Microsomal Subcellular Fractions Research Articles

The Benzene-induced Hepatic Cytochrome P450 2E1 Expression and Activity are Reduced by Quercetin Administration in Mice.

Benzene as an environmental and industrial agent induces adverse effects that are mainly metabolism-dependent. Effects of Quercetin (QCN) on Benzene (BNZ)-induced changes in the hepatic Cytochrome P450 2E1 expression and activity were investigated. Thirty-six adult male mice were divided into 6 groups (n = 6) and nominated as control, BNZ (exposed to BNZ: 30 ppm), QCN (received QCN: 50 mg/kg, orally), and the fourth, fifth and sixth groups were exposed to 30 ppm BNZ and received 10, 50 and 100 mg/kg QCN respectively, for 28 days. The microsomal subcellular fraction was isolated from the liver samples and the activity of CYP 2E1 was measured based on the hydroxylation rate of 4-nitrophenol. The hepatic activity of myeloperoxidase also was assessed. Total antioxidant capacity and nitric oxide contents of the liver were determined. Expression changes of CYP 2E1 at the mRNA level were examined by qPCR technique. QCN lowered significantly (p < 0.05) the BNZ-increased hepatic nitric oxide levels and restored the BNZ-reduced antioxidant capacity. The BNZ-elevated activity of myeloperoxidase was declined in QCN-received mice. QCN downregulated the expression and activity of hepatic CYP 2E1 in BNZ-exposed animals. Our results suggest that QCN could be a novel hepatoprotective compound for BNZ-induced hepatotoxicities, which is attributed to its capability in the down-regulation of CYP 2E1 expression and activity.

The role of carbonyl reductases in biotransformation of bupropion and 4-methylnitrozamino-1-(3-pyridyl)-1-butanone by human placenta

s / Drug and Alcohol Dependence 156 (2015) e102–e182 e161 The role of carbonyl reductases in biotransformation of bupropion and 4-methylnitrozamino-1-(3-pyridyl)-1-butanone by human placenta Tatiana Nanovskaya1, Valentina M. Fokina1,2, X. Wang1, Cheryl Oncken3, Mahmoud S. Ahmed1, Gary Hankins1 1 OBG the reaction is catalyzed primarily by 11 hydroxisteroid dehydrogenase (11 -HSD) and aldoketoreductases. Maternal cigarette smoking has been associated with prenatal exposure to NNK, which is the most abundant and potent carcinogen of cigarette smoke. One of the metabolic pathways of NNK is carbonyl reduction to 4-methylnitrosamino-1-(3-pyridyl)1-butanol (NNAL) that initiates NNK detoxification. The goal of the current investigation is to determine placental metabolism of NNK and to identify placental enzymes responsible for its reduction. Methods: Term placentas were collected from women who smoked during pregnancy and from non-smokers. NNK metabolism was determined in vitro using placental microsomal and cytosolic subcellular fractions; the formation of NNAL was quantified by HPLC-UV. Results: The apparent Km and Vmax values for the reaction in placental subcellular fractions were determined. The formation of NNAL in placentas of heavy smokers was lower than in placentas of non-smokers: 12.1±3.5 vs. 17±6.3nmolmgP−1 for cytosolic fraction (p methamphetamine>4-MePPP>4-MEC. Conclusions: The current findings demonstrated that three second-generation synthetic cathinones functioned as reinforcers in rats, providing some justification for permanent scheduling of these drugs by regulatory agencies. Future research in our laboratory will assess the reinforcing effectiveness of these three compounds with a behavioral economics approach. Financial support: This research was supported by the UMMC Drug Abuse Development Fund and R01 DA12970 to BB. http://dx.doi.org/10.1016/j.drugalcdep.2015.07.439 Serotonin 5-HT2C receptors in the ventral subiculum regulate cocaine-evoked hyperactivity in rats Harshini Neelakantan1, Sonja J. Stutz1, Marcy J. Bubar1, Robert M. Sears3, Ralph J. DiLeone3, Kathryn A. Cunningham1,2 1 Center for Addiction Research, University of Texas Medical Branch, Galveston, TX, United States 2 Department of Pharmacy, University of Texas Medical Branch, Galveston, TX, United States 3 Department of Psychiatry, Yale University, New Haven, CT, United States Aims: The ventral subiculum (vSUB) interconnects with limbiccorticostriatal circuit which regulates the behavioral effects of cocaine. Serotonin neurons innervate the vSUB, however, little is known about its role or the 5-HT receptors that are functionally relevant in this region. We investigated the vSUB localization of the 5-HT2C receptor (5-HT2CR) and its role in the control of cocaineevoked hyperactivity. Methods: The expression of the 5-HT2CR and co-localization with GAD 67 (marker for GABA) in the vSUB were evaluated using immunohistochemical analyses in rats (n=3).We employed virally mediated genetic knockdown strategy to test the hypothesis that knockdown of the 5-HT2CR in the vSUB of rats (n=5) will modulate cocaine-evoked hyperactivity. Motor activity was assessed after saline or cocaine (10–20mg/kg) in modified open-field chambers and analyzed using repeated measures two-way ANOVA. Results:Dense immunoreactivity (IR) for 5-HT2CRwasobserved throughout the vSUB. Only a small proportion of 5-HT2CR-IR was co-localized with GAD 67-IR that was restricted to the distal end

