Sex differences have been found in the pharmacokinetics of many drugs, and sex differences of drug-metabolizing enzymes have been considered one of the major factors of this issue. Cytochrome P450, a Phase I drug-metabolizing enzyme, consists of many isoforms having divergent substrate specificities. Some isoforms in rodents show sex-specific expression, and some in humans also show moderate differences, e.g., the activity of CYP2E1 and CYP1A2 is slightly higher in men than women. CYP3A4, the most clinically relevant isoform in humans, appears to have greater expression in women, as determined by mRNA and protein levels as well as the activities for more than ten clinically employed drugs, and the difference is increased by induction of the gene expression during pregnancy, implying the need to adjust the dose of a variety of drugs ingested by pregnant women. Regarding the mechanism of the sexually dimorphic expression of CYP3A genes, at least two hypotheses have been suggested. The first is that pregnane X receptor (PXR), activated by a higher concentration of female sex hormones, enhances the expression of PXR-target genes, including CYP3A. The second is that the different secretion patterns of growth hormone between men and women activate divergent sets of the signal transduction cascade discriminately, resulting in the sexually dimorphic expression of subsets of genes. In this review, we mainly introduce studies of female-specific CYP3A genes due to the recent progress of analysis.

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Molecularly targeted therapy has become widely used in clinical settings over the last decade. Although it was initially expected that molecularly targeted drugs have fewer side effects, it is becoming increasingly apparent that molecularly targeted drugs have an unanticipated repertoire of side effects. Some side effects are serious, some are manageable, some are rare, and some are frequent. Some may affect individuals with predisposing factors. In this minireview, we briefly summarize how the side effects of molecularly targeted drugs were discovered when new classes of drugs were introduced. We also summarize the clinical characteristics of these side effects focusing on progressive multifocal leukoencephalopathy (PML) associated with the use of an integrin antagonist, cardiotoxicity associated with the use of some tyrosine kinase inhibitors, and hypertension associated with the use of angiogenesis inhibitors. We also review the molecular mechanisms underlying these side effects. Research on the mechanisms underlying these side effects has revealed previously unknown physiological roles of targeted molecules. Awareness and understanding of the side effects of molecularly targeted drugs is important for those working in clinical practice and conducting basic research.

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Quinones are ubiquitously present in the environment. This review summarizes the cellular toxicity of 9, 10-phenanthraquinone (9, 10-PQ), a component of diesel exhaust particles, and the cytoprotective mechanism of the nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) system against oxidative stress exerted by 9, 10-PQ.

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Surfactin C, produced by acillus subtilis isolated from Korean soybean paste, was given to Sprague-Dawley rats of both sexes at dose of 500, 1000 or 2000 mg/kg for 28 days. There were no surfactin C-related toxicities in survival, clinical signs, and hematological parameters in the experimental period. The highest dose of surfactin C showed the decrease in body weight gain despite normal food and water consumptions and the increase in relative liver weight. alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels were increased in animals administered with surfactin C of 1000 or 2000 mg/kg. Zonal necrosis of hepatocyte around the hepatic vein was observed after administration of the same doses in a dose-dependent manner. In the present study, the no-observed-adverse-effect level (NOAEL) of surfactin C was 500 mg/kg following oral administration in rats.

