Katsuhito Nagai

Protective effects of taurine on doxorubicin-induced acute hepatotoxicity through suppression of oxidative stress and apoptotic responses.

The organ toxicity of doxorubicin (DOX), an anthracycline antineoplastic agent, narrows the therapeutic window despite its clinical usefulness. In the present study, we determined whether taurine protected against DOX-induced hepatic injury, and explored the molecular mechanisms underlying the suppressive effects of taurine in terms of alterations in oxidative stress and apoptotic responses. DOX-induced body weight loss was completely suppressed by taurine treatment. Elevations in the serum activity levels of lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase by DOX were also dose-dependently attenuated by a concurrent treatment with taurine. Superoxide dismutase activity and reduced glutathione content in the liver were decreased following the administration of DOX, whereas these changes were suppressed when 10 mg/kg taurine was given in combination with DOX. Taurine attenuated the increased expression of mRNAs for Fas and Bax after DOX exposure. Furthermore, the formation of cleaved caspase-3 protein in the group given DOX with taurine was lower than that in the group treated with DOX alone. Our results suggest that taurine can protect against DOX-induced acute hepatic damage, the underlying mechanism of which is attributable to the suppression of oxidative stress and apoptotic responses.

Theanine prevents doxorubicin-induced acute hepatotoxicity by reducing intrinsic apoptotic response.

Doxorubicin (DOX) is widely used as an antitumor agent with topoisomerase II inhibiting activity; however, its dosage and duration of administration have been strictly limited due to dose-related organ damage. The present study investigated whether theanine, an amino acid found in green tea leaves, could reduce DOX-induced acute hepatotoxicity and the apoptotic response in mice. Activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum, biomarkers of hepatic impairment, were markedly increased after the administration of 20 mg/kg DOX, whereas the degree of these elevations was significantly attenuated by 10 mg/kg theanine, which was consistent with histological hepatic images assessed by microscopic examination. The hepatic expression of Bax and Fas, representative intrinsic and extrinsic apoptotic molecules, respectively, was significantly increased by dosing with DOX. However, the elevation in the hepatic expression of Bax, but not Fas, was suppressed to control levels by theanine. The formation of cleaved caspase-3 protein in the group given DOX with theanine was significantly lower than that in the group treated with DOX alone. These results suggest that theanine can protect against acute hepatic damage induced by DOX, which is attributed to the suppression of intrinsic caspase-3-dependent apoptotic signaling.

Increased Gene Expression of Glucose Transporters in the Mouse Brain after Treatment with Fluoxetine and Pergolide

Glucose transporters play key roles in the homeostatic control of brain functions. In the present study, we examined the effect of fluoxetine, a selective serotonin reuptake inhibitor, and pergolide, a dopamine D receptor agonist, on the gene expression levels of glucose transporters in the mouse brain. mRNAs for 8 sodium-independent glucose transporters (GLUTs), other than GLUT4 and GLUT9, and sodium-dependent glucose transporter 1 (SGLT1) were confirmed to be expressed in brain tissue. Fluoxetine and pergolide significantly increased the expression levels of mRNAs for GLUT1 and GLUT10 in the brain. Furthermore, the expression of GLUT6 in tissue was increased by administering pergolide to mice. On the other hand, fluoxetine and pergolide had no effect on the expression levels of mRNAs for the other GLUTs and SGLT1. Therefore, we concluded that the gene expression of several GLUT isoforms in the mouse brain was affected by the treatment with fluoxetine and pergolide.

Effect of fluoxetine and pergolide on expression of nucleoside transporters and nucleic-related enzymes in mouse brain.

