslationally processed. The HDC cDNA hybridized to a 2.7 kilobase mRNA of mastocytoma cells. Homology was found between the sequences of mouse mastocytoma HDC and fetal rat liver HDC.

Purification and characterization of l-histidine decarboxylase from mouse mastocytoma P-815 cells.

Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.