Genotoxicity and 28-day oral toxicity studies of a functional food mixture containing maltodextrin, white kidney bean extract, mulberry leaf extract, and niacin-bound chromium complex.

Steady-fiber granule (SFG) is a functional food mixture that is composed of four major ingredients, resistant maltodextrin, white kidney bean (Phaseolus vulgaris) extract, mulberry leaf (Morus alba L.) extract, and niacin-bound chromium complex. This study focused on determining the safety of SFG. Genotoxicity and 28-day oral toxicity were evaluated. SFG did not induce mutagenicity in the bacterial reverse mutation assay using five Salmonella typhimurium strains (TA98, TA100, TA102, TA1535, and TA1537) in the presence or absence of metabolic activation (S9 system). SFG also did not induce clastogenic effects in Chinese hamster ovary cells with or without S9 treatment. Similarly, SFG did not induce genotoxicity in a micronucleus test conducted with mice. A dose-dependent 28-day oral toxicity assessment of SFG for rats revealed no significant effects on mortality, body weight, selected organ weights, and behavior. Evaluations of hematology, clinical biochemistry, and histopathology showed no adverse effects in rats treated with SFG. These results suggest that SFG has no significant mutagenic or toxic properties, and the no observed adverse effect level of SFG was defined as at least 5000 mg/kg/day orally for 28 days for male and female rats.

Genotoxicity and 28-day repeated dose oral toxicity study of garlic essential oil in mice.

Background and aim: Garlic essential oil (GEO) isolated from Garlic (Allium sativum L.) exerts biological activities in disease prevention, particularly in metabolic and liver diseases, and is used for a dietary therapy for centuries. However, due to the side effects associated with the excessive consumption of GEO, there is a need to evaluate the safety of the GEO. Experimental procedure: Ames test using five Salmonella typhimurium strains (TA98, TA100, TA102, TA1535, and TA1537) and Chinese hamster ovary (CHO-K1) cells with or without metabolic activation (S9 system), and mammalian erythrocyte micronucleus test were used to assess the genotoxicity and clastogenic effects of GEO. A repeated dose of GEO (15, 25, and 50 mg/kg body weight, p.o.) were administrated to ICR mice for 28 days to ascertain the subacute toxicity of GEO. Results and conclusions: The results of the Ames test with or without S9 system indicated that GEO did not induce mutagenicity nor have clastogenic effects in CHO-K1 cells with or without S9 activation. Furthermore, GEO did not affect the ratio of immature to total erythrocytes or the number of micronuclei in immature erythrocytes of ICR mice after 24 and 48 h. In a 28-day oral toxicity assessment, GEO (15, 25, and 50 mg/kg body weight, p.o.)-fed ICR mice exhibited normal behaviors, mortality, body weight, daily intake, hematology, clinical biochemistry, and organ weight. GEO shows no genotoxicity, and the no-observed-adverse-effect level (NOAEL) for GEO is considered to be greater than 50 mg/kg bw/day orally for 28 days in mice.

Composition of nanoclay supported silver nanoparticles in furtherance of mitigating cytotoxicity and genotoxicity.

Silver nanoparticle (Ag-NP) is well known for its high antibacterial efficacy. However, its toxicity toward mammalian cells is still a concern in clinical applications. The aim of our study was to evaluate the composition effects of Ag-NP supported by silicate nanoplatelet (NSP) with respect to the cytotoxicity and genotoxicity, and was in reference to the poly (styrene-co-maleic anhydride)-supported Ag-NP (Ag-NP/SMA). The NSP at the geometric dimension of averaged 80 x 80 x 1 nm3 was prepared from the exfoliation of natural clays and used to support different weight ratio of Ag-NP. The supporting limitation of NSP on Ag-NP was below the weight ratio of 15/85 (Ag-NP to NSP), and the detached Ag-NP from the Ag-NP/NSP (30/70) and Ag-NP/SMA hybrids were observed by TEM. Ames test was performed to assess the mutagenic potential of different compositions of Ag-NP/NSP, only Ag-NP/NSP (30/70) and Ag-NP/SMA hybrids exhibited mutagenicity when the concentration was 1.09 ppm or higher. In viewing of cytotoxicity using MTT tests toward HaCaT cells, the IC30 of Ag-NP/NSP (1/99, 7/93 and 15/85) were 1416.7, 243.6, and 148.9 ppm respectively, while Ag-NP/SMA was 64.8 ppm. The IC30 of Ag-NP/NSP (1/99, 7/93 and 15/85) were at least 833, 78 and 7 folds higher than their corresponding minimum inhibitory concentrations (MIC) respectively, and whereas Ag-NP/SMA was 6.4 folds. The Ag-NP/NSP and Ag-NP/SMA hybrids had been further investigated for genotoxicity by chromosomal aberrations and in vivo micronucleus assay within the concentration at IC10 and IC30, only Ag-NP/SMA showed a higher frequency of chromosomal aberrations. Our findings indicated that the viability of utilizing the NSP to maintain Ag-NP for antimicrobial activity, and the high-surface area of NSP served as an excellent support for associating Ag-NP and consequently rendering the mitigation of the inherent toxicity of Ag-NP in clinical uses.

