Postmitochondrial Supernatant Research Articles

Fabrication and evaluation of mannose decorated curcumin loaded nanostructured lipid carriers for hepatocyte targeting: In vivo hepatoprotective activity in Wistar rats

Curcumin is a well-recognized antioxidant phytoactive isolated from the rhizomes of Curcuma longa. Numerous landmark investigations have proved the antioxidant and hepatoprotective potential of curcumin. The aim of present study was to target curcumin loaded nanocarriers to hepatocytes using asialoglycoprotein receptors targeting strategy. Mannose, a water-soluble carbohydrate, was hydrophobized by anchoring stearylamine with an objective to conjugate mannose on the surface of curcumin loaded nanostructured lipid carriers for targeting asialoglycoprotein receptors on hepatocytes. Mannose conjugated stearylamine was synthesized and characterized using various analytical techniques. The synthesized targeting ligand was incorporated curcumin loaded nanostructured lipid carriers and characterized by photon correlation spectroscopy. Zeta potential measurement was used to confirm the conjugation of the synthesized ligand to the surface of drug-loaded nanostructured lipid carriers. CCl4 induced hepatotoxicity in male Wistar rats was used as an experimental animal model to evaluate the hepatoprotective potential of formulated drug encapsulated nanostructured lipid carriers. The hepatoprotective potential was assessed by measuring serum liver injury markers and oxidative stress parameters in the liver post–mitochondrial supernatant. Mannose conjugated nanostructured lipid carriers showed acceptable particle size which revealed its suitability for hepatocyte targeting. In addition to this, mannose conjugated nanocarriers revealed significantly better (p ​< ​0.05) reduction of serum liver injury markers and proinflammatory cytokines compared to the unconjugated one which confirmed hepatocytes targeting potential of the synthesized ligand. Asialoglycoprotein receptors targeting could be a landmark strategy for hepatocyte targeting. Thus, the synthesized mannose anchored stearylamine could be a promising novel targeting ligand having hepatocyte targeting potential.

Localization and distribution of connexin 43 in mitochondria of cervical cancer HeLa cells

Objective To study the distribution of connexin 43 (Cx43) in cervical cancer HeLa cells, and to verify the localization of Cx43 in mitochondria. Methods HeLa cells were segregated into cytoplasm, cell nucleus, mitochondria and supernatant after segregation by using the method of homogenate and centrifuge. Immunoelectron microscope was used to observe the morphology of mitochondria and the localization as well as the distribution of Cx43 in HeLa cells. Voltage-dependent anion channel 1 (VDAC1) was used to confirm the localization of mitochondria. Immunofluorescence was used to costain HeLa cells with Cx43 and mitochondrial marker VDAC1 to verify mitochondria localization of Cx43 in cervical cancer HeLa cells. Then Western blot was used to quantify the expression of Cx43 in fractions (cytoplasmic fraction, nuclear, mitochondria and post mitochondrial supernatant). Mitochondrial markers including VDAC1 and cytochrome c oxidase Ⅳ (COX Ⅳ) were used to confirm mitochondria. Plasma membrane marker (LHR) was used to confirm plasma membrane. Results Immunoelectron microscope confirmed that the normal mitochondria or cystic swollen one could be seen in the complete HeLa cells and the detached HeLa cells mitochondria, with the presence of Cx43 and VDAC1 in detached mitochondria. Immunofluorescence showed Cx43 colocalized with VDAC1. There was a significant difference in the Cx43 expressions of the subcellular structure in the HeLa cells [cytoplasm (1.23±0.11), cell nucleus (0.39±0.09), mitochondria (3.67±0.59), supernatant after segregation (0.16±0.06); F = 84.17, P < 0.05]. It also showed that the relative amount of Cx43 in mitochondria was enriched. Conclusions Cx43 is enriched in mitochondria in cervical cancer HeLa cells. Therefore, Cx43 in mitochondria might be a potential target in diagnosis, therapy and prognosis of cervical cancer. Key words: HeLa cells; Connexin 43; Mitochondria; Localization; Distribution

