Postmitochondrial Fraction Research Articles

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.

A 3-hydroxy %-end group in xanthophylls is preferentially oxidized to a 3-oxo ε-end group in mammals

We previously found that mice fed lutein accumulated its oxidative metabolites (3'-hydroxy-ε,ε-caroten-3-one and ε,ε-carotene-3,3'-dione) as major carotenoids, suggesting that mammals can convert xanthophylls to keto-carotenoids by the oxidation of hydroxyl groups. Here we elucidated the metabolic activities of mouse liver for several xanthophylls. When lutein was incubated with liver postmitochondrial fraction in the presence of NAD(+), (3'R,6'R)-3'-hydroxy-β,ε-caroten-3-one and (6RS,3'R,6'R)-3'-hydroxy-ε,ε-caroten-3-one were produced as major oxidation products. The former accumulated only at the early stage and was assumed to be an intermediate, followed by isomerization to the latter. The configuration at the C3' and C6' of the ε-end group in lutein was retained in the two oxidation products. These results indicate that the 3-hydroxy β-end group in lutein was preferentially oxidized to a 3-oxo ε-end group via a 3-oxo β-end group. Other xanthophylls such as β-cryptoxanthin and zeaxanthin, which have a 3-hydroxy β-end group, were also oxidized in the same manner as lutein. These keto-carotenoids, derived from dietary xanthophylls, were confirmed to be present in plasma of normal human subjects, and β,ε-caroten-3'-one was significantly increased by the ingestion of β-cryptoxanthin. Thus, humans as well as mice have oxidative activity to convert the 3-hydroxy β-end group of xanthophylls to a 3-oxo ε-end group.

The Effects of Curcumin on Aflatoxin B1- Induced Toxicity in Rats.

To evaluate the potential of curcumin on toxic and carcinogenic effects of Aflatoxin B1 (AFB1) in relation to AFB1 metabolism, we studied the effects of curcumin on hepatic AFB1-DNA adduct formation and glutathione S-transferase (GST) activity, and the toxic effects of AFB1 in male Fischer 344 rats. Oral administration of curcumin to 5-week-old male rats at a dose of 8 or 80 mg/kg for five consecutive days for three weeks resulted in reduction of AFB1-DNA adduct formation mediated by both liver microsomal and postmitochondrial fractions. The activity of liver GST toward a universal substrate, CDNB, was increased in curcumin-administered rats. As for the acute toxicity of AFB1, curcumin was orally administered to rats for 3 weeks and then AFB1 was given by intragastric intubation. The result showed a decrease of plasma AST and ALT activities in curcumin-treated rats compared with those which received AFB1 alone. Moreover, we have observed that curcumin also reduced glutathione S-transferase placental form positive single cells and foci caused by AFB1 treatment. These results demonstrate the potential of curcumin to reduce the toxic and carcinogenic effects of AFB1 by modulating hepatic drug metabolizing enzymes responsible for AFB1 metabolism.

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.

Influence of NAADP and bafilomycine A1 on activity of ATPase in liver postmitochondrial fraction

Influence of NAADP and bafilomycine A1 on activity of ATPase in liver postmitochondrial fraction

Активність АТФ-аз постмітохондріальної фракції клітин лімфоми NK/Ly за дії бафіломіцинута НААДФ