Self-administration of second-generation synthetic cathinones in rats

s / Drug and Alcohol Dependence 156 (2015) e102–e182 e161 The role of carbonyl reductases in biotransformation of bupropion and 4-methylnitrozamino-1-(3-pyridyl)-1-butanone by human placenta Tatiana Nanovskaya1, Valentina M. Fokina1,2, X. Wang1, Cheryl Oncken3, Mahmoud S. Ahmed1, Gary Hankins1 1 OBG the reaction is catalyzed primarily by 11 hydroxisteroid dehydrogenase (11 -HSD) and aldoketoreductases. Maternal cigarette smoking has been associated with prenatal exposure to NNK, which is the most abundant and potent carcinogen of cigarette smoke. One of the metabolic pathways of NNK is carbonyl reduction to 4-methylnitrosamino-1-(3-pyridyl)1-butanol (NNAL) that initiates NNK detoxification. The goal of the current investigation is to determine placental metabolism of NNK and to identify placental enzymes responsible for its reduction. Methods: Term placentas were collected from women who smoked during pregnancy and from non-smokers. NNK metabolism was determined in vitro using placental microsomal and cytosolic subcellular fractions; the formation of NNAL was quantified by HPLC-UV. Results: The apparent Km and Vmax values for the reaction in placental subcellular fractions were determined. The formation of NNAL in placentas of heavy smokers was lower than in placentas of non-smokers: 12.1±3.5 vs. 17±6.3nmolmgP−1 for cytosolic fraction (p methamphetamine>4-MePPP>4-MEC. Conclusions: The current findings demonstrated that three second-generation synthetic cathinones functioned as reinforcers in rats, providing some justification for permanent scheduling of these drugs by regulatory agencies. Future research in our laboratory will assess the reinforcing effectiveness of these three compounds with a behavioral economics approach. Financial support: This research was supported by the UMMC Drug Abuse Development Fund and R01 DA12970 to BB. http://dx.doi.org/10.1016/j.drugalcdep.2015.07.439 Serotonin 5-HT2C receptors in the ventral subiculum regulate cocaine-evoked hyperactivity in rats Harshini Neelakantan1, Sonja J. Stutz1, Marcy J. Bubar1, Robert M. Sears3, Ralph J. DiLeone3, Kathryn A. Cunningham1,2 1 Center for Addiction Research, University of Texas Medical Branch, Galveston, TX, United States 2 Department of Pharmacy, University of Texas Medical Branch, Galveston, TX, United States 3 Department of Psychiatry, Yale University, New Haven, CT, United States Aims: The ventral subiculum (vSUB) interconnects with limbiccorticostriatal circuit which regulates the behavioral effects of cocaine. Serotonin neurons innervate the vSUB, however, little is known about its role or the 5-HT receptors that are functionally relevant in this region. We investigated the vSUB localization of the 5-HT2C receptor (5-HT2CR) and its role in the control of cocaineevoked hyperactivity. Methods: The expression of the 5-HT2CR and co-localization with GAD 67 (marker for GABA) in the vSUB were evaluated using immunohistochemical analyses in rats (n=3).We employed virally mediated genetic knockdown strategy to test the hypothesis that knockdown of the 5-HT2CR in the vSUB of rats (n=5) will modulate cocaine-evoked hyperactivity. Motor activity was assessed after saline or cocaine (10–20mg/kg) in modified open-field chambers and analyzed using repeated measures two-way ANOVA. Results:Dense immunoreactivity (IR) for 5-HT2CRwasobserved throughout the vSUB. Only a small proportion of 5-HT2CR-IR was co-localized with GAD 67-IR that was restricted to the distal end