A multi-year study of hepatic biomarkers in coastal fishes from the Gulf of Mexico after the Deepwater Horizon Oil Spill

Following the 2010 Gulf of Mexico oil spill, concerns were raised regarding exposure of fish to crude oil components, particularly polycyclic aromatic hydrocarbons (PAHs). This three year study examined hepatic enzymes in post-mitochondrial supernatant fractions from red snapper (Lutjanus campechanus) and gray triggerfish (Balistes capriscus) collected in the north central Gulf of Mexico between 2011 and 2014. Biomarker activities evaluated included benzo(a)pyrene hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), glutathione transferase (GST), and glutathione peroxidase (GPx). Mean EROD activity was higher in gray triggerfish (12.97 ± 7.15 pmol/min/mg protein [mean ± SD], n = 115) than red snapper (2.75 ± 1.92 pmol/min/mg protein, n = 194), p < 0.0001. In both species, EROD declined over time between 2011 and 2014. Declines in GST and GPx activities were also noted over this time period for both species. Gray triggerfish liver was fatty, and heptane extracts of the liver fat contained fluorescent substances with properties similar to known PAHs, however the origin of these PAHs is unknown.

Bax mitochondrial relocation is linked to its phosphorylation and its interaction with Bcl-xL.

The heterologous expression of Bax, and other Bcl-2 family members, in the yeast Saccharomyces cerevisiae, has proved to be a valuable reporter system to investigate the molecular mechanisms underlying their interaction with mitochondria. By combining the co-expression of Bax and Bcl-xL mutants with analyzes of their localization and interaction in mitochondria and post-mitochondrial supernatants, we showed that the ability of Bax and Bcl-xL to interact is dependent both on Bax phosphorylation - mimicked by a substitution S184D - and by Bax and Bcl-xL localization. This, and previous data, provide the molecular basis for a model of dynamic equilibrium for Bax localization and activation, regulated both by phosphorylation and Bcl-xL.

The utility of metabolic activation mixtures containing human hepatic post-mitochondrial supernatant (S9) for in vitro genetic toxicity assessment.

In vitro genotoxicity assessment routinely employs an exogenous metabolic activation mixture to simulate mammalian metabolism. Activation mixtures commonly contain post-mitochondrial liver supernatant (i.e. S9) from chemically induced Sprague Dawley rats. Although Organization for Economic Cooperation and Development (OECD) test guidelines permit the use of other S9 preparations, assessments rarely employ human-derived S9. The objective of this study is to review and evaluate the use of human-derived S9 for in vitro genetic toxicity assessment. All available published genotoxicity assessments employing human S9 were compiled for analysis. To facilitate comparative analyses, additional matched Ames data using induced rat liver S9 were obtained for certain highly cited chemicals. Historical human and induced rat S9 quality control reports from Moltox were obtained and mined for enzyme activity and mutagenic potency data. Additional in vitro micronucleus data were experimentally generated using human and induced rat S9. The metabolic activity of induced rat S9 was found to be higher than human S9, and linked to high mutagenic potency results. This study revealed that human S9 often yields significantly lower Salmonella mutagenic potency values, especially for polycyclic aromatic hydrocarbons, aflatoxin B1 and heterocyclic amines (~3- to 350-fold). Conversely, assessment with human S9 activation yields higher potency for aromatic amines (~2- to 50-fold). Outliers with extremely high mutagenic potency results were observed in the human S9 data. Similar trends were observed in experimentally generated mammalian micronucleus cell assays, however human S9 elicited potent cytotoxicity L5178Y, CHO and TK6 cell lines. Due to the potential for reduced sensitivity and the absence of a link between enzyme activity levels and mutagenic potency, human liver S9 is not recommended for use alone in in vitro genotoxicity screening assays; however, human S9 may be extremely useful in follow-up tests, especially in the case of chemicals with species-specific metabolic differences, such as aromatic amines.