The system of active of transport of ions (ATPases) plays a critical role in cancer cells and is a potential target of anticancer therapy. A selective V-type H + -ATPase inhibitor – bafilomicine (Baf1A) was intensively studied as anticancer drug. It is known that H-ATPase creates the electrochemical gradients in the membranes of endo-lysosomal cells. These acidic stores are also Ca 2+ -containing organelles. The nicotinic acid adenine dinucleotide phosphate (NAADP) is a mediator of intracellular calcium signa­ling pathways. The role of NAADP-induced Ca 2+ transient in cancer cells is not understood. It’s correlation with activity of different ATPases in tumor cells is also poorly studied. The main goal of our study was to investigate the effects of Baf1A and NAADP on the activity of Na + /K + -ATPase, basal Mg 2+ -ATPase and Ca 2+ -ATPase of plasma membrane (PMCA) and endoplasmatic reticulum (SERCA) of NK/Ly murine lymphoma cells. All experiments were conducted on the postmitochondrial fraction of NK/Ly cells. We found that Baf1A (1 µМ) did not cause changes in activity of ATPase in postmitochondrial fraction of NK/Ly cells. It was revealed that NAADP (7 µМ) caused an increase of Na + /K + -ATPase activity by (54.18±7.84)% (n = 6, Р≤0.05) and PMCA by 3-fold (Р≤0.05) in subcellular fractions of NK/Ly cells. The activity of SERCA in membrane of cancer cells, as well as basal ATPase activity, were not changed upon NAADP application. Addition of Baf1A to the subcellular fraction of NK/Ly cells enhanced NAADP-induced increase in PMCA activity by 9.4-fold (n = 5, Р≤0.05), but NAADP-induced increase in Na + /K + -ATPase activity was slackened. We suggest that H + -ATPase functions independently on Na + /K + -ATPase as well as other ATPases identified in lymphoma membranes, due to its localization on cell surface. We found a correlation between the Н + -ATPase activity and pumps of PM (PMCA and Na + /K + -ATPase) only in presence of NAADP in subcellular fraction of NK/Ly cells. We consider that membranes of acidic stores are associated with PM in tumor cells. Keywords: tumor cells, NK/Ly lymphoma, ATPase, NAADP, Na + /K + -ATPase, PMCA, SERCA.

미생물복귀돌연변이(Ames)시험을 통한 탄산리튬의 변이원성 고찰

【Objectives: To evaluate the mutagenicity of lithium carbonate, a bacterial reverse mutation(Ames) test was carried out using four strains of S. typhimurium(TA1535; TA1537; TA98; and TA100) and one strain of E. coli(WP2uvrA). Materials: This was carried out in a dose range from 312.5 to $5,000{\mu}g/plate$ in triplicate with and without S9 activation, which is the most commonly used metabolic activation system supplemented by a post-mitochondrial fraction prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 or a combination of phenobarbitone and ${\beta}$ -naphthoflavone. Results: No significant increases in the number of revertants were observed under the conditions examined in this study. Conclusions: Based on the above observations, it can be concluded that lithium carbonate has no mutagenic activity. Despite the results, it can have an effect by inducing acute oral toxicity, eye irritation and acute aquatic toxicity. Based on this study, we suggest that future studies should be directed toward chronic, carcinogenic testing and other related areas.】

Induction of nuclear translocation of mutant cytoplasmic p53 by geranylgeranoic acid in a human hepatoma cell line

Mutant p53 proteins in human hepatoma cell lines such as HuH-7 (Y220C) and PLC/PRF/5 (R249S) accumulate in the cytoplasm, and lose their transcriptional function. Geranylgeranoic acid (GGA) is a naturally occurring acyclic diterpenoid that induces cell death in both cell lines, but not in HepG2 cells harboring wild-type p53. Here, we demonstrate that micromolar concentrations of GGA induce a rapid nuclear translocation of cytoplasmic p53 in both p53-mutant cell lines and p53 knockdown attenuates GGA-induced cell death in HuH-7 cells. Cell-free experiments demonstrate that GGA is able to release 670-kD p53-containing complexes from putative huge macromolecular aggregates in post-mitochondrial fractions as revealed on blue-native gradient PAGE. Among several p53-target genes tested, GGA upregulates PUMA gene expression, and ivermectin, an inhibitor for importin α/β, blocks GGA-induced nuclear translocation of cytoplasmic p53 and suppresses GGA-induced upregulation of PUMA mRNA levels in HuH-7 cells. Taken together, these data suggest that GGA treatment stimulates a nuclear translocation of mutant p53 through its dissociation from cytoplasmic aggregates, which may be essential for GGA-induced cell death.

