Fu-Chuo Peng

Evaluation on Cytotoxicity and Genotoxicity of the Exfoliated Silicate Nanoclay

The concern about toxicity for nanosilicate platelets (NSP) derived from natural montmorillonite clay is addressed. The NSP nanoclay was isolated from polyamine-salt exfoliation of the layered silicate clay into randomized individual plates, possessing multiple ionic charges on the surface of silicate plates with an average geometric dimension of ca. 80 x 80 x 1 nm(3). The material had been previously shown to be effective for antimicrobial and tendency for adhering onto the biomaterial surface based on the direct observation by using scanning electron microscope. The material safety on genotoxic effect was investigated by using three different test systems: the Comet assay test on Chinese Hamster Ovary (CHO) cells in vitro, micronucleus (MN) assay in vivo and the Salmonella gene mutation assay on strain TA98, TA100, TA102, TA1535 and TA1537. The Comet assay showed no DNA damage after 24 h of incubation with NSP of 1000 microg/mL. The MN test indicated no significant micronucleus induction in the CHO cells at the concentrations tested. With all five strains of Salmonella typhimurium, none of mutations was found. Furthermore, cytotoxicity of the same material was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release, showing a low cytotoxicity on CHO cells below 1000 microg/mL after 12 h incubation period and a dose-dependent effect after 24 h incubation. For feeding to rats, the acute oral toxicity was shown a low lethal dose (LD(50)) or greater than 5700 mg/kg body weight for both male and female Sprague-Dawley rats. Overall, the study has demonstrated the safety of the NSP for potential uses in biomedical areas.

Alterations in Oxidative Stress Status During Early Alcohol Withdrawal in Alcoholic Patients

BACKGROUND/PURPOSE Alcohol-induced oxidative stress is the result of the combined production of reactive oxygen species [ROS; e.g. malondialdehyde (MDA), an index of lipid peroxidation] and impairment of antioxidant defenses [e.g. superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), which are involved in the elimination of ROS]. Little is known about the oxidative stress markers among patients with alcohol dependence in Taiwan. This study aimed to investigate serial alterations of various oxidative stress markers during early detoxification in alcoholic patients. METHODS We enrolled 121 inpatients who fulfilled the DSM-IV-TR criteria for alcohol dependence, and 19 healthy controls. Fasting serum MDA level and antioxidant activity, including SOD, CAT and GPX, were measured at baseline in both groups, and after 1 and 2 weeks of detoxification in alcoholic patients. RESULTS MDA level in alcoholics was higher at baseline than in healthy controls. It decreased after 1 week of detoxification, and normalized at week 2. SOD and GPX activities remained significantly lower throughout the 2-week period. CAT activity in alcoholics was comparable to that in the controls at baseline, but decreased at week 1 of detoxification, and was significantly lower than that in the controls after 2 weeks. Moreover, baseline MDA level was correlated with baseline CAT activity in alcoholics; the magnitude of the decrease in MDA level was correlated with the decrease in CAT activity following the 1-week detoxification. CONCLUSION The findings suggest severe oxidative stress and weakened antioxidant activity in alcoholic patients, and limited changes in oxidative stress in the early stages of alcohol withdrawal.

Oxidative Status in Patients with Alcohol Dependence: A Clinical Study in Taiwan

The aim of this study is to examine the relationship between alcohol dependence and oxidative status. The biochemical parameters and antioxidants status were measured among 28 patients with alcohol dependence. Nineteen healthy persons without drinking problem were recruited as the control subjects. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyltransferase (γ-GT), and levels of cholesterol, triglyceride (TG), and uric acid were significantly increased in the specimen of patients compared with control. Serum malondialdehyde (MDA) levels of the patients were found to be significantly increased compared with controls and decreased after abstinence. Superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities were, respectively, 86% and 37% lower in alcoholic patients. After 14 d of abstinence, SOD activity was significantly reduced by 85%, CAT by 52%, and GPX by 54%, whereas no change was found in activity of glutathione reductase (GR). The duration of alcohol dependence is significantly correlated with the levels of MDA. In addition, the activity of CAT was significantly correlated with MDA levels. The results of this study suggest that oxidative stress occurred during alcohol dependence and subsequently affected the antioxidants mechanisms.

The correlation between early alcohol withdrawal severity and oxidative stress in patients with alcohol dependence.