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.

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.

Predicting the contribution of rat cytochrome P-450 3A1, 3A2 and human cytochrome P-450 3A4, 3A5 to territrem a 4beta-C hydroxylation using the relative activity factor.

The relative activity factor (RAF) was used to predict the contribution of different cytochrome P-450 (CYP) 3A isoforms (3A1 and 3A2 in rat liver microsomes and 3A4 and 3A5 in human liver microsomes) to 4beta-C hydroxylation of territrem A (TRA). Seven recombinant rat and eight recombinant human CYP450 isoforms, five rat liver microsomes, and seven human liver microsomes were assessed. In liver microsomes from five male Wistar rats, TRA 4beta-C hydroxylation activity significantly correlated with CYP3A1/2 activity, while, in liver microsomes from seven humans, there was marked correlation with CYP3A4 activity. Immunoinhibition confirmed that CYP3A2 and CYP3A4 were responsible for the hepatic metabolism of TRA 4beta-C hydroxylation. Using RAF, the percent contributions of CYP3A1 and CYP3A2 to 4beta-C hydroxylation of TRA in rat liver microsomes were estimated as 5 to 6 and 94 to 96, respectively, and those of CYP3A4 and CYP3A5 in human liver microsomes as 70 to 72 and 28 to 30%, respectively. These results suggest that CYP3A2 and CYP3A4 are the main form involved in the 4beta-C hydroxylation of TRA in rat and human liver microsomes.

Analysis of magnolol and honokiol in biological fluids by capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) method was used for analysis of magnolol and honokiol. Under the optimized condition, CZE with UV absorption detection provided that the limit of detection was at microM level. To enhance detection sensitivity of magnolol and honokiol, CZE separation system was coupled with a laser-induced fluorescence (LIF) detector for the first time. The limits of detection of magnolol and honokiol were 12 nM (3.20 ng ml(-1)) and 18 nM (4.79 ng ml(-1)), respectively, showing that the CZE-LIF system provides greater than 100-fold sensitivity improvements than does the CZE-UV system. The developed method was applied to analyze magnolol and honokiol in spiked human plasma samples, microsome incubation samples as a preliminary demonstration of its potential in pharmacokinetic studies.

Territrems B and C metabolism in human liver microsomes: major role of CYP3A4 and CYP3A5.

Human liver microsomes, supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms, and control and CYP3A4 cDNA-transfected V79 Chinese hamster cells were tested for their ability to metabolize territrem B (TRB) and territrem C (TRC). Two TRB metabolites, designated MB(2) and MB(4), and one TRC metabolite, designated MC, were formed by all of these preparations. Of the nine supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5), only those expressing CYP3A4 or CYP3A5 metabolized TRB and TRC. MB(2), MB(4), and MC were formed by CYP3A4 cDNA-transfected V79MZ Chinese hamster cells, but not by non-transfected cells. In order to investigate which CYP450 isoforms were responsible for MB(2), MB(4) and MC formation in human liver microsomal preparations, six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketoconazole, and diethyldithiocarbamate) and antibodies against CYP3A4 were used. MB(2), MB(4), and MC formation was markedly inhibited by ketoconazole, but less affected by quinidine and sulfaphenazole. Anti-CYP3A4 antibody markedly inhibited MB(2), MB(4), and MC formation and also 6 beta-hydroxytestosterone formation from testosterone. The CYP3A-dependent reaction of testosterone 6 beta-hydroxylation showed a high correlation with 4 beta-C hydroxylation of TRB (r(2)=0.97, P<0.0001), O-demethylation of TRB (r(2)=0.95, P<0.0001), and 4 beta-C hydroxylation of TRC (r(2)=0.99, P<0.0001). Immunoblotting and RT-PCR showed that CYP3A4 and CYP3A5 were expressed in all four human liver microsomal preparations tested (HLM1-HLM4). The amount of MB(2), MB(4), and MC formed using different HLM preparations was related to the 6 beta-testosterone hydroxylase activity of the preparations. However, the extent of MB(2), MB(4), and MC formation was not related to the age or gender of the person from whom the microsomal sample was prepared. It was therefore suggest that CYP3A4 and CYP3A5 are the major enzymes responsible for TRB and TRC metabolism by human liver microsomes.