The role of placental carbonyl-reducing enzymes in biotransformation of bupropion and 4-methylnitrozamino-1-(3-pyridyl)-1-butanone (NNK)

Background: Bupropion (BUP) has a potential to be an effective pharmacotherapy for smoking cessation during pregnancy. Smoking during pregnancy stimulates placental carbonyl reductases that catalyze the biotransformation of BUP. 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen of cigarette smoke. Carbonyl reduction of NNK into 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) constitutes a major step in NNK detoxification. Thus, placentas of pregnant smokers on BUP therapy can become a site of drug-drug interaction. Therefore, we investigated the effect of continuous exposure to BUP and cigarette smoke on the activity of placental carbonyl reductases in the formation of NNAL from NNK. Methods: The reductive metabolism of NNK was determined using microsomal and cytosolic subcellular fractions of placentas obtained from non-smoking women treated with BUP for depression, and women not exposed to BUP: non-smokers (control) and smokers. The effect of BUP and its metabolites on the reductive metabolism of NNK was investigated using subcellular fractions of control placentas. Results: The formation of NNAL from NNK by placental cytosolic fractions of heavy smokers (≥20 cigarettes per day) was lower than that of control (12.1±3.5 nmol.mgP-1 vs 16.5±6.0 nmol.mgP-1, P<0.05). While exposure to BUP did not affect the activity of placental carbonyl reductases, the formation of NNAL in the placental cytosolic fraction was decreased only in the presence of high concentrations of BUP metabolites. Conclusion: Smoking during pregnancy decrease the detoxifying capacity of soluble carbonyl reductases towards NNK. Given the experimental conditions, exposure to BUP and its metabolites should not impede the reductive metabolism of NNK by placenta in vivo.

Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori

Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD- dependent reaction.Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation.

Characterizing the in vitro hepatic biotransformation of the flame retardant BDE 99 by common carp

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardant chemicals known to biomagnify in aquatic foodwebs. However, significant biotransformation of some congeners via reductive dehalogenation has been observed during in vivo and in vitro laboratory exposures, particularly in fish models. Little information is available on the enzyme systems responsible for catalyzing this metabolic pathway in fish. This study was undertaken to characterize the biotransformation of one primary BDE congener, 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99), using in vitro techniques. Hepatic sub-cellular fractions were first prepared from individual adult common carp ( Cyprinus carpio) to examine metabolism in both microsomal and cytosolic sub-cellular fractions. Debromination rates (i.e. BDE-99 biotransformation to BDE-47) were generally higher in the microsomal fraction than in the cytosolic fraction, and some intra-species variability was observed. Further experiments were conducted to determine the biotransformation kinetics and the influence of specific co-factors, inhibitors and competitive substrates on metabolism using pooled carp liver microsomes. The apparent K m and V max values were 19.4 μM and 1120 pmoles h −1 mg protein −1, respectively. Iodoacetate (IaC) and the two thyroid hormones, reverse triodothyronine (rT3) and thyroxine (T4), significantly inhibited the debromination of BDE-99 in microsomal sub-cellular fractions with IC 50 values of 2.2 μM, 0.83 μM, and >1.0 μM, respectively. These results support our hypothesis that deiodinase enzymes may be catalyzing the metabolism of PBDEs in fish liver tissues. Further studies are needed to evaluate metabolic activity in other species and tissues that contain these enzymes.