Oxidative stress is enhanced in alcoholic patients. This clinical study aimed to explore the correlation between alcohol withdrawal severity and two oxidative stress markers, malondialdehyde (MDA) and superoxide dismutase (SOD). Seventy-six inpatients fulfilled the DSM-IV-TR criteria for alcohol dependence and 19 healthy controls were enrolled. Serum MDA level and SOD activity were measured within 24 h of alcohol detoxification. The severity of alcohol withdrawal was evaluated by the Chinese version of the revised Clinical Institute Withdrawal Assessment for Alcohol Scale (CIWA-Ar-C) every 8 h. Average and highest scores of the CIWA-Ar-C at the first day were recorded as the baseline withdrawal severity. We compared the differences of MDA and SOD between groups, and examined the correlation between baseline withdrawal severity and oxidative stress markers. Compared to controls, serum MDA levels were significantly elevated and SOD activity was significantly lowered in alcoholic patients. In stepwise multiple regression analysis, MDA was the only variable significantly correlated with the average (beta=0.48, p<0.0001) and highest (beta=0.47, p<0.0001) CIWA-Ar-C scores at the first day of detoxification. In agreement with previous studies, alcoholic patients encountered high oxidative stress. Although there was a correlation between early withdrawal severity and MDA levels, the meanings of the correlation are worth further studies in the future.

Rapid determination of aristolochic acids I and II in herbal products and biological samples by ultra-high-pressure liquid chromatography-tandem mass spectrometry

Aristolochic acids (AAs) are a mixture of structural-related compounds, in which aristolochic acid I (AA I) and aristolochic acid II (AA II) are reported to be correlated with Aristolochic acid nephropathy (AAN). In this work, a rapid and sensitive ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to determine AA I and AA II in herbal products and biological fluids. By using gradient elution with a mobile phase composed of a mixture of 10mM ammonium formate buffer (pH 3.0) and acetonitrile, AAs could be determined within 10 min. Under optimum UHPLC-MS/MS conditions, the limit of detections was 0.14 and 0.26 ng mL(-1) for AA I and AA II, respectively. Run-to-run repeatability and intermediate precision of peak area for AA I and AA II were less than 5.74% relative standard deviation (RSD). Accuracy was tested by spiking 10, 100 and 1000 ng mL(-1) in rat serum and the recoveries were within 76.5-92.9%. Matrix effects were within 78.8-127.7%. The developed method was successfully applied to determine AA I and AA II in several herbal products and to investigate their pharmacokinetic behavior in female Wister rats. The result shows that the developed UHPLC-MS/MS method is efficient, sensitive, and accurate for the determination of AA I and AA II in herbal products and biological samples.

Toxicity and anticholinesterase activity of the fungal metabolites territrems to the corn earworm, Helicoverpa zea

The toxicity and anticholinesterase activity of tremorgenic fungal metabolites, territrems, to the corn earworm, Helioverpa zea (Boddie) (Lepidoptera, Noctuidae) were examined. In oral toxicity studies, territrem A significantly inhibited growth by 40% at 25 ppm and by 89% at 250 ppm. Territrem B and an epoxy‐derivative significantly inhibited growth by ca. 45% at 250 ppm. Piperonyl butoxide administered orally synergized the toxicity of the territrems tested. In topical toxicity studies, the epoxy‐derivative caused 26% mortality and 25% growth retardation at 10 mg/gm insects. Territrem A and B were not significantly lethal, but did reduce growth by 15–20% at 10 mg/gm insect. Paraoxon tested in the same way caused 100% mortality at 25 ppm orally and 10 mg/gm topically. However, all territrems tested in vitro as inhibitors of H. zea head acetylcholinesterase were at least comparable to or were more active than paraoxon. Topically administered epoxy‐territrem B also inhibited H. zea head acetylcholinest‐erase. The potential for development of new insecticides from a territrem lead structure is discussed.

Nanohybrids of Silver Particles Immobilized on Silicate Platelet for Infected Wound Healing

Silver nanoparticles supported on nanoscale silicate platelets (AgNP/NSP) possess interesting properties, including a large surface area and high biocide effectiveness. The nanohybrid of AgNP/NSP at a weight ratio 7/93 contains 5-nm Ag particles supported on the surface of platelets with dimensions of approximately 80×80×1 nm3. The nanohybrid expresses a trend of lower cytotoxicity at the concentration of 8.75 ppm Ag and low genotoxicity. Compared with conventional silver ions and the organically dispersed AgNPs, the nanohybrid promotes wound healing. We investigated overall wound healing by using acute burn and excision wound healing models. Tests on both infected wound models of mice were compared among the AgNP/NSP, polymer-dispersed AgNPs, the commercially available Aquacel, and silver sulfadiazine. The AgNP/NSP nanohybrid was superior for wound appearance, but had similar wound healing rates, vascular endothelial growth factor (VEGF)-A levels and transforming growth factor (TGF)-β1 expressions to Aquacel and silver sulfadiazine.