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Intestinal condition and ethanol toxicity have been discussed as predictors of alcoholic liver damage. In this study, we investigated the association of hepatic antioxidant enzymes and cecal condition, including intestinal bacteria estimated by terminal restriction fragment length polymorphism (T-RFLP), in the early stage of alcoholic fatty liver. Three liquid isocaloric diets, control (CT) diet, ethanol (ET) diet, or ethanol diet including modified beet fiber (MBF), were prepared and administered to rats for 4 weeks. At 4 weeks, the plasma alanine aminotransferase (ALT) levels in the ET group were higher and that in MBF groups tended to be higher than the CT group, but endotoxin was not detected in the portal vein in any of the samples. The hepatic thiobarbituric acid reactive substances (TBARS) concentration in the MBF group was higher than that in the CT group. Hepatic cytochrome P450 2E1 (CYP2E1) was induced in ethanol-fed rats. The hepatic catalase levels in the ET and MBF groups were lower than that in the CT group. The hepatic superoxide dismutase-2 (SOD-2) level did not differ among the groups. Plasma ALT level and/or hepatic TBARS level correlated positively with CYP2E1 and negatively with catalase level and SOD-2 level. Indicators of cecal condition, including the bacterial population estimated by T-RFLP, may not give any explanation for the hepatic damage. In conclusion, the hepatic CYP2E1 induced by ethanol, rather than the cecal conditions, may influence the early stage of alcoholic hepatic damage in rats; hepatic catalase may provide protective effects.

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Reactions of sunscreen agents, octyl dimethyl-p-aminobenzoate (ODPABA) and octyl-p-methoxycinnamate (OMC), with hypochlorite in aqueous solution were investigated under the conditions that simulate swimming pool disinfection sites. Chlorination byproducts were determined by GC-MS. At a concentration of 9 μM, ODPABA reacted rapidly with free chlorine in the buffered solution at pH 7.0, OMC reacted with hypochlorite reasonably slowly under the same condition. ODPABA and OMC produced chlorine-substituted compounds as intermediates, which were decomposed to cleavaged products of ester-bond during the aqueous chlorination process. The chlorination intermediates of OMC exhibited weak mutagenic on Salmonella typhimurium TA100 strain without the S9 mix. The extent of the reactions depended on the chlorine dose, solution pH, and compound structures.

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Pleurocybella porrigens (P. porrigens) is a traditional food consumed in Japan. Toxicity was first reported in 2004, following which a series of poisonings were reported in 2007. More than 59 people who consumed P. porrigens suffered from similar severe cryptogenic encephalitis, with an overall death rate of approximately 29%. P. porrigens is believed to be a major etiological agent of this disease, but the mechanism of pathogenesis is not clear. To elucidate the toxic properties of P. porrigens in the 2004 and 2007 poisonings, we compared the oligosaccharide constituents of mushroom samples collected in these years with those collected in other years. Water extracts (90°C and 4°C) of P. porrigens were dialyzed, and the oligosaccharides obtained from the high-molecular-weight fraction (>7.8 kDa) were subjected to acid hydrolysis for modification and labeling. Resultant saccharides were analyzed by high performance liquid chromatography on an octadecyl silane (ODS) column. Our analysis revealed that the concentration of N-acetylneuraminic acid (NeuAc) was abundant in all samples, however, N-glycolylneuraminic acid (NeuGc) was present only in significant amounts in the P. porrigens samples collected in 2004 and 2007.

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Di(2-ethylhexyl) phthalate (DEHP) used as a common plasticizer additive in the manufacture of plastics, such as polyvinyl chloride (PVC). This study examined the effect of DEHP on steroidogenesis or spermatogenesis in the testes of Sprague-Dawley male rats treated orally with 250, 500, 750 mg/kg over a 30-day period. The expression levels of the steroidogenic- or spermatogenic-related genes were analyzed in the testis using a reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. High doses of DEHP (500 and 750 mg/kg) significantly decreased the testicular sperm counts and daily sperm production (DSP). In addition, serum testosterone levels were significantly lower in the DEHP treatment groups than in the control. The mRNA levels of SR-B1, StAR, PBR and CYP17 increased in a dose-dependent manner. These increases were significant at 500 and 750 mg/kg. In the other hand, the mRNA levels of CYP19 decreased significantly in testes of rats exposed to DEHP 500 and 750 mg/kg. Dose-dependent decreases in Spag4 and LDHA mRNA in the testis were observed after DEHP exposure, while there was a significant decrease in thyroid hormone receptor (TR)α1 protein levels. High doses of DEHP significantly increased the expression of peroxisome proliferator-activated receptor (PPAR)-r and retinoid X receptor (RXR)-α protein but markedly decreased the expression of RXR-r. These results suggest that DEHP exposure can alter the expression of the spermatogenic- or steroidogenic-related genes resulting in a decrease in sperm production in the testis. This study is expected to be helpful in research examining the mechanisms for how DEHP reduces the expression pattern of the genes involved steroid hormone synthesis after chronic exposure to DEHP.

