ß3-adrenoceptor-mediated increased circulating transaminase levels in mice treated with its agonist BRL 37344.

Treatment with the selective ß(3)-adrenoceptor agonist BRL 37344 increased circulating levels of alanine transaminase (ALT) and aspartate transaminase (AST) in mice without causing hepatocellular injury. To clarify whether this was a ß(3)-adrenoceptor-mediated effect, the inhibitory effect of the selective ß(3)-adrenoceptor antagonist SR 59230A on the increase in circulating transaminase levels induced by BRL 37344 was examined. A single intraperitoneal dose of BRL 37344 alone initially increased insulin and non-esterified fatty acid (NEFA) dose-proportionally at 0.5 hr post-dose, findings considered attributable to ß(3)-adrenoceptor-stimulating effects. Levels of the gluconeogenic precursors pyruvate (PA) and lactate (LA) were increased corresponding to the change in insulin. Thereafter, glucose (GLU) level was decreased at 4 and 8 hr post-dose, suggesting disruption of glucose homeostasis. In association with these changes in glucose metabolism, transaminase levels were increased maximally at 4 hr post-dose. The transaminase changes were not accompanied by increases in circulating levels of other hepatocellular enzymes, including guanine deaminase (GUA), glutamate dehydrogenase (GLDH), and lactate dehydrogenase (LDH), or any morphological hepatocellular injury. Intraperitoneal pre-treatment with SR 59230A partly inhibited the effects of BRL 37344 alone, indicating that the increase in levels of circulating ALT by BRL 37344 was attributable to a ß(3)-adrenoceptor-stimulating effect. In conclusion, the ß(3)-adrenoceptor agonist BRL 37344 was shown to increase circulating transaminase levels in mice accompanied with dynamic changes in glucose metabolism. These findings suggest the possibility that circulating transaminase levels are increased as pharmacological effects of drugs disrupting glucose metabolism, and that hepatotoxic markers should be selected considering these effects to distinguish between acceptable pharmacology and toxicity.

Adrenomedullin in sinusoidal endothelial cells play protective roles against cold injury of liver.

Donor organ damage caused by cold preservation is a major problem affecting liver transplantation. Cold preservation most easily damages liver sinusoidal endothelial cells (LSECs), and information about the molecules modulating LSECs function can provide the basis for new therapeutic strategies. Adrenomedullin (AM) is a peptide known to possess anti-apoptotic and anti-inflammatory properties. AM is abundant in vascular endothelial cells, but levels are comparatively low in liver, and little is known about its function there. In this study, we demonstrated both AM and its receptors are expressed in LSECs. AM treatment reduced LSECs loss and apoptosis under cold treatment. AM also downregulated cold-induced expression of TNFalpha, IL1beta, IL6, ICAM1 and VCAM1. AM reduced apoptosis and expression of ICAM1 and VCAM1 in an in vivo liver model subjected to cold storage. Conversely, apoptosis was exacerbated in livers from AM and RAMP2 (AM receptor activity-modifying protein) knockout mice. These results suggest that AM expressed in LSECs exerts a protective effect against cold-organ damage through modulation of apoptosis and inflammation.

Endogenous alpha-calcitonin gene-related peptide mitigates liver fibrosis in chronic hepatitis induced by repeated administration of concanavalin A.

BACKGROUND: Alpha-calcitonin gene-related peptide (alphaCGRP) is a 37-amino acid pleiotropic peptide that we previously showed to exert a hepatoprotective effect during concanavalin A (Con A)-induced acute hepatitis. In the present study, we used alphaCGRP(-/-) mice to further investigate the antifibrogenic and hepatoprotective effects of endogenous alphaCGRP in Con A-induced chronic hepatitis. METHODS: Chronic hepatitis was induced in alphaCGRP(-/-) and wild-type mice by repeated administration of Con A. Serum transaminases were measured to assess hepatic injury. The severity of fibrosis and the activation of hepatic stellate cells (HSCs) were analysed by Masson trichrome staining and immunohistochemical staining of alpha-smooth muscle actin (alpha-SMA) respectively. Altered expression of fibrosis- and inflammation-related genes was evaluated using a quantitative real-time polymerase chain reaction. Activation and proliferation of HSCs were analysed using both primary cultured HSCs from the mice and the LI90 HSC cell line. RESULTS: alphaCGRP(-/-) mice showed more severe liver fibrosis than wild-type mice in a Con A-induced chronic hepatitis model. In histological and gene expression analyses, alphaCGRP(-/-) mice showed greater inflammatory and fibrotic changes, greater HSC activation and a higher incidence of apoptosis among nonparenchymal cells than wild-type mice. CONCLUSIONS: Endogenous alphaCGRP mitigates liver fibrosis in chronic hepatitis induced by repeated administration of Con A. alphaCGRP could be a useful therapeutic target for the treatment of chronic hepatitis.