Lycopene abrogates Aβ(1–42)-mediated neuroinflammatory cascade in an experimental model of Alzheimer’s disease

BackgroundNeuroinflammation characterized by glial activation and release of proinflammatory mediators is considered to play a critical role in the pathogenesis of Alzheimer's disease (AD). β-Amyloid1–42 (Aβ1–42)-induced learning and memory impairment in rats is believed to be associated with neuronal inflammation. ObjectivesThe present study was designed to investigate the effect of lycopene, a potent antioxidant and anti-inflammatory carotenoid, in intracerebroventricular (ICV) Aβ1–42-induced neuroinflammatory cascade along with learning and memory impairment in rats. Material and methodsICV Aβ1–42 was injected bilaterally followed by treatment with lycopene or rivastigmine for 14 days. Morris water maze and elevated plus maze tests were used to assess the memory function. Rats were sacrificed and brains harvested to evaluate various biochemical parameters and mitochondrial complex activities in postmitochondrial supernatant fractions of cerebral cortex and hippocampus of rat brains. The levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), tumor growth factor β (TGF-β), nuclear factor-κB (NF-κB) and caspase-3 were assessed by enzyme-linked immunosorbent assay analysis. ResultsLycopene remediated Aβ-induced learning and memory deficits in a dose-dependent manner. Aβ1–42-induced mitochondrial dysfunction along with surge of proinflammatory cytokines TNF-α, TGF-β and IL-1β as well as NF-κB and caspase-3 activity in rat brain was significantly reduced with lycopene treatment. ConclusionThe amelioration of Aβ1–42-induced spatial learning and memory impairment by lycopene could be linked, at least in part, to the inhibition of NF-κB activity and the down-regulation of expression of neuroinflammatory cytokines, suggesting that lycopene may be a potential candidate for AD treatment.

Renal and Hepatic Antioxidant Status of Hyperglycemic Rats Treated with Single and Combinatorial Herbal Formulations.

Background/Aim: The present study ascertained the comparative capacities of single and combinatorial herbal formulations of Acanthus montanus, Emilia coccinea, Hibiscus rosasinensis and Asystasia gangetica to exert glycemic control and ameliorate oxidative stress in renal and hepatic tissues of alloxan-induced hyperglycemic rats (HyGR) following 30 days treatment. Materials and Methods: Phytochemical composition of the herbal extracts and fasting plasma glucose concentration (FPGC) of the rats were measured by standard methods. The post mitochondrial supernatant fractions (PMSF) of renal and hepatic tissues homogenates were measured for activity levels of glutathione peroxidase (GPOx), superoxide dismutase (SOD) and catalase (CAT) as well as glutathione (GSH) concentration. Results: Comparatively, flavonoids were the most abundant phytochemical present in the four herbal extracts. FPGC of treated HyGR was with in the range of 66.30±0.71-133.00±0.63 mg/dL compared to the untreated hyperglycemic rat group=368.20±0.72 mg/dL (p<0.05). At the end of the experimental time, treated HyGR liver SOD activities were lower than that of normal group (p<0.05). Liver and kidney GPOx activities of HyGR were 2.36 folds and 3.93 folds lower than the normal rats. Furthermore, liver and kidney CAT activities of the treated HyGR were significantly (p<0.05) lower than the normal rats, whereas liver and kidney GSH/GSSG ratios were significantly (p<0.05) increased when compared with treated HyGR. Conclusion: Combinatorial herbal formulations exerted greater glycemic control than the single herbal formulations and caused a sparing effect on renal and hepatic antioxidants against reactive oxygen species.