Toxicokinetics and tissue depletion of Fusarenon-X and its metabolite nivalenol in piglets

Fusarenon-X (FX) is one of the trichothecene mycotoxins mainly produced by Fusarium crookwellense, which naturally occurs in agricultural commodities such as wheat and barley. To investigate the toxicokinetics of FX and its metabolite nivalenol (NIV), FX was then administered intravenously or orally to piglets at a dosage of 1mg/kg body weight. The concentrations of FX and NIV in the plasma and various tissues were measured using LC-MS/MS. The plasma concentrations of FX in the piglets were determined up to 24h and 48h after iv and po administration, respectively, and the concentration of NIV was detected up to 12h after both types of administration. The Cp0 of FX was 580.28±140.81ng/ml after iv administration. The values of t1/2β, Vss and Foral were 1.71±0.74h, 0.009±0.002ml and 74.40±18.96%, respectively. FX and NIV were detectable in the vital organs up to 24h after po administration. The peak level of FX in the liver, the kidney, and the spleen, respectively, were 165.95±9.68ng/g, 66.29±8.48 and 7.35±0.69ng/g at 3h following po administration. In vitro of liver postmitochondrial fractions with FX demonstrated that the liver and kidney are capable of FX-to-NIV metabolism.

Effects of Oral Administration of Non-genotoxic Hepato-hypertrophic Compounds on Metabolic Potency of Rat Liver

It remains uncertain why non-genotoxic compounds that result in liver hypertrophy cause liver tumors. In an effort to resolve this issue, we examined whether liver post-mitochondrial fraction (S9) prepared from rats treated with non-genotoxic compounds affected the genotoxicity of pro-mutagens. Known hepatotoxic compounds, such as piperonyl butoxide (PBO), decabromodiphenyl ether (DBDE), beta-naphthoflavone (BNF), indole-3-carbinol (I3C) and acetaminophen (AA), were orally administered to male and female F344 rats at doses sufficient to cause liver hypertrophy. Rats received diets containing each test compound for 3 days, 4 weeks or 13 weeks, and were then kept for 4 weeks without the test chemical. S9 prepared from the livers of each group was used for the Ames test with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), benzo[a]pyrene (BaP) and N-nitrosodimethylamine (NDMA). In both sexes, liver hypertrophy was observed following administration of all test compounds, and was then reversed to the control state when administration ceased. The mutagenicity of MeIQx, BaP and NDMA increased with the use of S9 derived from rats treated with non-genotoxic compounds other than AA. DBDE administration had a marked effect on the mutagenicity of BaP (over a 30-fold increase in females) and NDMA (about a 20-fold increase in males). To estimate the involvement of metabolic enzymes in the alteration of mutagenicity, we measured the activity of ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-demethylase (MROD) (phase I enzymes), and UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) (phase II enzymes) in each S9 sample. The activity of phase I enzymes increased, even at the 3rd day following administration, and then decreased gradually, except in the case of AA, while the activity of phase II enzymes increased slightly. These results suggest that non-genotoxic hepato-hypertrophic compounds may be partly involved in carcinogenesis by modulating the metabolism of pre-carcinogens incorporated from the environment, in a manner that is dependent on sex and pre-incorporated chemicals.

Exploring the protective role of apocynin, a specific NADPH oxidase inhibitor, in cisplatin-induced cardiotoxicity in rats

Despite the clinical reports, few studies have focused on reducing the cardiotoxicity of cisplatin. In the present study, cardiotoxicity was examined after a single ip injection of cisplatin (7mg/kg) in rats. Apocynin was given in drinking water (600mg/L) for five successive days before and after cisplatin injection. At the end of the experiment, hemodynamic parameters were recorded, animals were sacrificed and serum creatine kinase-MB activity was determined. The whole ventricle was isolated for estimation of tumor necrosis factor-alpha (TNF-α) content, NADPH oxidase, myeloperoxidase and caspase-3 activities in addition to nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and nuclear factor kappa B (NF-κB) gene expressions. Furthermore, oxidative stress markers and antioxidant enzymes were measured in postmitochondrial and mitochondrial fractions. Mitochondrial membrane potential, nuclear DNA fragmentation and cardiomyocyte cross-sectional area were also evaluated. Apocynin was effective against cisplatin-induced decrement in heart rate and blood pressure. Moreover, pretreatment with apocynin notably ameliorated the state of oxidative stress, mitigated inflammation and preserved mitochondrial membrane potential. Apocynin provided also a significant cardioprotection as revealed by alleviating the overexpression of Nrf2, HO-1 and NF-κB, the elevation of caspase-3 activity, the prominent nuclear DNA fragmentation and the decreased cardiomyocyte cross-sectional area. This study highlights the potential role of apocynin in inhibiting cisplatin-induced hemodynamic changes, postmitochondrial and mitochondrial damage as indicated by improvement in the state of oxidative stress, inflammation and apoptosis.