Unfractionated bone marrow cells attenuate paraquat-induced glomerular injury and acute renal failure by modulating the inflammatory response.

The aim of this study was to evaluate the efficacy of unfractionated bone marrow cells (BMCs) in attenuating acute kidney injury (AKI) induced by paraquat (PQ) in a mouse model. PQ (55 mg/kg BW) was intraperitoneally injected into C57BL/6 female mice to induce AKI, including renal function failure, glomerular damage and renal tubule injury. Glomerular podocytes were the first target damaged by PQ, which led to glomerular injury. Upon immunofluorescence staining, podocytes depletion was validated and accompanied by increased urinary podocin levels, measured on days 1 and 6. A total of 5.4 × 10(6) BMCs obtained from the same strain of male mice were injected into AKI mice through the tail vein at 3, 24, and 48 hours after PQ administration. As a result, renal function increased, tubular and glomerular injury were ameliorated, podocytes loss improved, and recipient mortality decreased. In addition, BMCs co-treatment decreased the extent of neutrophil infiltration and modulated the inflammatory response by shifting from pro-inflammatory Th1 to an anti-inflammatory Th2 profile, where IL-1ß, TNF-α, IL-6 and IFN-γ levels declined and IL-10 and IL-4 levels increased. The present study provides a platform to investigate PQ-induced AKI and repeated BMCs injection represents an efficient therapeutic strategy.

Acquisition of tumorigenic potential and enhancement of angiogenesis in pulmonary stem/progenitor cells through Oct-4 hyperexpression.

Cancer stem cells, also known as cancer initiating cells (CICs), are considered to be responsible for tumor growth and chemoresistance. Different hypotheses have been proposed to explain the origin of CICs, including mutations in adult stem/progenitor cells or the acquisition of stem-like characteristics in differentiated cells; however, studies have yielded conflicting identification for CICs and have little information for the origin to generate CICs. Part of the difficulty in identifying CICs may stem from the fact that the CICs studied have been largely derived from cancer cell lines or well-developed tumors. In previous studies, we have reported the enrichment of mouse pulmonary stem/progenitor cells (mPSCs) by using serum-free primary selection culture followed by FACS isolation using the coxsackievirus/adenovirus receptor (CAR) as the positive selection marker. Here, we demonstrated that overexpression of the pluripotent transcription factor Oct-4 is sufficient to induce CAR+/mPSCs transformation, which we name CAR+/mPSCsOct-4_hi. These transformed cells possess cancer initiating and chemoresistance potential, as well as exhibiting remarkable expression of certain proangiogenic factors, including angiopoietins (ANGs) and VEGF, and enhanced angiogenic potential. Moreover, CAR+/mPSCsOct-4_hi actively participated in tumor blood vessel formation and triggered a novel angiogenic mechanism, the angiopoietins/Tie2 signaling pathway. These study provide critical evidence supporting the possible origin to generate CICs, and help elucidate the pathways responsible for CICs-mediated blood vessel formation.

Profile of territrem metabolism and cytochrome P-450 3A expression in liver microsomes from Wistar rats of both genders as a function of age.