Ubiquinone Synthesis and its Regulation in Pneumocystis carinii

The opportunistic pathogen Pneumocystis causes a type of pneumonia in individuals with defective immune systems such as AIDS patients. Atovaquone, an analog of ubiquinone (coenzyme Q [CoQ]), is effective in clearing mild to moderate cases of the infection. Rat-derived Pneumocystis carinii was the first organism in which CoQ synthesis was clearly demonstrated to occur in both mitochondrial and microsomal subcellular fractions. Atovaquone inhibits microsomal CoQ synthesis with no effect on mitochondrial CoQ synthesis. We here report on additional studies evaluating CoQ synthesis and its regulation in the organism. Buparvaquone also inhibited CoQ synthesis and it reduced the synthesis of all four CoQ homologs in the microsomal but not the mitochondrial fraction. Glyphosate, which inhibits a reaction in the de novo synthesis of the benzoquinone moiety of CoQ reduced cellular ATP levels. Bacterial and plant quinones, and several chemically synthesized phenolics, flavanoids, and naphthoquinones that inhibit electron transport in other organisms were shown to reduce CoQ synthesis in P. carinii. The inhibitory action of naphthoquinone compounds appeared to depend on their molecular size and structural flexibility rather than redox potential. Results of experiments examining the synthesis of the polyprenyl chain of CoQ were consistent with negative feedback control of CoQ synthesis. These studies on P. carinii suggest that cellular sites and the control of CoQ synthesis in different organisms and cell types might be more diverse than previously thought.

The v-SNARE Vti1a Regulates Insulin-stimulated Glucose Transport and Acrp30 Secretion in 3T3-L1 Adipocytes

Regulated exocytosis in adipocytes mediates key functions, exemplified by insulin-stimulated secretion of peptides such as adiponectin and recycling of intracellular membranes containing GLUT4 glucose transporters to the cell surface. Using a proteomics approach, the v-SNARE Vti1a (vps10p tail interacting 1a) was identified by mass spectrometry in purified GLUT4-containing membranes. Insulin treatment of 3T3-L1 adipocytes decreased the amounts of both Vti1a and GLUT4 in these membranes, confirming that Vti1a is a component of insulin-sensitive GLUT4-containing vesicles. In the basal state, endogenous Vti1a colocalizes exclusively with perinuclear GLUT4. Although Vti1a has previously been reported to be a v-SNARE localized in the trans-Golgi network, treatment with brefeldin A failed to significantly modify Vti1a or GLUT4 localization while completely dispersing Golgi and trans-Golgi network marker proteins. Furthermore, depletion of Vti1a protein in cultured adipocytes through small interfering RNA-based gene silencing significantly inhibited both adiponectin secretion and insulin-stimulated deoxyglucose uptake. Taken together, these results suggest that the v-SNARE Vti1a may regulate a step common to both GLUT4 and Acrp30 trafficking in 3T3-L1 adipocytes.

Ubiquinone Synthesis in Mitochondrial and Microsomal Subcellular Fractions of Pneumocystis spp.: Differential Sensitivities to Atovaquone

The lung pathogen Pneumocystis spp. is the causative agent of a type of pneumonia that can be fatal in people with defective immune systems, such as AIDS patients. Atovaquone, an analog of ubiquinone (coenzyme Q [CoQ]), inhibits mitochondrial electron transport and is effective in clearing mild to moderate cases of the infection. Purified rat-derived intact Pneumocystis carinii cells synthesize de novo four CoQ homologs, CoQ7, CoQ8, CoQ9, and CoQ10, as demonstrated by the incorporation of radiolabeled precursors of both the benzoquinone ring and the polyprenyl chain. A central step in CoQ biosynthesis is the condensation of p-hydroxybenzoic acid (PHBA) with a long-chain polyprenyl diphosphate molecule. In the present study, CoQ biosynthesis was evaluated by the incorporation of PHBA into completed CoQ molecules using P. carinii cell-free preparations. CoQ synthesis in whole-cell homogenates was not affected by the respiratory inhibitors antimycin A and dicyclohexylcarbodiimide but was diminished by atovaquone. Thus, atovaquone has inhibitory activity on both electron transport and CoQ synthesis in this pathogen. Furthermore, both the mitochondrial and microsomal fractions were shown to synthesize de novo all four P. carinii CoQ homologs. Interestingly, atovaquone inhibited microsomal CoQ synthesis, whereas it had no effect on mitochondrial CoQ synthesis. This is the first pathogenic eukaryotic microorganism in which biosynthesis of CoQ molecules from the initial PHBA:polyprenyl transferase reaction has been unambiguously shown to occur in two distinct compartments of the same cell.

Heme oxygenase expression in selected regions of term human placenta.