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The C-type natriuretic peptide stimulates osteoblastic functions through the B-type natriuretic receptor (NPR-B). In this study, we examined the signaling pathway behind the regulation of NPR-B expression through the prostaglandin E₂ (PGE₂) receptor, EP1 subtype using rat calvarial osteoblasts. A23187 as a Ca²⁺ ionophore increased NPR-B expression dose-dependently. PGE₂ or 17-phenyl-ω-trinor PGE₂ (EP1A), an EP1 agonist, increased NPR-B expression, and the potentiating effects were blocked by treating with BAPTA-AM as an intracellular Ca²⁺ chelator. Activators of protein kinase C (PKC), 1-oleoyl-2-acetyl-sn-glycerol, a membrane-permeable diacylglycerol, and 12-o-tetradecanoyl-phorbol-13-acetate, also increased NPR-B expression, and the potentiating effects were blocked by treating with BAPTA-AM. The treatment of cells with GF109203X, a PKC inhibitor, blocked the PGE₂- and EP1A-induced increase in NPR-B expression. From these results, we concluded that EP1-mediated increase in the expression of NPR-B requires not only Ca²⁺ mobilization but also PKC activation through the activation of phosphatidylinositol-specific phospholipase C.

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To clarify the mechanism underlying the enhancing effect of 17β-estradiol (E2) on contact hypersensitivity (CHS) and the expression of interferon (IFN)-γ in mice, the mRNA expression levels of interleukin (IL)-18 were evaluated. Female BALB/c mice aged 3 weeks were ovariectomized, administered 3.2 μg of E2, and sensitized by 50 μl of 3% 4-ethoxymethylene-2-phenyl-2-oxazolin-one (OXA). Seven days later, CHS was elicited by the application of 7.5 μl of 1% OXA on the ear auricles. The auricles, cervical lymph nodes and spleens were excised, and gene expression was evaluated by reverse transcription-polymerase chain reaction. E2 enhanced the expression of IL-18 mRNA in the spleen on the following day and in the ear auricles on days 4 and 7 after sensitization with OXA. The preadministration of an antibody against IL-18 receptor suppressed the CHS and reduced IFN-γ mRNA expression in E2-administered mice. IL-18 was present in the dermis of the ear skin and absent in the epidermis. E2 also enhanced the expression of IFN-γ and IL-18 mRNAs in splenocytes cultured with lipopolysaccharide (LPS). IL-18 protein was detected by flow cytometry in CD4⁺, CD8⁺ and NKG2⁺ lymphocytes among splenocytes cultured with LPS. These results suggest that E2 enhances lymphocyte activation in the sensitization phase of CHS, and that IFN-γ mRNA expression is enhanced in the elicitation phase of CHS.

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To understand the physiological significance of endogenous zinc release under excess excitation in the hippocampus, in the present study, high K⁺-induced increase in extracellular glutamate and endogenous zinc action against its increase were examined in young rats fed a zinc-deficient diet for 2 weeks. When the ventral hippocampus was perfused by 100mM KCl, the extracellular concentration of glutamate was more increased in zinc-deficient rats than the control rats. Calcium orange signal in mossy fiber boutons was more increased in slices from zinc-deficient rats after delivery of tetanic stimuli (100 Hz, 5 sec) to the dentate granule cell layer. The decrease in FM4-64 signal, which is a measurement of exocytosis, in mossy fiber boutons was also enhanced in slices from zinc-deficient rats. These results suggest that the abnormal increase in extracellular glutamate in the hippocampus induced with high K⁺ in zinc deficiency is due to the enhancement of exocytosis associated with affected Ca²⁺ mobilization. The expression of GLT-1, a glial glutamate transporter, in the hippocampus was higher in zinc-deficient rats, suggesting that GLT-1 protein increased serves to maintain the basal concentration of extracellular glutamate, which was not different between the control and zinc-deficient rats. On the other hand, the increase in extracellular glutamate concentration induced with high K⁺ was enhanced in the presence of 1 mM CaEDTA, a membrane-impermeable zinc chelator, in both the control and zinc-deficient rats. It is likely that zinc released from glutamatergic neurons serves to suppress glutamate release under excess excitation in the hippocampus.