This study determined territrem metabolites after incubation of territrem A, B, or C with NADPH and liver microsomes from Wistar rat of both genders aged 2 to 76 wk. The liver microsomal cytochrome P-450 content, NADPH-cytochrome P-450 reductase activity, and CYP3A1 and CYP3A2 protein and mRNA levels were also analyzed. Male rats had significantly higher liver microsomal cytochrome P-450 content and NADPH-cytochrome P-450 reductase activities than females at 14 to 26 wk. Microsomal cytochrome P-450 content was decreased in senescence in both genders compared with postpubertal and adulthood stages. The activity of 6beta-testosterone hydroxylase in male rats, which was significantly higher than those in females at all ages, decreased after 52 wk. After 26 wk, the levels of CYP3A1 protein markely declined in both genders, which resulted in a large gender difference (male greater than female). The protein levels and mRNA of CYP3A2 were constitutively expressed in 2- to 52-wk-old male rats, but they decreased after 76 wk, and decreased in females after 6 wk. The expression of CYP3A1 or CYP3A2 in males are generally higher than in females. The metabolites of territrems MA1, MAX, MA2, MB2, MB4, and MC were measured by high-performance chromatography (HPLC). Formation of MA1, MAX, and MA2 decreased after 52 wk in males, and MAX and MA2 were not formed after 6 wk in females. The amount of MB2 formed in females was less than in males, but the amount of MC (TRC metabolites) formed in females was higher than in males. The gender differences in metabolism of TRA were related to the protein and mRNA expression of CYP3A2. The protein levels and mRNA expression of CYP3A2 and efficiency of territrems metabolism were decreased after 76 wk. The results suggested that the effects of age and gender on territrem metabolism are due to differences in CYP3A1 and CYP3A2 expression in the liver microsomes.

Metabolism of territrem B and C in liver microsomes from 14-wk-old Wistar rats is catalyzed by cytochrome P-450 3A.

The metabolism of territrem B (TRB) and territrem C (TRC) in liver microsomes of 14-wk-old male and female Wistar rats was investigated. Metabolism of TRB to 4beta-hydroxylmethyl-4beta-demethylterritrem B (MB2), O-demethylation of the methoxy group of the aromatic moiety of TRB to form MB4 (same structure as TRC), and metabolism of TRC to 4beta-hydroxylmethyl-4beta-demethylterritrem C (MC) were observed in both genders. However, the amounts of MB2, MB4, and MC formed in females were much lower than in males. To investigate which cytochrome P-450 (CYP450) isoforms were involved in each step, four CYP450 isotype-specific inhibitors (furafylline, orphenadrine, cimetidine, and troleandomycin) and antibodies against CYP1A1, CYP2B1, CYP2C11, or CYP3A2 were used. Formation of MB2, MB4, and MC was markedly inhibited by cimetidine and troleandomycin, but less by furafylline and orphenadrine. Anti-CYP3A2 antibody completely inhibited MB, MB, and MC formation, while antibodies against CYP1A1, CYP2B1, or CYP2C11 produced no marked effect. Of the seven tested supersomes from baculovirus-transformed insect cells expressing rat CYP450 isoforms (1Al, 1A2, 2B1, 2C11, 2C12, 3A1, and 3A2), only those expressing CYP3A1 and CYP3A2 metabolized TRB and TRC. The amounts of MB2, MB4, and MC formed by male and female rat liver microsome preparations were related to the testosterone 6beta-hydroxylase activity and CYP3A1/2 protein content of the preparation. Immunoblotting showed that CYP3A1 was expressed in both genders, but at different levels, while CYP3A2 was only expressed in males. These results suggest that the formation of MB2, MB4, and MC in liver microsomes from 14-wk-old rats of either gender is mediated by CYP3A1 and CYP3A2.

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 (gamma-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.

Gastroprotective potential against indomethacin and safety assessment of the homology of medicine and food formula cuttlebone complex.

Cuttlebone complex (CBC), a homology of medicine and food formula, is comprised of five herbal medicines (Endoconcha Sepiae, Radix Paeoniae Rubra, fresh ginger, Fructus Amomi, and Radix Glycyrrhizae) and two food ingredients (Zingiber zerumbet and chitosan). Herein, the gastroprotective potential against indomethacin and a safety assessment of CBC were investigated. In a gastroprotective model, CBC effectively decreased the indomethacin-increased gastric ulcerous lesions, and increased the indomethacin-decreased prostaglandin E2 levels in the gastric mucosa. In genotoxicity tests, CBC treatment did not increase the numbers of revertant colonies in five Salmonella typhimurium strains and chromosome aberrations in Chinese hamster ovary CHO-K1 cells, with or without S9 metabolic activation. The oral supplementation of CBC did not increase micronucleus formation in the peripheral blood of mice. In a subacute toxicity study, the body weight and blood biochemical parameters observed in CBC-treated rats were normal. In conclusion, CBC was considered as a non-toxic formula and could be used to remedy indomethacin-induced gastric damage.