Antioxidant Status of Liver and Kidney Homogenates from Hyperglycemic Rats Administered with Single and Combinatorial Herbal Formulations

Aims: The present study ascertained the comparative capacities of single and combinatorial herbal formulations of Acanthus montanus, Emilia coccinea, Hibiscus rosasinensis and Asystasia gangetica to exert glycemic control and ameliorate oxidative stress in renal and hepatic tissues of alloxan-induced hyperglycemic rats (HyGR) following 30 days treatment. Materials and Methods: Phytochemical composition of the herbal extracts and fasting plasma glucose concentration (FPGC) of the rats were measured by standard methods. The post mitochondrial supernatant fractions (PMSF) of renal and hepatic tissues homogenates were measured for activity levels of glutathione peroxidase (GPOx), superoxide dismutase (SOD) and catalase (CAT) as well as glutathione (GSH) concentration. Results: Comparatively, flavonoids were the most abundant phytochemical present in the four herbal extracts. FPGC of treated HyGR was within the range of 66.30 ± 0.71133.00 ± 0.63 mg/dL as against the untreated hyperglycemic rat group=368.20 ± 0.72 mg/dL (p<0.05). At the end of the experimental time, treated HyGR liver SOD activities were lower than that of normal group (p<0.05). Liver and kidney GPOx activities of HyGR were 2.36 folds and 3.93 folds lower than the normal rats. Furthermore, liver and kidney CAT activities of the treated HyGR were significantly (p<0.05) lower than the normal rats, whereas liver and kidney GSH/ GSSG ratios were significantly ( p<0.05) increased when compared with treated HyGR. Conclusion: Combinatorial herbal formulations exerted greater glycemic control than the single herbal formulations and caused a sparing effect on renal and hepatic antioxidants against reactive oxygen species.

Glycemic Indices/Renal and Hepatic Antioxidant Status of Hyperglycemic Rats Treated with Single and Combinatorial Herbal Formulations

The present study ascertained the comparative capacities of single and combinatorial herbal formulations of Acanthus montanus, Emilia coccinea, Hibiscus rosasinensis and Asystasia gangetica to exert glycemic control and ameliorate oxidative stress in renal and hepatic tissues of alloxan-induced hyperglycemic rats (HyGR) following 30 days treatment. Phytochemical composition of the herbal extracts and fasting plasma glucose concentration (FPGC) of the rats were measured by standard methods. The post mitochondrial supernatant fractions (PMSF) of renal and hepatic tissues homogenates were measured for activity levels of glutathione peroxidase (GPOx), superoxide dismutase (SOD) and catalase (CAT) as well as glutathione (GSH) concentration. Comparatively, flavonoids were the most abundant phytochemical present in the four herbal extracts. FPGC of treated HyGR was within the range of 66.30 ± 0.71 - 133.00 ± 0.63 mg/dL as against the untreated hyperglycemic rat group=368.20 ± 0.72 mg/dL (p<0.05). At the end of the experimental time, treated HyGR liver SOD activities were lower than that of normal group (p<0.05). Liver and kidney GPOx activities of HyGR were 2.36 folds and 3.93 folds lower than the normal rats. Furthermore, liver and kidney CAT activities of the treated HyGR were significantly (p<0.05) lower than the normal rats, whereas liver and kidney GSH/GSSG ratios were significantly (p<0.05) increased when compared with treated HyGR. Combinatorial herbal formulations exerted greater glycemic control than the single herbal formulations and caused a sparing effect on renal and hepatic antioxidants against reactive oxygen species.

Metabolism of Gamma-tocopherol in the Liver of Male Sprague-Dawley Rat

Vitamin E (VE) plays important roles in the physiological and biochemical processes of animals and humans. However, VE is an unique vitamin that has not been understood thoroughly because the metabolites of VE are very complicated and hard to be analysed. This experiment was conducted to study the metabolism of γ-tocopherol, one of 8 VE isomers, in the liver post-mitochondrial supernatant (S9) of male Sprague-dawley rat (Rattus norvegiens, SD-rat) using a novel approach. The results showed that an optimal reactive system of γ-tocopherol metabolism in vitro was composed of 5 mg/ml of S9, 8 μg/ml of γ-tocopherol, 1 mmol/ml of nicotine amide adenine dinucleotide phosphate (NADPH) and 45 min of reactive time at 35°C. The metabolites of γ-tocopherol could be successfully analyzed using gradient elution and reverse-phase high-performance liquid chromatography (RP-HPLC). A main metabolite was identified as ω-hydroxylate, a side chain of γ-tocopherol, when the structure of γ-tocopherol metabolites was determined using gas chromatography-mass spectrometry (GC-MS). A conclusion was drawn that ω-hydroxylate, a main metabolite of γ-tocopherol, could be most utilized by SD-rat and other mammals.