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The present study was undertaken to evaluate the efficacy of freeze dried extract of Sida rhomboidea. roxb leaves (FESR) on alteration in lipid and cholesterol metabolism in high fat diet induced hyperlipidemia in experimental rats. Plasma and hepatic lipid profiles, lipid and cholesterol metabolizing enzymes in target tissues and fecal total lipids and bile acid contents were evaluated in FESR treated normolipidemic and hyperlipidemic rats. These results were compared with synthetic hypolipidemic drug lovastatin (LVS). Results indicate that FESR was able to positively regulate induced experimental hyperlipidemia by significant alteration in plasma and tissue lipid profiles. These results can be attributed to reduced absorption, effective elimination and augmented catabolism of lipids and cholesterol possibly due to high content of saponin and phytosterols in S. Rhomboidea. roxb (SR). Use of SR extract as a potential therapeutic agent against hyperlipidemia is indicated.

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Based upon the Framework Convention on Tobacco Control (FCTC), the World Health Organization (WHO) has recommended that health authorities disclose toxicological properties of cigarette mainstream smoke (MSS) obtained not only according to US Federal Trade Commission (FTC)/International Organization for Standardization (ISO) conditions but also by more intense conditions such as the Health Canada Intense (HCI) condition. This is because smokers are believed to smoke more intensely than machine smoking under the ISO regimen. Because there are no previous reports on the toxicological properties of MSS of Japanese cigarettes under the HCI condition, we determined nicotine and water contents by gas chromatography (GC-MS and GC/Thermal Conductivity Detector (TCD)) for three product lots each of the ten bestselling brands of Japanese cigarettes following the WHO protocol. One of the three lots of each MSS condensate was also resolved in dimethylsulfoxide and investigated by Ames preincubation assay using Salmonella typhimurium TA100, TA98, and YG1024 strains with and without metabolic activation (rat liver S9 mix). Nicotine and tar yields with the HCI regimen were higher than those with ISO, the latter being very close to the values described on the packages of each cigarette brand. Mutagenicity was mainly observed in TA98 and YG1024 with metabolic activation. Mutagenic activity of MSS with the HCI regimen was 1.4-9 times higher than that with the ISO regimen. Based on YG1024 with activation, high activities were observed in several “low yield” brands. The activity of “low-yield” brands with the HCI regimen was not always lower than that of regular-yield brands with the ISO regimen. These results suggest that “low yield” cigarettes do not result in reduced exposure or reduced risk to humans.

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We studied changes in protein expressions using human monoblastic leukemia cells (U937) exposed to carbon black (CB) to explore the possibility of a new safety evaluation method using a proteomic approach. The cells exposed to CB with a particle size of approximately 85 nm did not show cell growth inhibition by 96 hr. However, when we performed two-dimensional electrophoresis with U937 cell lysates four times and analyzed the gel images, we found that the CB-exposed cells had changed significantly in 14 protein spots, with expression amounts being different by more than two-fold those at 96 hr. Only one protein was up-regulated while the others were downregulated. Ten of the 14 spots were identified using peptide mass fingerprinting: transportin 1, thioredoxin domain containing 5, annexin A2, otubain 1, 14-3-3 protein γ, etc. The functions of these proteins are associated with metabolism, responses to stress, signal transduction, and cell differentiation. According to our research, CB undoubtedly causes biological responses, but those responses are not detectable by conventional methods. Regarding safety risk, the proteomic approach could possibly detect biological responses more sensitively than conventional in vitro evaluation methods.