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.

Production of polyclonal antibodies against territrem B and detection of territrem B in the conidia of Aspergillus terreus 23-1 by immunoelectron microscopy.

Territrem B, a fungal metabolite isolated from Aspergillums terreus 23-1, is a tremorgenic mycotoxin. Immunoelectron microscopy using anti-territrem B polyclonal antibody was used to detect territrem B in the fungal body of A. terreus 23-1 at different times of culture without shaking on potato dextrose (PD) agar medium. The anti-territrem B serum was produced by immunization of rabbits with 4beta-hydroxymethyl-4beta-demethylterritrem B-sccinate bound by a linker to keyhole limpet hemocyanin. This antiserum recognized territrems and immunoelectron microscopy using this antiserum, and colloidal gold-conjugated anti-rabbit IgG antibodies showed that territrem B was localized to the fungal body of A. terreus 23-1. Territrem B was first seen in the cytoplasm of the conidia after 4 days' culture on PD agar medium. Maximal territrem B production in the conidia was seen on the 14th day of culture; however, territrem B was not formed in the hyphae at any stage of culture. These results are consistent with the previous finding that the formation of territrems is related to fungal sporulation.

Metabolism of territrem a in liver microsomes from male wistar rats: 3. Cytochrome p-450 isoforms catalyzing tra metabolism.

The cytochrome P-450 isoforms involved in territrem A (TRA) metabolism in liver microsomes of male Wistar rats have been characterized. Pretreatment with phenobarbital (PB) or dexamethasone (DEX) resulted in a similar significant increase in TRA metabolic activity. Although PB treatment resulted in a significant elevation in CYP2B, CYP2C11, and CYP3A levels, only CYP3A levels were significantly increased by DEX treatment. Cimetidine markedly reduced the formation of the TRA metabolites 4beta-hydroxymethyl-4beta-demethylterritrem A (MA(1)), 4beta-oxo-4beta-demethylterritrem A (MAX) and 2-dihydro-4beta-demethylterritrem A (MA(2)) in liver microsomes from 2-wk-old rats (mainly containing CYP3A2) and 7-wk-old rats (containing CYP2B, CYP2C11, and CYP3A2). SKF 525A, which inhibits CYP2B, CYP2C11, and CYP3A2, and orphenadrine, which inhibits CYP2B, also decreased MA(2) formation in liver microsomes from 7-wk-old phenobarbital-pretreated rats. The formation of MA(1) and MAX was not affected. Furthermore, an immunoinhibition study demonstrated that anti-CYP3A2 antibody reduced MA(1), MAX, and MA(2) formation to nondetectable levels in liver microsomes from 2- and 7-wk-old rats, whereas anti-CYP2C11 or anti-CYP2B antibody, respectively, had no marked effect on MA(1), MAX, and MA(2) formation in liver microsomes from 7-wk-old untreated or PB-treated rats. These results suggest that the CYP3A isoform is mainly responsible for MA(1), MAX, and MA(2) formation in liver microsomes in male Wistar rats.

Cytochrome P-4503A1 catalyzes the formation of MA1 from territrem a in liver microsomes of 7-week-old female Wistar rats.

Liver microsomal territrem A (TRA) metabolism was studied in 7-wk-old female Wistar rats. Pretreatment with phenobarbital (PB) or dexamethasone (DEX) resulted in a significant increase in 4 beta-hydroxymethyl-4 beta-demethylterritrem A (MA1) production. SKF 525A (0.025 and 0.05 mM), a general cytochrome P-450 (CYP450) inhibitor, blocked MA1 formation in liver microsomes from PB-pretreated female rats. Anti-CYP2B antibody had no marked effect on MA1 formation, although orphenadrine (0.5 mM), which inhibits CYP2B, blocked MA1 formation in liver microsomes from PB-treated female rats. An immunoinhibition study showed that anti-CYP3A2 antibody reduced MA1 formation to nondetectable levels in liver microsomes from PB-treated female rats. Furthermore, immunoblotting showed that CYP3A1 protein was expressed in 7-wk-old female rat and only MA1 was formed from TRA using supersomes from CYP3A1-expressing baculovirus-infected insect cells. Further, Western blot analysis indicated that CYP3A2 protein was expressed in 2-wk-old rats of both sexes and 7-wk-old male rats, and 3 metabolites of TRA, such as MA1, MAX, and MA2, were formed using supersomes from CYP3A2-expressing baculovirus-infected insect cells. These results suggest that MA1 formation in liver microsomes of 7-wk-old female Wistar rats is mediated by CYP3A1.