Carbon monoxide (CO), formed during heme oxygenase (HO)-catalyzed oxidation of heme, has been proposed to play a complementary role with nitric oxide in the regulation of placental hemodynamics. The objective of this study was to elucidate HO enzymatic activity and HO-1 (inducible) and HO-2 (constitutive) protein content in the microsomal subcellular fraction of homogenate of selected regions of placenta from normotensive and mild pre-eclamptic pregnancies. HO enzymatic activity was measured under optimized conditions by gas chromatography using CO formation as an index of activity, and HO-1 and HO-2 protein content were determined by Western immunoblot analysis. Microsomal HO activity in each of the four placental regions was not different between normotensive and mild pre-eclamptic pregnancies. Microsomal HO-2 protein content was not different between normotensive and mild pre-eclamptic pregnancies, whereas there was increased expression of microsomal HO-1 protein in chorionic villi and fetal membranes from pre-eclamptic pregnancy compared with normotensive pregnancy. Microsomal HO enzymatic activity correlated with HO-2, but not HO-1, protein content.

Changes in global gene and protein expression during early mouse liver carcinogenesis induced by non-genotoxic model carcinogens oxazepam and Wyeth-14,643.

We hypothesized that the mouse liver tumor response to non-genotoxic carcinogens would involve some common early gene and protein expression changes that could ultimately be used to predict chemical hepatocarcinogenesis. In order to identify a panel of genes to test, we analyzed global differences in gene and protein expression in livers from B6C3F1 mice following dietary treatment with two rodent carcinogens, the benzodiazepine anti-anxiety drug oxazepam (2500 p.p.m.) and the hypolipidemic agent Wyeth (Wy)-14,643 (500 p.p.m.) compared with livers from untreated mice. Male mice were exposed for 2 weeks and 1, 3 or 6 months to oxazepam or Wy-14,643 in an age-matched study design. By histopathological evaluation, no liver preneoplastic foci or tumors were detected at 6 months in treated or control groups. By cDNA microarray analysis [NIEHS Mouse Chip (8700 genes); n = 3 individual livers/group, four hybridizations/sample], expression of 36 genes or 220 genes were changed relative to control livers following 6 months of oxazepam or Wy-14,643 treatment, respectively. To obtain a more comprehensive picture of gene/protein expression changes, we also conducted a proteomics study by 2D-gel electrophoresis followed by matrix assisted laser desorption/ionization-mass spectrometry on cytoplasmic, nuclear, and microsomal subcellular fractions of the same liver samples utilized for the cDNA microarray analysis. Real-time PCR, western blot analysis and immunohistochemistry were utilized for validation and to expand the results to other time points. Cyp2b20, growth arrest- and damage-inducible gene beta (Gadd45beta), tumor necrosis factor alpha-induced protein 2 and insulin-like growth factor binding protein 1 (Igfbp5) genes and proteins were upregulated by oxazepam, and Cyp2b20, Cyclin D1, proliferating cell nuclear antigen, Igfbp5, Gadd45beta and cell death-inducing DNA fragmentation factor alpha subunit-like effector A exhibited higher expression after Wy-14,643 treatment. Most of these genes/proteins were also deregulated at 2 weeks. There appeared to be more distinct than common changes in the expression of carcinogenesis-related genes/proteins between the two compounds, suggesting that the major carcinogenic pathways are different for these compounds and may be distinct for different chemical classes.

Characterization of the Drosophila ortholog of mouse eIF-3p48/INT-6

The mouse mammary tumor virus (MMTV) has been shown to integrate frequently into INT-6 in MMTV-induced mouse mammary tumors. The INT6 gene has been highly conserved through evolution and has recently been shown to encode the p48 component of the eucaryotic translation initiation factor 3 (eIF-3) complex. We report here the isolation of the Drosophila eIF-3p48/INT-6. The gene comprises three exons within 1.8kb of genomic DNA located at cytogenetic position 73C2 in the Drosophila genome. The 1.5kb eIF-3p48/INT-6 RNA species encodes a protein composed of 364 amino-acid residues whose sequence is 71% similar to that of the mouse/human eIF-3/INT-6 amino-acid sequence. eIF-3p48/INT-6 RNA is expressed throughout development in Drosophila and the encoded protein is associated with the microsomal subcellular fraction.

Effects of di-isononyl phthalate (DINP) on peroxisomal markers in the marmoset-DINP is not a peroxisome proliferator.