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We performed a prospective study to assess the optimal blood concentration of tacrolimus in Japanese patients with rheumatoid arthritis (RA). The dose of tacrolimus (1-3mg/day) was administered orally once daily after the evening meal, and the blood concentration of tacrolimus 12 hr after administration was quantified. A total of 23 patients were enrolled. Clinical efficacy was assessed using the Disease Activity Score in 28 joints and European League Against Rheumatism (EULAR) response criteria at each outpatient visit during months 1-6. The ratio of patients who showed a moderate or good response was 47.8% (11/23). The mean blood concentrations of tacrolimus in EULAR responders and EULAR non-responders were 5.5±3.6 (mean±S.D.) and 3.1±1.9 ng/ml, respectively (p=0.069). Interestingly, although tacrolimus had lower mean blood concentrations (<5 ng/ml) compared with transplant fields (10-20 ng/ml), six (35.3%) of 17 patients showed sufficient response to tacrolimus. In addition, in five patients, tacrolimus was co-administered with methotrexate (MTX). On the other hand, the mean blood concentrations of tacrolimus in patients who did or did not develop adverse events were 4.8±4.4 (n=8) and 4.0±2.1 (n=15) ng/ml, respectively (p=0.624). We failed to clear the optimal blood concentration of tacrolimus in RA patients, but one of the most remarkable findings was the observation that patients in whom tacrolimus was combined with MTX reached a positive response at much lower tacrolimus concentrations compared to patients not co-treated with MTX.

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Catechins show various biological activities, for which beneficial as well as adverse effects have been observed. Unfavorable properties of catechins are often attributed to reactive oxygen species (ROS) production. Our aim was to investigate whether catechins affect protein via ROS generation. We examined the effects on bovine serum albumin (BSA) of (+)-catechin with Cu²⁺ as well as ascorbate with Cu²⁺. The ascorbate/Cu²⁺ system is a metal-catalyzed oxidation (MCO) system and can produce ROS. Both systems altered spectroscopic properties of albumin, increased protein carbonyl content and resulted in several conformational changes. Catalase and hydroxyl radical scavengers markedly suppressed protein modifications by the ascorbate/Cu²⁺ systembut not those by the (+)-catechin/Cu²⁺ system. As far as we observed, no protein alteration induced by the (+)-catechin/Cu²⁺ system correlated with ROS formation.

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Ferrous ferric chloride (FFC^®) is a distinct form of aqueous iron made up of a complex of ferrous chloride and ferric chloride that participates in both oxidation and reduction reactions. Although FFC is known to stimulate cellular function, its precise mechanism of action is largely unknown. My previous study showed that FFC stimulated the proliferation of cultured epidermal keratinocytes and melanocytes derived from newborn mice, suggesting that FFC stimulates their function of mice. However, it is not known whether FFC stimulates the proliferation of keratinocytes and melanocytes derived from human skin. Moreover, it is unknown whether FFC stimulates the proliferation of human skin fibroblasts. Pairogen^® (Akatsuka Co., Tsu, Japan) is a drink comprising FFC, vinegars, and vitamins, Pairogen Gold^® (Akatsuka) contains herbal medicines in addition to FFC, vinegars, and vitamins, while Noncalorie Pairogen^® (Akatsuka) contains low molecular weight collagen and hyaluronic acid in addition to FFC, vinegars and vitamins. To clarify whether FFC in addition to the natural factors included in these drinks stimulates the proliferation of human skin cells, FFC and these different drinks were added to culture media and tested for their proliferation-stimulating activity on skin cells. FFC, Pairogen, and Pairogen Gold equally doubled the growth rate of cultured human keratinocytes, melanocytes, and fibroblasts. Non-calorie Pairogen tripled the growth rate of human fibroblasts, but Non-calorie Pairogen only doubled the growth rate of human keratinocytes and melanocytes. FFC plus low molecular weight collagen tripled the growth rate of human fibroblasts, whereas FFC plus low molecular weight hyaluronic acid only doubled the growth rate of human fibroblasts. Low molecular weight collagen, with or without hyaluronic acid failed to stimulate the proliferation of fibroblasts. These results suggest that FFC stimulates the proliferation of human skin keratinocytes and melanocytes. Moreover, FFC markedly stimulates the proliferation of human skin fibroblasts synergistically with low molecular weight collagen.