Effect of co-existent components in CO2 supercritical fluid extract of Angelica Sinensis Radix on metabolism of Z-ligustilide after oral administration in rats

ObjectiveTo establish a basis for Angelica Sinensis Radix (ASR) as a dietary supplement for colorectal cancer chemoprevention, the effect of co-existent components in supercritical fluid extract (SFE) of ASR on the pharmacokinetics of Z-ligustilide after oral administration was investigated in vitro and in vivo. MethodsIncubation in gastrointestinal contents and incubation in rat liver tissue homogenates post-mitochondrial supernatant (PMS) experiments were used to study changes in the levels of Z-ligustilide in vitro. ResultsWithin 4 hours, the level of Z-ligustilide in SFE declined at a slower rate than in its pure form. Clearance of Z-ligustilide after administration in its pure form was significantly slower than that of SFE of ASR (CL, 0.96 ± 0.16 mL·min/kg versus 1.24 ± 0.21 mL·min/kg P < 0.05; AUC, 243.37 ± 16.84 versus 176.69 ± 12.59 mg·min/L). ConclusionThese phenomena may be attributed to the interactions between the co-existent components in SFE of ASR and Z-ligustilide enhancing the stability of Z-ligustilide. These results suggest that the bioavailability of Z-ligustilide in SFE of ASR is improved. However, stabilization of plasma concentration was not sustained, so that the efficacy of active components could not be maintained. Thus, further processing of SFE of ASR is required.

Testicular effect of a mixture of 2-methoxyethanol and 2-ethoxyethanol in rats

Background2-Methoxyethanol (ME) and 2-ethoxyethanol (EE) represent a large group of chemicals which are used separately or as mixtures. These compounds exert multidirectional toxic effects. The present studies aimed to demonstrate the effects of ME and EE alone and their mixture on the reproductive organs in the rats. MethodsMale Wistar rats were treated subcutaneously with ME and EE alone (1.25–5.0mM/kg/day) or with their mixture (1:1) for 4 weeks. After completion of the experiment, the testes, epididymides, and prostate were weighed. In post-mitochondrial supernatant of the testes, the level of total protein, non-protein and protein sulfhydryl groups, malondialdehyde, total antioxidant status, and glutathione peroxidase and glutathione reductase activities were determined. ResultsExposure to ME alone resulted in a dose-dependent decrease in the organ weights, the total protein, non-protein and protein sulfhydryl groups. EE alone led to less marked alterations. Co-exposure to ME and EE caused alterations similar as in the rats treated with ME alone. ConclusionsMarked testicular atrophy, decrease in epididymis and prostate weights are predominant effects of the repeated exposure to relatively low doses of ME and EE. A decrease in the total protein level, and protein sulfhydryl groups may be responsible for testicular atrophy. A significant depletion of non-protein sulfhydryl groups and occasionally elevated glutathione peroxidase activity may indicate that ME and EE resulted in disturbances of pro-oxidant/antioxidant balance. The study suggests that testicular toxicity in male rats co-exposed to ME and EE is mainly caused by the former compound.