IL-6 Regulates Mcl-1L Expression through the JAK/PI3K/Akt/CREB Signaling Pathway in Hepatocytes: Implication of an Anti-Apoptotic Role during Liver Regeneration

Aims To investigate the role and the regulation of the long variant of myeloid cell leukemia-1 protein (Mcl-1L) during liver regeneration. Background Liver regeneration is an important phenomenon after liver injury. The rat partial hepatectomy (PH) model was used to characterize liver regeneration and Mcl-1L expression after PH. Methods Male Wistar rats were subjected to 70% PH. The expression of mcl-1L mRNA was determined by quantitative RT-PCR, and protein levels were analyzed by Western blot analysis and immunohistochemistry during liver regeneration. Functional evaluations of Mcl-1L were tested using chemical inhibition (flavopiridol), genetic inhibition (siRNA) of Mcl-1L production, and by assaying for annexin V levels and DNA ladder formation. Serum IL-6 levels were determined by enzyme immunoassays; signal transduction of IL-6-regulated Mcl-1L expression was verified by chemical inhibitors and decoy double-stranded oligodeoxynucleotides. Results High levels of Mcl-1L were observed in remnant tissue at 4 h after PH. Administration of flavopiridol decreased Mcl-1L accumulation and also inhibited liver regeneration. IL-6 administration promoted the accumulation of Mcl-1L in rat hepatocytes, an effect that was impaired by siRNA treatments that reduced Mcl-1L production. Chemical inhibition and decoy oligonucleotide competition demonstrated that IL-6-induced Mcl-1L production required signaling mediated by JAK kinase, phosphoinositide 3-kinase (PI3K), and cAMP response-element-binding (CREB) proteins. Conclusion Mcl-1L is an anti-apoptotic protein induced during liver regeneration after PH in rats. The expression of Mcl-1L is induced by IL-6 through the JAK/PI3K/Akt/CREB signaling pathway. Chemotherapy drugs that depend on Mcl-1L- or IL-6-related signaling should be considered carefully before use in patients undergoing hepatectomy for malignant tumor resection.

Structure-based development of bacterial nitroreductase against nitrobenzodiazepine-induced hypnosis

Nitrobenzodiazepine (NBDZ) is an addictive drug of the abused substances that causes severe neurological effects and even death. Bacterial type I nitroreductase NfsB (EC 1.5.1.34) has been reported to catalyze NBDZ into inactive metabolite 7-amino-benzodiazepine (7ABDZ) with promising activity, so as to become an attractive candidate for treatment of NBDZ overdose and addiction. Here, we investigate the nitroreduction of an NBDZ, flunitrazepam (FZ), by various mutants of NfsB designed from the solved crystal structure and characterize their in vitro and in vivo potency. Conformational changes occurred in the active site of N71S/F124W in contrast to the wild-type, including the flipping on the aromatic rings of W124 and F70 as well as the extension on the hydrogen bond network between flavin mononucleotide (FMN) and S71, which allow the significant enlargement in the active site pocket. In the complex structure of N71S/F124W and nicotinamide (NIA), stacking sandwich attractions of W124-FMN-NIA were also found, implying the importance of W124 in substrate accessibility. Consequently, N71S/F124W exhibited increased 7AFZ production in vitro with nearly no toxicity and reduced 50% sleeping time (hypnosis) in vivo. Taken together, we demonstrate for the first time that N71S/F124W can serve as an effective antidote for NBDZ-induced hypnosis and provide the molecular basis for designing NfsB and the like in the future.

Lysophosphatidic acid alters the expression profiles of angiogenic factors, cytokines, and chemokines in mouse liver sinusoidal endothelial cells

Background and Aims Lysophosphatidic acid (LPA) is a multi-function glycerophospholipid. LPA affects the proliferation of hepatocytes and stellate cells in vitro, and in a partial hepatectomy induced liver regeneration model, the circulating LPA levels and LPA receptor (LPAR) expression levels in liver tissue are significantly changed. Liver sinusoidal endothelial cells (Lsecs) play an important role during liver regeneration. However, the effects of LPA on Lsecs are not well known. Thus, we investigated the effects of LPA on the expression profiles of angiogenic factors, cytokines, and chemokines in Lsecs. Methods Mouse Lsecs were isolated using CD31-coated magnetic beads. The mRNA expression levels of LPAR’s and other target genes were determined by quantitative RT-PCR. The protein levels of angiogenesis factors, cytokines, and chemokines were determined using protein arrays and enzyme immunoassay (EIA). Critical LPAR related signal transduction was verified by using an appropriate chemical inhibitor. Results LPAR1 and LPAR3 mRNA’s were expressed in mouse LPA-treated Lsecs. Treating Lsecs with a physiological level of LPA significantly enhanced the protein levels of angiogenesis related proteins (cyr61 and TIMP-1), cytokines (C5/C5a, M-CSF, and SDF-1), and chemokines (MCP-5, gp130, CCL28, and CXCL16). The LPAR1 and LPAR3 antagonist ki16425 significantly inhibited the LPA-enhanced expression of cyr61, TIMP-1, SDF-1, MCP-5, gp130, CCL28, and CXCL16, but not that of C5/C5a or M-CSF. LPA-induced C5/C5a and M-CSF expression may have been through an indirect regulation mechanism. Conclusion LPA regulated the expression profiles of angiogenic factors, cytokines, and chemokines in Lsecs that was mediated via LPAR1 and LPAR3 signaling. Most of the factors that were enhanced by LPA have been found to play critical roles during liver regeneration. Thus, these results may prove useful for manipulating LPA effects on liver regeneration.