In the present study the systemic toxic potential of di-isononyl phthalate (DINP) was assessed in a 13-week study in marmosets. Particular attention was given to its potential for hepatic peroxisome proliferation. Three groups of four male and four female marmosets received DINP, by oral gavage administration, at dosages of 100, 500 or 2500 mg/kg/day for 13 weeks. A fourth group served as a concurrent Control group and received the vehicle (1% methylcellulose and 0.5% Tween) only. A fifth group received clofibrate at a dosage of 500 mg/kg/day to provide a positive Control for liver peroxisome activity. At the end of the treatment period, the animals were killed and their livers were removed. 3000 x g supernatant and microsomal subcellular fractions were prepared from homogenised liver by differential centrifugation. The peroxisomal marker enzyme activity, cyanide-insensitive palmitoyl CoA oxidase, was assayed in the former, while cytochrome P450 concentration and lauric acid 11- and 12-hydroxylase activities (selective for CYP2E1 and 4A, respectively) were assayed in the microsomes. No statistically significant changes were seen in any of these parameters measured following DINP treatment, compared with the Control. Clofibrate treatment resulted in an approximately 100% increase (p < 0.01) in both male and female marmoset cyanide-insensitive palmitoyl CoA oxidase activity and a similar increase (p < 0.05) in male (only) lauric acid 11-hydroxylase activity. No other changes were statistically significant at the 5% level. These data provide no evidence that DINP was acting as a peroxisome proliferator when administered to marmosets under the conditions of the study.

The coffee-specific diterpenes cafestol and kahweol protect against aflatoxin B1-induced genotoxicity through a dual mechanism.

The diterpenes cafestol and kahweol (C&K) have been identified in animal models as two potentially chemoprotective agents present in green and roasted coffee beans. It has been postulated that these compounds may act as blocking agents by producing a co-ordinated modulation of multiple enzymes involved in carcinogen detoxification. In this study, we investigated the effects of C&K against the covalent binding of aflatoxin B1 (AFB1) metabolites to DNA. Male Sprague-Dawley rats were treated with increasing amounts of a mixture of C&K in the diet (0-6200 p.p.m.) for 28 and 90 days. A dose-dependent inhibition of AFB1 DNA-binding was observed using S9 and microsomal subcellular fractions from C&K-treated rat liver in an in vitro binding assay. Significant inhibition was detected at 2300 p.p.m. and maximal reduction of DNA adduct formation to nearly 50% of the control value was achieved with 6200 p.p.m. of dietary C&K. Two complementary mechanisms may account for the chemopreventive action of cafestol and kahweol against aflatoxin B1 in rats. A decrease in the expression of the rat activating cytochrome P450s (CYP2C11 and CYP3A2) was observed, as well as a strong induction of the expression of the glutathione-S-transferase (GST) subunit GST Yc2, which is known to detoxify highly the most genotoxic metabolite of AFB1. These data and the previously demonstrated effects of C&K against the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis at various tissue sites suggest the potential widespread effect of these coffee components against chemical carcinogenesis.

Effect of cancer chemopreventive agents on microsome-mediated DNA adduction of the breast carcinogen dibenzo[ a,l]pyrene

Due to the large and expanding number of potential cancer chemopreventive agents, there is an increasing need for short term tests to study the efficacy and mechanisms of these agents. In this study, we have employed a microsome-mediated test system to study the effect of several suspected chemopreventive agents on the DNA adduct formation capacity of the potent mammary carcinogen, dibenzo[ a,l]pyrene (DBP). Bioactivation of DBP by Aroclor 1254-induced rat liver microsomes in the presence of calf thymus DNA (300 μg/ml) resulted in the formation of one major and six other prominent DNA adducts (324 adducts/10 7 nucleotides). These adducts were previously determined to be deoxyadenosine (dA) and deoxyanosine (dG)-derivatives of both anti- and syn-DBP-11,12-diol-13,14-epoxides (DBPDE). Intervention with ellagic acid, chlorophyllin, benzyl isocyanate (BIC), oltipraz or genistein (150 μM) strongly diminished DBP–DNA adduction by ≥75%. Linoleic acid, curcumin and butylated hydroxytoluene (BHT) also significantly inhibited DBP–DNA adduction (26–46%) while N-acetylcysteine (NAC) had no effect. Moreover, nonenzymatic studies with anti- and syn-DBPDE isomers revealed that chlorophyllin, ellagic acid, BIC and BHT may be inhibiting DBP–DNA adduction in an enzymatic-independent manner since these agents diminished DBPDE–DNA adduction by 30–75%. Genistein, oltipraz and curcumin did not diminish DBPDE–DNA adduction and therefore most likely require the presence of the microsomal subcellular fraction to inhibit DBP–DNA adduction.