Oral Toxicity of Okadaic Acid in Mice: Study of Lethality, Organ Damage, Distribution and Effects on Detoxifying Gene Expression

In vivo, after administration by gavage to mice and rats, okadaic acid has been reported to produce lesions in liver, small intestine and forestomach. Because several reports differ in the damage detected in different organs, and on okadaic acid distribution after consumption, we determined the toxicity of this compound after oral administration to mice. After 24 hours, histopathological examination showed necrotic foci and lipid vacuoles in the livers of intoxicated animals. By immunohistochemical analysis, we detected this toxin in the liver and kidneys of intoxicated animals. Okadaic acid induces oxidative stress and can be activated in vitro into reactive compounds by the post-mitochondrial S9 fraction, so we studied the okadaic effect on the gene expression of antioxidant and phase II detoxifying enzymes in liver. We observed a downregulation in the expression of these enzymes and a reduction of protein expression of catalase and superoxide dismutase 1 in intoxicated animals.

Extramitochondrial OPA1 and adrenocortical function

We have previously described that silencing of the mitochondrial protein OPA1 enhances mitochondrial Ca2+ signaling and aldosterone production in H295R adrenocortical cells. Since extramitochondrial OPA1 (emOPA1) was reported to facilitate cAMP-induced lipolysis, we hypothesized that emOPA1, via the enhanced hydrolysis of cholesterol esters, augments aldosterone production in H295R cells. A few OPA1 immunopositive spots were detected in ∼40% of the cells. In cell fractionation studies OPA1/COX IV (mitochondrial marker) ratio in the post-mitochondrial fractions was an order of magnitude higher than that in the mitochondrial fraction. The ratio of long to short OPA1 isoforms was lower in post-mitochondrial than in mitochondrial fractions. Knockdown of OPA1 failed to reduce db-cAMP-induced phosphorylation of hormone-sensitive lipase (HSL), Ca2+ signaling and aldosterone secretion. In conclusion, OPA1 could be detected in the post-mitochondrial fractions, nevertheless, OPA1 did not interfere with the cAMP – PKA – HSL mediated activation of aldosterone secretion.

Evaluation of Level of Precursors of N-Nitrosamine in Vitro in Wistar Rats Fed Different Levels of Dietary Protein

This study compares the level of Nitrite in urine, protein concentration and nitrite concentration in post mitochondrial fraction of rats fed different levels of dietary protein with concurrent administration of precursors of N-nitrosamine; dimethylamine hydrochloride (DMA-HCl) and sodium nitrite (NaNO2). Thirty Male Wistar rats were divided into three groups and were kept for four weeks. Group one was given high protein diet (64%), group two was given a normal protein diet (27%) and group three was given low protein diet (3.5%). All the groups were administered with 3mg NaNO2 and 20mg DMA-HCL/kg, using the application of spectrophotometric analysis, centrifugation, as well as colorimetric methods. Following administrations of the chemicals to the test animal groups, the concentration of 24 hours urinary excretion of nitrite was 7.417µg/ml in high protein fed rats, 2.063µg/ml in normal protein fed rats and 0.569 µg/ml in low protein fed rats. There was a significant difference (p<0.05) in the nitrite concentration in the group fed high protein diet with concurrent administration of NaNO2 and DMA-HCl. The wistar rats fed with high protein diet, excreted 5.8 to 7 times more nitrite in urine than the severely protein deprived animals. The protein and nitrite concentration of the post mitochondrial fraction of liver was highest in rats that were fed high protein diet. This study has revealed that nutrition status affects metabolism of foreign compounds including nitrites and dimethylamine hydrochloride.