English

Pathogenesis of several chronic liver diseases has been attributed to overwhelmed antioxidant protective system against reactive oxygen species (ROS). The present study ascertained the capacity of short-term administration of ethanolic extract of Allium sativa to neutralize ROS and ameliorate hyperlipidemia. Hyperlipidemia was induced in rats by single intra-peritoneal injection of CCl4 (dosage = 2.0 mL/kg), followed by treatment with ethanolic extract of A. sativa (dosage: 200 and 400 mg/kg) at a regular interval of 16 for 64 h. Blood samples were drawn from the rats at t = 0 h and t = 76 h, that is, 12 h after the end of 64 h treatment with CCl4/A. sativa extract, to ascertain for hepatic function and serum lipid profile (SLP). In addition, liver post mitochondrial supernatant (PMS) fraction was measured for oxidative stress indicators: lipid peroxidation (LPOx), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and reduced glutathione (GSH). On the average, short-term administration of ethanolic extract of A. sativa caused reduction of SLP in the following magnitude: total cholesterol (TC) = 19.48%, triacylglycerol (TAG) = 48.59%, VLDL-C = 48.57%, LDL-C = 19.49% and increase in HDL-C = 32.43%. Also, improvement in oxidative stress indicators gave SOD = 10.20%, GPx = 30.92%, CAT = 18.18%, LPOx = 35.92% and GSH = 51.09%. Although the administration of A. sativa extract to the rats did not restore full therapeutic benefits within the experimental time (t = 76 h), the capacity of the plant extract to ameliorate oxidative stress and hyperlipidemia in the animals was fairly at par with the standard hepatic drug-hepaticum. &nbsp; Key words: Allium sativa, hepatocyte, hyperlipidemia, lipid profile, oxidative stress.

Ameliorative effect of polyphenols from Padina boergesenii against ferric nitrilotriacetate induced renal oxidative damage: With inhibition of oxidative hemolysis and in vitro free radicals.

The aim of this study was to evaluate the antioxidant activities of diethyl ether (DEE) and methanol (M) extracts from brown alga Padina boergesenii using in vitro and in vivo antioxidant assay, which may help to relate the antioxidant properties with the possible outline of its ameliorative effect. M extract showed higher radical scavenging activity through ferric reducing antioxidant power 139.11 µmol tannic acid equivalent/g; DPPH 71.32 ± 0.56%; deoxyribose radical 88.31 ± 0.47%, and total antioxidant activity 0.47 ± 0.02 mg ascorbic acid equivalents/g. Oxidative red blood cell (RBC) hemolysis inhibition rate was significantly higher in M extract (150 mg/kg body weight) in reference to total phenolic content (r = 0.935). Rats administered with DEE and M extracts (150 mg/kg body weight) for seven days before the administration of ferric nitrilotriacetate (9 mg of Fe/mg/kg bodyweight). Rats pretreated with extracts significantly changed the level of renal microsomal lipid peroxidation, glutathione, and antioxidant enzymes in post-mitochondrial supernatant (P < 0.05). Ameliorative effect of extracts against renal oxidative damage was evident in rat kidney through changes in necrotic and epithelial cells. HPTLC technique has identified the presence of rutin with reference to retardation factor (Rf ) in both the extracts. These findings support the source of polyphenols (rutin) from P. boergesenii had potent antioxidant activity; further work on isolation of bioactive compounds can be channeled to develop as a natural antioxidant.