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In recent years, the use of silver nanoparticles (SNPs) is gaining ground especially on its use as disinfectant or antiseptic. The application of SNPs in the manufacture of medical devices and products is attributed to its high antibacterial efficacy. But while most of the studies on SNPs focus on its direct effect on bacteria, investigations have not been done on the effect of SNPs on mammalian innate immune molecules that possess bacterial killing capacity. Thus, in this report, we determined whether SNPs could affect naturally occurring antimicrobial molecules. We used here a silver nanoparticle, termed S (Atomyball S^®), which has a size of 5 nanometers (nm), and treated it to lung epithelial cell lines A549 and Calu-3. A low concentration of nanoparticle S (5 μg/ml) did not induce cell toxicity as determined by lactate dehydrogenase assay. Interestingly, we observed that S increased the mRNA and protein expression levels of the antimicrobial peptide lysozyme, which is considered as one of the most important host immune defense molecules in human epithelial tissues. Quantification of the secreted lysozyme in the apical surface fluid obtained from air-liquid interface cultures of Calu-3 cells also revealed a significant increase of lysozyme in cells treated with nanoparticle S. Although the mechanism of its action on lysozyme is yet to be elucidated, these findings firstly suggest that the silver nanoparticle S (Atomyball S^®) positively regulated the expression of antimicrobial peptide lysozyme in mammalian cells.

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Naringin is a flavonoid commonly found in citrus fruits. Previous studies have supported a positive association between fruit consumption and health including bone quality. To clarify the role of naringin in bone turnover, we examined the effect of naringin on bone mass in mice, and on osteoclast formation induced by bone-resorbing cytokine. When mice were orally administered naringin for 2 weeks, femoral bone mineral density (BMD) was clearly elevated compared with control mice. BMD in distal and middle portion was significantly enhanced, but proximal BMD was not changed by naringin administration. In soft-X-ray analysis, a marked accumulation of cancellous bone could be detected in distal femoral metasphysis in mice treated with naringin. In cocultures of bone marrow cells and osteoblasts, naringin dose-dependently suppressed the number of osteoclasts formed by treatment with interleukin-1 (IL-1). The size of osteoclasts formed in the presence of naringin was smaller than that induced by IL-1. Naringin enhances bone mass possibly by suppression of osteoclast formation in mice, suggesting the role of the citrus flavonoid on bone health and to prevent bone diseases such as osteoporosis.

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Transforming growth factor-β (TGF-β) is a potent growth factor that contributes to wound healing. TGF-β is usually secreted in a latent form complexed with its propeptide, latency-associated peptide (LAP), and LAP covalently binds to a molecule of latent TGF-β binding protein (LTBP). Fibrillin-1 sequesters TGF-β within connective tissue microfibrils through interaction with LTBP-1. However, it is not clear whether TGF-β bound to LTBP-1 is available during wound healing. Therefore, we further characterized LTBP-1, the extracellular regulator of TGF-β in wound healing. LTBP-1 fragments were released from skin by plasmin treatment. The LTBP-1 fragment that is similar to plasmin treatment was also detected in a wound surface. The enzymatic activity of plasmin was also detected in wound surfaces. Immunoblotting analyses showed that the LTBP-1 fragment was preferentially detected in a wound surface with proliferating granulation tissues. These results suggest that proteolytic release of LTBP-1 from a wound surface is physiological and important in regulating wound healing.

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