Nucleoside transporter (NT) and nucleic‐related enzyme (NRE) play key roles in the physiology of nucleosides and the pharmacology of its analogs in mammals. In this study, we examined the effect of fluoxetine, a selective serotonin reuptake inhibitor, and pergolide, a dopamine D receptor agonist, on the expression of NTs and NREs in mouse brain. It was confirmed by the detection of corresponding mRNAs that three equilibrative nucleoside transporter (ENT1‐3) isoforms, concentrative nucleoside transporter 2 (CNT2), CNT3, adenosine kinase (AK), and apyrase, but not CNT1, were expressed in brain tissue. Based on an assessment by mRNA determination, the cerebral expression of CNT2 was found to be increased by administration of fluoxetine and pergolide to mice. Furthermore, pergolide increased the expression of ENT2. However, fluoxetine and pergolide had no significant effect on the expression of mRNA for other NTs, AK, and apyrase. Therefore, we concluded that the expression of several NT isoforms, but not NREs, in mouse brain was affected by treatment with fluoxetine and pergolide.

Difference in nephrotoxicity of vancomycin administered once daily and twice daily in rats

Abstract We compared the degree of nephrotoxicity of vancomycin (VCM) administered once daily and twice daily in rats. VCM was intraperitoneally administered once daily to rats at a dose of 400 mg/kg (VCM-1-treated) or administered at a dose of 200 mg/kg twice daily at 12-hour intervals (VCM-2-treated) for 7 consecutive days. Creatinine clearance was decreased more markedly in VCM-1 rats relative to VCM-2 rats, although there was no significant difference in renal accumulation of VCM between the two groups. Renal superoxide dismutase activity was lower in VCM-1 rats than that in VCM-2 rats. The magnitude of histological change in kidney tissue was in agreement with the degree of alterations in the abovementioned biochemical values. These results suggest that the nephrotoxic effect of once-daily VCM administration is more pronounced than that of the twice-daily treatment. Our findings provide fundamental evidence for the advantage in choosing a divided VCM administration to attenuate nephrotoxicity.

Underestimation of rat serum vancomycin concentrations measured by an enzyme‐multiplied immunoassay technique and the strategy for its avoidance

An enzyme-multiplied immunoassay technique (EMIT) has been widely adopted for the measurement of serum concentrations of vancomycin (VCM) in clinical practice. Because of the growing demand for its application to fundamental pharmacokinetic studies, we examined whether VCM concentrations in rat serum were accurately measured by EMIT. It was found that measured values of known amounts of VCM spiked to rat serum were markedly underestimated with a large analytical variance. When ultrafiltrated rat serum was used as the sample matrix, interference was significantly improved, and the degree of underestimation was attenuated also by diluting samples with physiological saline. These results suggest that endogenous substances of a high molecular weight in rat serum interfere with the analysis of VCM concentrations by EMIT. However, measured values of rat serum VCM concentrations by EMIT were restored to theoretical levels by exposing samples to 70°C for 3-7 min. A likely explanation for the avoidance of interference is that an appropriate thermal force eliminated the immunological function of endogenous substances falsely recognizing VCM without affecting the VCM molecule itself. Regarding serum samples collected from rats that were administered VCM, values measured by EMIT following the heat-treatment agreed well with those by the high performance liquid chromatography (HPLC) method. This is the first report showing interference by endogenous high-molecular substances in the measurement of drug concentrations in rat serum using EMIT. Our findings will contribute to the appropriate use of VCM based on evidence provided by clinical-oriented rat experiments requiring the measurement of serum VCM concentrations by EMIT.

Altered tolbutamide pharmacokinetics by a decrease in hepatic expression of CYP2C6/11 in rats pretreated with 5-fluorouracil

Abstract 1. We investigated the change in the pharmacokinetic profile of tolbutamide (TB), a substrate for CYP2C6/11, 4 days after single administration of 5-fluorouracil (5-FU), and the hepatic gene expression and activity of CYP2C6/11 were also examined in 5-FU-pretreated rats. 2. Regarding the pharmacokinetic parameters of the 5-FU group, the area under the curve (AUC) was significantly increased, and correspondingly, the elimination rate constant at the terminal phase (ke) was significantly decreased without significant change in the volume of distribution at the steady state (Vdss). 3. The metabolic production of 4-hydroxylated TB in hepatic microsomes was significantly reduced by the administration of 5-FU. 4. The expression level of mRNAs for hepatic CYP2C6 and CYP2C11 was significantly lower than in the control group when the rats were pretreated with 5-FU. 5. These results demonstrated that the pharmacokinetic profile of TB was altered by the treatment with 5-FU through a metabolic process, which may be responsible for the decreased CYP2C6/11 expression at mRNA levels.