Expression and Localization of sPLA2-III in the Rat CNS

Phospholipases A(2) (PLA(2)) are enzymes that cleave the sn-2 bond of membrane phospholipids to yield free fatty acids and lysophospholipids. Secretory PLA2-III (sPLA(2)-III) has been suggested to be important for neuronal differentiation, growth and survival, and is highly expressed in the spinal cord. The aim of this study is to elucidate its expression and distribution in different regions of the adult rat CNS. Quantitative RT-PCR analyses showed high levels of sPLA(2)-III mRNA expression in the brainstem and spinal cord and low expression in the olfactory bulb. Western blot analyses showed high level of expression in the brainstem, spinal cord and cerebral neocortex. A dense band corresponding to the catalytically active, mature/cleaved form, and a faint band corresponding to the full length sPLA(2)-III were detected in post-mitochondrial supernatants, from different parts of the CNS. Subcellular fractionation of spinal cord homogenates showed that sPLA(2)-III protein is present in the 'light membrane/cytosol' fraction, but not the nucleus, synaptosomal membrane or synaptic vesicle-enriched fractions. sPLA(2)-III was immunolocalized to neurons in the cerebral neocortex, Purkinje neurons in the cerebellar cortex, periaqueductal gray, red nucleus, spinal trigeminal nucleus and dorsal horn of the spinal cord. Electron microscopy of the spinal cord and cerebral neocortex showed that sPLA(2)-III was localized in dendrites or dendritic spines, that formed asymmetrical synapses with unlabeled, putatively glutamatergic, axon terminals. The localization of mature/cleaved form of sPLA(2)-III in postsynaptic structures suggest a physiological role of the enzyme in neurotransmission or synaptic plasticity.

Decreased cardiac mitochondrial tetrahydrobiopterin in a rat model of pressure overload

Sustained cardiac pressure overload induces mitochondrial dysfunction and apoptosis of cardiomyocytes leading to pathological cardiac hypertrophy and dysfunction. Mitochondrial nitric oxide synthase (NOS) appears to cause uncoupling, which produces reactive oxygen species (ROS) instead of nitric oxide (NO), by a decrease in the cofactor tetrahydrobiopterin (BH4). This study focused on examining the changes in mitochondrial BH4 levels during cardiac pressure overload. Chronic cardiac pressure overload was generated by abdominal aortic banding in rats. Levels of BH4 and its oxidized form were measured in the mitochondria isolated from the left ventricle (LV) and the post-mitochondrial supernatants. Chronic aortic banding increased blood pressure, and induced cardiac hypertrophy and fibrosis. Notably, the BH4 levels were decreased while its oxidized forms were increased in LV mitochondria, but not in the post-mitochondrial supernatants containing the cytosol and microsome. Anti-neuronal NOS antibody-sensitive protein was detected in the cardiac mitochondria. Moreover, continuous administration of BH4 to rats with pressure overload increased mitochondrial BH4 levels and reduced cardiac fibrosis and matrix metallopeptidase activity, but not cardiac hypertrophy. These findings show the possibility that NOS uncoupling by decreased cardiac mitochondrial BH4 levels is implicated, at least in part, in the development of cardiac fibrosis, leading to cardiac dysfunction induced by pressure overload.

The effect of a novel tobacco process on the in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter

Some of the toxic effects of smoking have been attributed to the combustion of nitrogenous protein in tobacco. The effects of a treatment which reduces tobacco’s protein nitrogen level, on the in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter (PM), were measured. PMs were tested in the Neutral Red Uptake (NRU) test; the Salmonella mutagenicity assay (SAL); the mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT). PMs from all of the cigarettes were cytotoxic and genotoxic. PM obtained from smoking treated tobacco, showed a small, consistent and statistically significant reduced mutagenicity (revertants/μg) in TA98 with post-mitochondrial supernatant (S9). No consistent quantitative or qualitative differences were detected in the other tests. The data are discussed in relation to published information on smoke chemistry obtained from cigarettes made of tobacco treated using this technique. The observations confirm that the method did not give rise to any new qualitative or quantitative cytotoxic or genotoxic effects, and may have reduced PM’s bacterial mutagenicity in TA98 with S9. Further toxicity testing is warranted, to investigate the effects of the tobacco treatment in more detail and add to the data already obtained.