Strain differences in hepatic cytochrome P450 1A and 3A expression between Sprague-Dawley and Wistar rats.

Expression of hepatic cytochrome P450 (CYP) isoforms was compared in Sprague-Dawley (SD) and Wistar (WI) rats, which are commonly used strains in preclinical studies. Basal CYP1A1, CYP1A2, and CYP3A2 mRNA levels were higher in WI rats than in SD rats (by 8-, 3- and 2-fold, respectively). Treatment with phenobarbital, a potent CYP inducer, increased the predominance of expression of these three mRNAs in WI rats (by 26-, 4-, and 2-fold, respectively) along with the predominance of increased microsomal total P450 contents and smooth-surface endoplasmic reticulum in the centrilobular hepatocytes. CYP1A enzymatic activity was also higher in WI rats than in SD rats. No strain differences were observed in phenobarbital induction of CYP2B1/2, CYP2C6, or CYP3A1. CYP3A2 mRNA was more strongly induced by dexamethasone, a typical inducer of CYP3A, together with CYP3A1 mRNA, in WI rats than in SD rats (by 2-fold), whereas the CYP1A1 and CYP1A2 mRNA expression induced by beta-naphtoflavone, a typical inducer of CYP1A, did not differ between the two strains. Furthermore, WI rats exhibited predominantly arylhydrocarbon receptor, pregnane X receptor, and constitutive androstane receptor mRNAs, responsible for CYP1A or CYP3A induction, with phenobarbital or dexamethasone induction. In conclusion, significant, predominant expression of hepatic CYP1A and CYP3A mRNAs in WI rats was observed, possibly related to nuclear receptor-mediated induction. Considering the pharmacokinetic and toxicological importance of CYP1A and CYP3A, different outcomes might arise depending on the rat strains used in preclinical studies of drugs metabolized typically or mainly by both isoforms.

Diluted isoflurane as a suitable alternative for diethyl ether for rat anaesthesia in regular toxicology studies.

Despite its explosive properties and toxicity to both animals and humans, diethyl ether is an agent long used in Japan in the anaesthesia jar method of rat anaesthetises. However, in response to a recent report from the Science Council of Japan condemning diethyl ether as acceptable practice, we searched for an alternative rat anaesthesia method that provided data continuous with pre-existing regular toxicology studies already conducted under diethyl ether anaesthesia. For this, we examined two candidates; 30% isoflurane diluted with propylene glycol and pentobarbitone. Whereas isoflurane is considered to be one of the representatives of modern volatile anaesthetics, the method of propylene glycol-diluted 30% isoflurane used in this study was our modification of a recently reported method revealed to have several advantages as an inhalation anaesthesia. Intraperitoneal pentobarbitone has long been accepted as a humane method in laboratory animal anaesthesiology. These 2 modalities were scrutinized in terms of consistency of haematology and blood chemistry with previous results using ether. We found that pentobarbitone required a much longer induction time than diethyl ether, which is suspected to be the cause of fluctuations in several haematological and blood chemical results. Conversely, only calcium ion concentration showed a slight difference from traditional results in the case of 30% isoflurane. Additionally, serum prolactin and corticosterone levels indicated that 30% isoflurane induced less stress than ether, confirming that 30% isoflurane can both provide results consistent with diethyl ether, while at the same time remove its disadvantages. As such 30% isoflurane appears to be a strong alternative anaesthetic agent for future regular toxicology studies in Japan.

Endogenous alphaCGRP protects against concanavalin A-induced hepatitis in mice.

To evaluate hepatoprotective effect of alpha-calcitonin gene-related peptide (alphaCGRP), we compared the susceptibilities of alphaCGRP-/- and wild-type mice to concanavalin A (Con A)-induced hepatitis. Twelve hours after Con A administration, serum transaminases were markedly higher in alphaCGRP-/- than wild-type mice, and much more extensive TUNEL-positive lesions and DNA fragmentation were detected in the livers of alphaCGRP-/- mice. Notably, expression of IL-6 was selectively diminished in alphaCGRP-/- mice, suggesting that induction of IL-6 during acute inflammatory responses is blunted in alphaCGRP-/- mice. In addition, primary cultured alphaCGRP-/- hepatocytes were more susceptible to IFN-gamma-induced cell death than hepatocytes from wild-type mice. Administration of exogenous alphaCGRP reduced the incidence of apoptosis among hepatocytes and endothelial cells. It thus appears that alphaCGRP exerts a hepatoprotective effect by modulating cytokine expression and preventing apoptosis.

[Toxicity profile of silodosin (KMD-3213)].