Evaluation of the Effects of Risedronate on Hepatic Microsomal Drug Metabolizing Enzyme Activities Following Administration to Rats for 14 Days: Lack of an Induction Response

ABSTRACTRisedronate ([1-hydroxy-2-(3-pyridinyl)-ethylidene[bis]phosphonic acid] monosodium salt) was evaluated for induction of hepatic microsomal drug metabolizing enzymes in male and female Sprague Dawley rats (N-4/sex/dose group). Main study animals received water (vehicle control), risedronate (0.1, 0.8, 4, or 16 mg/kg/day) or phenobarbital (80 mg/kg/day, positive control) by daily oral gavage for 14 consecutive days. Recovery study animals received water, risedronate (16 mg/kg/day) or phenobarbital (80 mg/kg/day) by daily oral gavage for 14 consecutive days and then were maintained drug-free for 14 days to evaluate the reversibility of any observed effects. At the conclusion of each study the animals were sacrificed, the liver removed, weighed and the microsomal subcellular fraction prepared. the hepatic microsomal fraction was then evaluated for protein content, cytochrome P450, and the activities of aniline hydroxylase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase and p-nitrophenol UDP-glucuronosyltransferase. Risedronate was well tolerated during the dosing phase of the study as evidenced by clinical observations, body weight gain and food consumption which were not significantly different from the vehicle controls. Risedronate did not significantly increase (P>0.05) liver weight, liver/body weight ratio, protein content, P450, aniline hydroxylase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase or p-nitrophenol UDP-glucuronosyltransferase in rats of either sex when compared to vehicle controls. As expected, the hepatic microsomal enzyme inducer phenobarbital significantly increased (P>0.05) liver weight, liver/body weight ratio, protein content (males only), P450, aniline hydroxylase (males only), aminopyrine N-demethylase (males only), ethoxycoumarin O-deethylase and p-nitrophenol UDP-glucuronosyltransferase in rats relative to vehicle controls. Following the 14 day drug-free recovery period the induction parameters increased by phenobarbital reversed to vehicle control levels. the results obtained in this well controlled study indicate that risedronate is not an inducer of hepatic microsomal drug metabolizing enzymes in the rat.

Pleiotropic effect of LEC mutation: a rodent model of Wilson's disease

Metabolic studies with 67Cu were undertaken to identify the site of the cellular defect in copper metabolism in the Long-Evans Cinnamon (LEC) rat. The apparent rate of copper uptake by LEC primary hepatocytes was increased [maximal velocity (Vmax) = 259 pmol.min-1.mg protein-1] compared with controls (Vmax = 161 pmol.min-1.mg protein-1); however, Michaelis-Menten constant (Km) values were comparable (11.8 and 12.7 microM, LEC and control, respectively). Efflux of copper from LEC and control hepatocytes was similar from 0 to 15 min, but was reduced from 15 to 60 min in the former. Although hepatic copper contents were markedly elevated in LEC rats compared with controls (658 +/- 199 vs. 21.5 +/- 6.6 micrograms/g dry wt), biliary copper concentration was reduced in LEC rats compared with controls (0.187 vs. 1.39 +/- 0.66 microgram/ml). Subcellular fractionation of LEC liver homogenates revealed approximately 75% of copper to be present in cytosol, with gradients of copper concentration from cytosol to either lysosome or microsomal subcellular fractions. LEC rat bile and hepatic microsome and lysosome fractions contained smaller fractions of 67Cu administered intravenously as cupric acetate compared with control rats. However, recovery of 67Cu in bile and in lysosomal subcellular fractions were similar for LEC and controls following administration of 67Cu-labeled asialoceruloplasmin, which is targeted to lysosomes. This discordance suggests a possible defect in the entry of copper into lysosomes but normal delivery of lysosomal copper to bile. Based on these findings, we conclude that the mutation in LEC rats alters copper transport at more than one cellular site.