Use of Enzyme Inhibitors to Evaluate the Conversion Pathways of Ester and Amide Prodrugs: A Case Study Example with the Prodrug Ceftobiprole Medocaril

An approach was developed that uses enzyme inhibitors to support the assessment of the pathways that are responsible for the conversion of intravenously administered ester and amide prodrugs in different biological matrices. The methodology was applied to ceftobiprole medocaril (BAL5788), the prodrug of the cephalosporin antibiotic, ceftobiprole. The prodrug was incubated in plasma, postmitochondrial supernatant fractions from human liver (impaired and nonimpaired), kidney, and intestine as well as erythrocytes, in the presence and absence of different enzyme inhibitors (acetylcholinesterase, pseudocholinesterase, retinyl palmitoyl hydrolase, serine esterases, amidases, and cholinesterase). Hydrolysis was rapid, extensive, and not dependent on the presence of β-nicotinamide-adenine dinucleotide phosphate (reduced form) in all matrices tested, suggesting the involvement of carboxylesterases but not P450 enzymes. Hydrolysis in healthy human plasma was rapid and complete and only partially inhibited in the presence of paraoxonase inhibitors or in liver from hepatic impaired patients, suggesting involvement of nonparaoxonase pathways. The results demonstrate the utility of this approach in confirming the presence of multiple conversion pathways of intravenously administered prodrugs and in the case of BAL5788 demonstrated that this prodrug is unlikely to be affected by genetic polymorphisms, drug interactions, or other environmental factors that might inhibit or induce the enzymes involved in its conversion.

Antioxidant potential and radioprotective effect of soy isoflavone against gamma irradiation induced oxidative stress

The in vitro antioxidant potential and in vivo radioprotective ability of soy isoflavones was studied. Male Wistar rats were orally administered with soybean isoflavones (60mg/kg) for 21days followed by gamma irradiation exposure. Survival studies in rats exposed at 10Gy and endogenous spleen colony forming unit assay (CFU) at 6.0Gy were performed in order to find radioprotective and immunomodulatory nature of the compound. The rat liver post mitochondrial supernatant and erythrocytes were used to measure lipid peroxidation (LPO) and glutathione (GSH) content along with various antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) after gamma irradiation exposure at 2.0Gy. Pretreatment with soy isoflavone, prior to gamma irradiation resulted in the increased survival rate of the animals as compared to irradiated group. CFU counts in the isoflavone treated group followed by gamma irradiation at 6 Gy were significantly high as compared to control and the irradiated group, showing immunomodulatory nature of the isoflavones. Pretreatment with isoflavones also significantly reduced the LPO, enhanced the activity of antioxidant enzymes and improved haematological and histological parameters. The present study suggests that supplementation with isoflavone has potent antioxidant activity and act as probable radioprotector against gamma radiation induced oxidative damage.

Mammalian ACSF3 Protein Is a Malonyl-CoA Synthetase That Supplies the Chain Extender Units for Mitochondrial Fatty Acid Synthesis

The objective of this study was to identify a source of intramitochondrial malonyl-CoA that could be used for de novo fatty acid synthesis in mammalian mitochondria. Because mammalian mitochondria lack an acetyl-CoA carboxylase capable of generating malonyl-CoA inside mitochondria, the possibility that malonate could act as a precursor was investigated. Although malonyl-CoA synthetases have not been identified previously in animals, interrogation of animal protein sequence databases identified candidates that exhibited sequence similarity to known prokaryotic forms. The human candidate protein ACSF3, which has a predicted N-terminal mitochondrial targeting sequence, was cloned, expressed, and characterized as a 65-kDa acyl-CoA synthetase with extremely high specificity for malonate and methylmalonate. An arginine residue implicated in malonate binding by prokaryotic malonyl-CoA synthetases was found to be positionally conserved in animal ACSF3 enzymes and essential for activity. Subcellular fractionation experiments with HEK293T cells confirmed that human ACSF3 is located exclusively in mitochondria, and RNA interference experiments verified that this enzyme is responsible for most, if not all, of the malonyl-CoA synthetase activity in the mitochondria of these cells. In conclusion, unlike fungi, which have an intramitochondrial acetyl-CoA carboxylase, animals require an alternative source of mitochondrial malonyl-CoA; the mitochondrial ACSF3 enzyme is capable of filling this role by utilizing free malonic acid as substrate.