The toxicity profile of silodosin, a selective alpha(1A)-adrenoceptor antagonist, was evaluated. The lethal doses were 800 mg/kg in rats and 1500 mg/kg in dogs. Repeated-dose studies revealed fatty degeneration of hepatocytes and an induction of drug-metabolizing enzymes at 15 mg/kg/day or more in male rats, mammary gland hyperplasia at 60 mg/kg/day or more in female rats, and degeneration of the seminiferous tubular epithelium at 25 mg/kg/day or more only in young dogs. Silodosin was negative in all mutagenicity studies, except for a weak positive in a chromosomal aberration assay conducted without metabolic activation. In carcinogenicity studies, mammary gland tumors and pituitary adenomas were increased in female mice given 150 mg/kg/day or more and 400 mg/kg/day respectively, while thyroid follicular cell carcinoma was increased in male rats given 150 mg/kg/day. Reproductive studies in rats revealed a decreased male fertility at 20 mg/kg/day or more and a prolonged estrous cycle at 60 mg/kg/day or more. Silodosin did not exhibit any teratogenic potential in either rats or rabbits, and had no effects on the postnatal development of rat offspring. In safety pharmacology studies, silodosin produced no severe effects on the central nervous, cardiovascular, or respiratory systems. In conclusion, silodosin exhibited adequate safety margins between the clinically recommended dose and those at which toxic effects or safety pharmacological changes were detected. As a new therapeutic drug for the micturition difficulties caused by benign prostatic hyperplasia, silodosin should have few serious side effects in clinical use.

Altered cytokine expression in mesenteric lymph nodes in a rat strain (Matsumoto Eosinophilic Shinshu) that spontaneously develops hypereosinophilia.

The Matsumoto Eosinophilic Shinshu (MES) rat is an inbred mutant strain that spontaneously develops systemic hypereosinophilia with eosinophilic inflammatory lesions similar to those associated with hypereosinophilic syndrome in humans and other mammals. To elucidate the pathogenic mechanisms that underlie these features of MES rats, we examined the pattern of cytokine gene expression in mesenteric lymph nodes (MLNs), the thymus, and peripheral blood mononuclear cells as well as the blood clinicopathology and MLN lymphocytic subsets of these animals. MES rats exhibited both leucocytosis, attributable in large part to hypereosinophilia and neutrophilia, and immunoglobulin M (IgM) and IgA gammaglobulinaemia, with increased titres of IgM autoantibodies to nuclear antigens. Reverse transcription and polymerase chain reaction analysis revealed that the amounts of interleukin (IL)-5, IL-4, eotaxin, and interferon-gamma mRNAs were increased in the MLN lymphocytes of MES rats compared with the corresponding values for Sprague-Dawley rats. Intraperitoneal administration of a monoclonal antibody specific for IL-5 resulted in an immediate suppression of hypereosinophilia and a delayed suppression of neutrophilia in MES rats. Flow cytometry revealed an increased percentage of CD3+ CD4- CD8- T lymphocytes in MLNs of MES rats. Our results suggest that the hypereosinophilia of MES rats results from an increased production of IL-5, and that the eosinophilic inflammatory lesions of these animals, which are largely restricted to the gut, may be related both to cytokine overexpression in MLNs and to T helper 1 and 2 immunological responses.

Liver protection by bis(maltolato)zinc(II) complex.

The aim of this study was to perform screening of a novel drug for treating liver injury. Bis(maltolato)zinc(II) complex [Zn(Mal)(2)], which was previously reported to possess insulinomimetic activity, was found to have potency against experimentally induced liver injury both in vitro and in vivo. Cultured rat hepatocytes were treated with bromobenzene for 24 h to induce cellular injury. Zn(Mal)(2) of various concentrations was added along with bromobenzene in order to evaluate the hepatoprotective activity of Zn(Mal)(2) in vitro. The number of viable hepatocytes decreased by 42% in the culture with bromobenzene. However, hepatocyte viability was maintained when Zn(Mal)(2) was added to the bromobenzene culture. The hepatoprotective activity of Zn(Mal)(2) in vivo was investigated using a concanavalin A-induced liver injury model in BALB/c mice. Changes in serum aminotransferase activities and the secretion of several cytokines were measured. The hepatoprotective effect of Zn(Mal)(2) was also demonstrated in vivo by the suppression of serum aspartate aminotransferase and alanine aminotransferase elevation. No significant changes in serum cytokines associated with the induction of hepatic damage were observed in the concanavalin A-induced injury model. However, examination of concanavalin A-treated mouse splenocytes revealed a dose-dependent suppression of cytokine secretions by Zn(Mal)(2). Zn(Mal)(2) possessed hepatoprotective activity and might exert its effect by a number of mechanisms.