Decreased elimination clearance of midazolam by doxorubicin through reductions in the metabolic activity of hepatic CYP3A in rats.

Abstract 1. We examined the effects of doxorubicin (DOX) on the expression level and metabolic activity of CYP3A in the liver as well as on the pharmacokinetics of midazolam (MDZ), a probe for CYP3A, in rats. Changes in the hepatic status of DOX-treated rats were confirmed. 2. Serum levels of the biomarkers of hepatic impairment were elevated by the DOX treatment, which was consistent with the results obtained from a histopathological evaluation of the liver. 3. No significant difference was observed in the expression of proteins for hepatic CYP3A1 and CYP3A2 between the DOX and control groups. The metabolic production of 1′-hydroxylated and 4′-hydroxylated MDZ by hepatic microsomes was significantly lower in DOX-treated rats than in control rats. 4. The area under the curve (AUC) and the half-life (t1/2) of intravenously administered MDZ were significantly increased, and the total clearance (CLtot) and the elimination rate constant at the terminal phase (ke) were significantly decreased without significant changes in the volume of distribution at a steady state (Vdss). 5. These results indicated that a DOX-induced depression in the metabolic activity, but not expression level of CYP3A contributed to a decrease in the elimination clearance of MDZ, and also that reduced CYP3A function may be associated with the hepatotoxicity of DOX.

Change in pharmacokinetic behavior of intravenously administered midazolam due to increased CYP3A2 expression in rats treated with menthol.

Menthol is used widely as a constituent of functional foods and chemical drugs. The present study investigated changes in the pharmacokinetic behavior of intravenously administered midazolam (MDZ), a probe for CYP3A, when rats were treated with menthol. The study also examined which isoforms of CYP3A1 and 3A2 were menthol‐inducible and contributed to the altered disposition of midazolam. Menthol was administered intraperitoneally to rats once daily for 3 days at a dose of 10 mg/kg, while the control rats received vehicle alone. The pharmacokinetic examination of i.v. administered midazolam revealed that serum midazolam concentrations at each sampling point were lower in the menthol‐treated rats than in the control rats. Regarding the pharmacokinetic parameters of the menthol‐treated group, the area under the curve (AUC) was decreased significantly and, correspondingly, the elimination rate constant at terminal phase (ke) was increased significantly without significant changes in the volume of distribution at steady state (Vdss). The metabolic production of the 1′‐hydroxylated and 4′‐hydroxylated forms of MDZ by hepatic microsomes was significantly greater in the menthol‐treated rats than in the control rats. The expression levels of mRNA and protein for hepatic CYP3A2 were more than 2.5‐fold higher than the control levels when the rats were treated with menthol, whereas no changes were observed in the expression levels of CYP3A1. These results indicate that menthol enhanced the elimination clearance of midazolam by inducing hepatic CYP3A2 and that careful attention should be paid when menthol is ingested in combination with drugs that act as substrates for CYP3A. Copyright

Prevention of Doxorubicin-Induced Renal Toxicity by Theanine in Rats

Doxorubicin (DOX) is a highly potent anti-neoplastic agent widely used in clinical practice, but its dosage and duration of administration are strictly limited due to dose-related organ damage. In the present study, we examined whether theanine, an amino acid derivative found in green tea leaves, can protect against DOX-induced acute nephrotoxicity in rats. Decreases in the creatinine clearance by DOX administration were attenuated by concurrent treatment with theanine, which was consistent with the change in histological renal images assessed by microscopic examination. Theanine had no effect on the distribution of DOX to the kidney. The production of lipid peroxide in the kidney after DOX administration was suppressed by concurrent treatment with theanine. Reduced glutathione content, but not superoxide dismutase activity, was decreased following DOX administration, whereas this change was suppressed when theanine was given in combination with DOX. These results suggest that theanine prevents DOX-induced acute nephrotoxicity through its antioxidant properties.