NADPH-cytochrome P-450 reductase is involved in flunitrazepam reductive metabolism in Hep G2 and Hep 3B cells.

Flunitrazepam (FNTZ), like other benzodiazepines, has a high affinity for the benzodiazepine receptor within the gama-aminobutyric acid (GABA) complex. These affinities correlate with the pharmacological and therapeutic potencies of the drug. FNTZ is a drug commonly abused by young adults. In humans, FNTZ is oxidized to the major metabolites N-demethylflunitrazepam (DM FNTZ) and 3-hydroxyflunitrazepam (3-OH FNTZ) and reduced to 7-aminoflunitrazepam (7A FNTZ). Human CYP2C19 and CYP3A4 are the principal P-450 cytochromes involved in DM FNTZ and 3-OH FNTZ formation. However, it is not clear which enzyme is responsible for the reduction of FNTZ to 7-aminoflunitrazepam (7A FNTZ). In this study, the involvement of NADPH-cytochrome P-450 reductase in the conversion of FNTZ to 7A FNTZ was investigated in two human hepatoma cell lines, human lymphoblast microsomes specifically expressing human NADPH-cytochrome P-450 reductase and purified recombinant human HADPH-cytochrome P-450 reductase. Significantly more FNTZ was converted to 7A FNTZ in Hep G2 than in Hep 3B cells, and this difference was associated with the catalytic activity and protein levels of NADPH-cytochrome P-450 reductase in these cells. In Hep G2 cells, conversion of FNTZ to 7A FNTZ was effectively inhibited by alpha-lipoic acid, an NADPH-cytochrome P-450 reductase inhibitor. In addition, formation of 7A FNTZ by the microsomal fraction of Hep G2 cells was specifically inhibited by antibody against NADPH-cytochrome P-450 reductase. Under hypoxia (N2 85%; CO2 5%; H2 10%), human lymphoblast microsomes specifically expressing human NADPH-cytochrome P-450 reductase and purified recombinant human NADPH-P-450 reductase catabolized FNTZ to 7A FNTZ in a concentration-dependent manner. These results suggest that NADPH-cytochrome P-450 reductase is involved in the reductive metabolism of FNTZ to 7A FNTZ under hypoxic conditions.

Role of human hepatic cytochrome P-450s in territrem A metabolism.

The ability of human liver microsomal preparations (HLM1, 2, 3, and 5), microsomes from human lymphoblasts expressing different cytochrome P-450 (CYP450) isoforms, and CYP3A4 cDNA-transfected V79 Chinese hamster cells to metabolize territrem A (TRA) was studied. The only metabolite generated by any of these preparations was 6beta-hydroxymethyl-6beta-demethylterritrem A (MA(1)). MA(1) formation was observed with all four human liver microsomal samples. Of the eight microsomal preparations from human lymphoblasts expressing different cytochrome P-450 enzymes (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) examined, only those expressing CYP2C9, CYP2D6, or CYP3A4 metabolized TRA, with that expressing CYP3A4 being the most active. No TRA metabolites were formed by control V79MZ cells, but MA(1) was formed by CYP3A4 cDNA-transfected V79 Chinese hamster cells. In order to investigate which CYP450 isoforms were involved in MA(1) formation in the human liver microsomal preparations, the effects of six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketaconazole, and diethyldithiocarbamate) and anti-3A4, anti-2C9, and anti-2D6 antibodies on TRA metabolism by HLM2 and HLM5 were examined. MA(1) formation was markedly inhibited by ketaconazole, with quinidine and sulfaphenazole having less of an effect. Anti-CYP3A4 antibody markedly inhibited MA(1) formation, while antibodies against CYP2C9 or CYP2D6 had little effect. The amount of MA(1) formed using different HLM preparations was related to the 6beta-testosterone hydroxylase activity and CYP3A4 protein content of the preparations. These results suggest that CYP3A4 is the major enzyme involved in TRA metabolism by human liver microsomes, with CYP2C9 and CYP2D6 playing a minor role.