H7-resistant protein kinase C substrates in two-dimensional gels of proestrous rat anterior pituitary gland

The presence of Ca 2+-dependent and -independent protein kinase C (PKC) activities which were stimulated by phorbol-12,13-dibutyrate (PDBu) and sensitive to the kinase inhibitor staurosporine (IC 50 values approx. 100 nM) was demonstrated in proestrous rat anterior pituitary gland. These PDBu-induced activities were completely abolished by the PKC-specific inhibitors Ro31-8220 and GF109203X (3 μM). The Ca 2+-independent activity was more resistant (IC 50 = 61 μM) to the kinase inhibitor H7 than the Ca 2+-dependent activity (IC 50 approx. 20 μM), however, this (unusual) resistance to H7 was not observed in the brain regions, hypothalamus and hippocampus. Possible substrates for the Ca 2+-independent PKC in anterior pituitary were identified by two-dimensional gel electrophoresis and autoradiography following incubation in vitro with [ 32P]phosphate and 300 nM PDBu ± 300 nM staurosporine or 30 μM H7. The phosphorylation of six proteins (16, 16, 25, 36, 65 and 69 kDa) was found to be stimulated by PDBu and inhibited by staurosporine, but not H7, in whole tissue, and another two such phosphorylated proteins (each 76 kDa) were observed in microsomal subcellular fractions. These phosphoproteins may be substrates for an H7-resistant PKC isoform previously shown to mediate a number of cellular responses in rat anterior pituitary gland.

Cholesterol metabolism in the rat lactating mammary gland: The role of cholesteryl ester hydrolase

An acid cholesteryl ester hydrolase activity associated with a fraction containing mitochondria and lysosomes from rat lactating mammary glands was found to have a pH optimum of 5.0. Its sedimentation pattern was closely related to that of the lysosomal enzyme markers acid phosphatase and beta-glucuronidase, suggesting that the activity is associated with the lysosomes. The enzyme was strongly inhibited by Cu2+, but was inhibited little by other divalent metal ions. Acid cholesteryl ester hydrolase activity was almost completely abolished by p-hydroxy-mercuribenzoate, but this effect was reversed in the presence of an equimolar concentration of reduced glutathione (GSH), indicating that the enzyme requires free sulfhydryl groups for activity. These properties are similar to those of acid, lysosomal cholesteryl ester hydrolases found in other tissues. Acid cholesteryl ester hydrolase activity was 8-14 fold higher in mammary tissue from lactating as compared to virgin rats. Neutral cholesteryl ester hydrolase activities associated with the microsomal and cytosolic subcellular fractions were also increased in lactating glands, but to a lesser extent. In addition, a 2-fold increase in the activities of both the acid and microsomal neutral enzymes was seen during the first few days of lactation, while the cytosolic neutral activity remained constant. These results suggest that mammary gland cholesteryl ester hydrolases have a role in the regulation of cholesterol metabolism in mammary cells, and in the provision of cholesterol for secretion into milk.

Sialidase in the Guinea Pig Pulmonary Parenchyma. Increased Activity in the Cytosolic and Microsomal Subcellular Fractions after Stimulation withBacillus Calmette Guérin

The sialidase activity was assayed in the guinea pig pulmonary parenchyma after removal of bronchoalveolar cells by washing. After differential centrifugation of the crude tissue homogenate, sialidase activities were measured in the subcellular fractions using the fluorogenic substrate 2-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminate. Sialidase activities were found in the lysosomal-enriched (17,000 x g pellet), in the microsomal (105,000 x g pellet) and in the cytosolic (105,000 x g supernatant) fractions. Microsomal and lysosomal forms of sialidase had an optimum activity at pH 3.6-3.8, whereas the optimum for the cytosolic form was pH 4.6. The activity of all three forms was inhibited by Cu2+, whereas 1 mM Zn2+ and 0.5 mM Ca2+ activated the lysosomal and the cytosolic forms, respectively. In the crude homogenate taken from lungs of Bacillus Calmette Guérin-(BCG-) stimulated guinea pigs, the sialidase activity was increased by 43% (p = 0.025) 3 weeks after the end of the treatment. The cytosolic (+246%) and microsomal (+51%) sialidase activities were significantly increased, whereas the lysosomal sialidase activity was not changed significantly by BCG stimulation.