Background & Goals: Reliable non-invasive biomarkers to assess the histological

-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, -tively). Treatment with phenobarbital, a potent CYP inducer, increased the predominance of expression increased microsomal total P450 contents and smooth-surface endoplasmic reticulum in the centrilobular -rats exhibited predominantly arylhydrocarbon receptor, pregnane X receptor, and constitutive androstane 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.

ABSTRACT:An in vitro system for liver organogenesis from murine embryonic stem (ES) cells has been recently established. This system is expected to be applied to the development of a new drug metabolism assay system that uses ES cells as a substitute for animal experiments. The objective of this study was to elucidate the drug metabolism profiles of the murine ES cell-derived hepatic tissue system compared with those of primary cultures of murine adult and fetal hepatocytes. The expression of the genes of the cytochrome P450 (P450) family, such as Cyp2a5, Cyp2b10, Cyp2c29, Cyp2d9, Cyp3a11, and Cyp7a1, was observed in the murine ES cell-derived hepatic tissue system at 16 days and 18 days after plating (A16 and A18). To investigate the activities of these P450 family enzymes in the murine ES cell-derived hepatic tissue system at A16 and A18, testosterone metabolism in this system was analyzed. Testosterone was hydroxylated to 6␤-hydroxytestosterone (6␤-OHT), 16␣-OHT, 2␣-OHT, and 2␤-OHT in this system, and was not hydroxylated to 15␣-OHT, 7␣-OHT, and 16␤-OHT. This metabolism profile was similar to that of fetal hepatocytes and different from that of adult hepatocytes. Furthermore, pretreatment with phenobarbital resulted in a 2.5-and 2.6-fold increase in the production of 6␤-OHT and 16␤-OHT. Thus, evidence for drug metabolic activities in relation to P450s has been demonstrated in this system. These results in this system would be a stepping stone of the research on the development and differentiation to adult liver.Embryonic stem (ES) cells are pluripotent and can differentiate in vitro and in vivo. There have been several reports on the differentiation of murine or human ES cells into hepatocyte-like or albuminproducing cells and their isolation (Chinzei et al., 2002;Jochheim et al., 2004;Shirahashi et al., 2004); these cells also differentiate into a variety of other cell lineages. Thus far, in all of the above researches, the ES cells were differentiated into a single cell lineage by the addition of specific growth factors and chemicals to the culture. Limiting these differentiation systems during in vivo liver development is considered difficult because of the multiple functions and complex structure of the liver. However, we recently succeeded in establishing an in vitro system of liver morphogenesis by using murine ES cells (Ogawa et al., 2005). This system consists of not only hepatocytes but also cell lineages such as cardiomyocytes and endothelial cells that support liver-specific functions and differentiations. The system is more efficient with respect to hepatic functions such as albumin production and ammonia degradation. Furthermore, the expression of the transthyretin, ␣-fetoprotein, ␣1-antitrypsin, and tyrosine aminotransferase genes is higher in this system than in the cultures of hepatic cell lines and murine primary cultures of adult hepatocytes. This system is expected to have many practical applications. It can be used in the development of new drugs and in drug metabolism assays as an alterna...

-Acute and chronic inflammatory diseases are associated with the induction of inducible nitric oxide synthase (iNOS) and inducible heme oxygenase (HO-1). These inducible enzymes are upregulated in macrophages subjected to inflammatory stimuli and oxidative stress. β 2 -Adrenoceptor (AR) agonists, which function as bronchial dilators, are widely used for the treatment of asthma and chronic obstructive pulmonary disease (COPD). We examined whether salbutamol, a classical β 2 -AR agonist, inhibits the induction of proinflammatory cytokines and stress inducible proteins. Rat macrophages obtained from the abdominal cavity were incubated with lipopolysaccharide (LPS) with or without salbutamol. Induction by LPS of tumor necrosis factor (TNF)-α and interleukin (IL)-6 was significantly inhibited (P < 0.05) by salbutamol treatment. Induction by LPS of iNOS mRNA and protein was also significantly inhibited (P < 0.05) by salbutamol. LPS-mediated increases in HO-1 mRNA and protein were not appreciably affected by salbutamol. One of the anti-inflammatory mechanisms of salbutamol was thus found to be inhibition of induction by LPS of extracellular stimulus-responsive kinase (ERK) 1/2 in macrophages. These findings suggest that salbutamol has the potential for use as an anti-inflammatory agent due to its suppression of LPS-induced TNF-α, and IL-6 and iNOS via ERK pathway without affecting HO-1 expression.Key words: Salbutamol, TNF-α, IL-6, iNOS, LPS Correspondence: Satoru Tanaka (E-mail: satoru_tanaka@pharm.kissei.co.jp) Original ArticleThe Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.35, No.3, 327-334, 2010 Vol. 35 No. 3 327 enzymes play roles in the pathogenesis of and may therefore be useful as targets in the treatment of asthma and COPD. β 2 -adrenoceptor (AR) agonists such as salbutamol are mainstay bronchodilators in the treatment of asthma and COPD. Malfait et al. (1999) demonstrated that salbutamol exhibited therapeutic effects against rheumatoid arthritis model rats with collagen-induced arthritis. In a clinical study, inhalation of a long-acting β 2 -AR agonist by patients with mild asthma yielded a novel antineutrophilic effect (Jeffery et al., 2002).Based on these findings we examined whether β 2 -AR agonists inhibit proinflammatory cytokine and stressinducible protein production by in vitro cultured abdominal macrophages, in an attempt to determine novel targets in the treatment of inflammatory diseases. MATERIALS AND METHODS MaterialsSalbutamol (hemisulfate, minimum purity 98%) and PD98059, an extracellular stimulus-responsive kinase (ERK) 1/2 inhibitor, were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). LPS from Escherichia coli and ICI118551, a selective β 2 -adrenoceptor antagonist, were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). The rat IL-6 Immunoassay KIT and rat TNF-α ELISA KIT were purchased from BioSource International Inc. (Camarillo, CA, USA). Primer sets, including those for iNOS oligo (RA008296), HO-1 oligo (RA008256), and β-actin oligo (RA006...

To develop beta2-adrenergic receptor (AR) agonists with higher selectivity, it is essential to evaluate the cardiac side effects which are the most serious side effects of this class of drugs. We studied receptor occupancy of beta1-ARs in rats as a possible cause for the side effect of beta2-AR agonists, namely myocardial fibrosis. Myocardial fibrosis in rats was observed on Day 7 after the administration of salbutamol and terbutaline, both of which are selective beta2-AR agonists, at higher dose levels. To evaluate receptor occupancy, plasma concentrations of (R)-salbutamol and (R)-terbutaline, plasma protein binding and the EC50 for chronotropic effects in rats were determined. Based on the plasma concentrations, the plasma protein binding and EC50, receptor occupancy-time profiles were constructed. The relationship between the receptor occupancy-time profile under the curve, the AUCphi, and the degree of myocardial fibrosis was evaluated with a multiple correlation analysis. Myocardial fibrosis was significantly correlated (r2 > 0.78) to the AUCphi with the threshold above approximately 50%, but not to plasma concentrations. These results indicate that the receptor occupancy theory is also useful for the evaluation of the chronotropic side effects of beta2-AR agonists.