Curcumin and vitamin E modulate hepatic antioxidant gene expression in PTU-induced hypothyroid rats

In the present study, regulatory role of vitamin E and curcumin on antioxidant gene (AOG) expression in hypothyroid rat liver is reported. Adult male rats were rendered hypothyroid by administration of 0.05 % 6-propyl-thiouracil in their drinking water, while vitamin E (200 mg/kg body weight) and curcumin (30 mg/kg body weight) were supplemented orally for 30 days. Expression of antioxidant genes (Cu/Zn-superoxide dismutase; SOD1, Mn superoxide dismutase; SOD2, catalase; CAT, glutathione peroxidase; GPx1 and glutathione reductase; GR) was evaluated using RT-PCR and Western blot analyses. The activities of antioxidant enzymes were measured in mitochondrial fraction (MF) and post-mitochondrial fraction (PMF) of rat liver. In addition measurement of glutathione redox status was also carried out in both the fractions. The enhanced transcripts of CAT, GPx1 and GR in hypothyroid rat liver were alleviated by administration of vitamin E and curcumin. Elevated levels of translated product of all AOGs in hypothyroid group were remained unchanged after antioxidant administration. However, enhanced SOD1, GPx1 and decreased GR activities in PMF were normalized by vitamin E and curcumin. Similarly the increased SOD2, GPx1 and decreased CAT activities in MF were also normalized by vitamin E and curcumin supplementation. Administration of vitamin E and curcumin enhanced mitochondrial GSH level; whereas the enhanced GSH level in PMF of hypothyroid rats was alleviated by vitamin E. Thus it can be concluded that besides the antioxidant role of vitamin E and curcumin, they also regulate hepatic antioxidant gene expression in hypothyroid rats.

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.

In vitro reduction of zearalenone to β-zearalenol by rainbow trout (Oncorhynchus mykiss) hepatic microsomal and post-mitochondrial subfractions

Summary Mycoestrogen zearalenone (ZEA) is found in human foods and animal feeds. Its estrogenic potency mainly depends on its biotransformation fate. The hepatic biotransformation of ZEA in rainbow trout was investigated in this study. Various concentrations of ZEA were separately incubated with the hepatic microsomal and post-mitochondrial sub-fractions in the presence of NADPH, and the metabolites were determined by means of HPLC. Moreover, the rate of glucuronidation for ZEA and its reduced metabolites were estimated in the presence of uridine diphosphate glucuronic acid. β-zearalenol (β-ZOL) was found to be the major metabolite of ZEA by both sub-cellular fractions. The enzymatic kinetics analyses indicated that the α-ZOL and β-ZOL production by microsomal fraction were 8- and 2-fold higher than those by postmitochondrial fraction, respectively. High percentages of ZEA and its metabolites are conjugated with glucuronic acid at the lower concentrations. Data suggest that the hepatic biotransformation of ZEA in rainbow trout resulted in its detoxification as the main metabolite tends to be β-ZOL with weak estrogenic property. Moreover, at certain concentrations, the produced metabolites are entirely conjugated with glucuronic acid, which may consequently cause a prolonged duration of action due to entero-hepatic cycle.

Activity of endogenous aldehydes catabolism enzymes in subcellular fractions of liver, heart and brain of rats at pubertal age under stress

Activities of enzymes involved in redox transformation of endogenous aldehydes have been investigated in subcellular fractions of liver, heart, and brain of pubertal rats exposed to prolonged immobilization stress. In the liver aldo-keto reductase (AKR) activity in the postmitochondrial fraction and aldehyde dehydrogenase (ALDH) acivity of the mitochondrial fraction demonstrated a more pronounced decrease in 2-month-old rats. Rat heart postmitochondrial AKR and ALDH demonstrated opposite changes in their enzymatic activities, while activity of mitochondrial ALDH remained unchanged. Brain cells create conditions that favor effective utilization of endogenous aldehydes in metabolic redox pathways.