Rat Liver Microsomal Fraction Research Articles

Immobilization of cytochrome P450 enzymes onto magnetic beads: An approach to drug metabolism and biocatalysis

Cytochrome P450 (CYP, P450) presents a wide range of applicability in drug metabolism studies and in biocatalysis as a great alternative to synthesizing compounds. Nonetheless, their lack of stability is one of their major drawbacks. Aiming the possibility of enhancing the catalytic activity and promoting higher stability, liver microsomal fractions have been immobilized on magnetic beads (Mb). The immobilized procedure was modulated by using rat liver microsomal fractions (RLM-Mb). The optimal condition achieved was further employed for the immobilization of the human microsomal fractions on magnetic beads (HLM-Mb). In vitro metabolism assays were conducted using albendazole (ABZ) as a model drug, and the formation of albendazole sulfoxide (ABZSO) was monitored. Biotransformation reactions applying the produced HLM-Mb were examined for the best temperature to increase the production of metabolites and their reuse cycles. A kinetic study was carried out for HLM-Mbs monitoring the production of ABZO by the oxidation reaction of ABZ by CYP3A4. The Km value was 25.6 µmol L−1 and Vmax was 121.0 µmol L−1. Inhibition assays were conducted in the presence of ketoconazole and the production of ABZSO decreased by 46.8 ± 2.5%. Enzymatic activities for CYP2C9 and CYP2D6 on HLM-Mbs were evaluated by monitoring the hydroxylation reactions of diclofenac and bufuralol as substrates. The immobilization of CYP P450 on magnetic beads increased not only the production of ABZSO metabolites but also the stability of CYP. The use of HLM-Mbs jointly with immobilized glucose-6-phosphate dehydrogenase (G6PDH-Mbs) as a unique dual generator system to produce NADPH has established the one-pot conditions for biocatalysis in a greener approach with reuse of the biocatalyst (G6PDH-HLM-Mbs). To this end, the herein reported HLM-Mbs and G6PDH-HLM-Mbs are excellent analytical tools to be explored either in biocatalysis reactions or in in vitro metabolism studies.

INTENSITY OF LIPID PEROXIDATION IN THE MICROSOMAL FRACTION OF RAT LIVER UNDER THE EFFECTS OF SODIUM BENZOATE AND ASCORBIC ACID

The effect of food preservatives on the state of free radical processes in rat liver cells are studied in the paper. The changes of lipid peroxidation (LPO) products (the level of primary products – diene conjugates (DK), secondary products – ketodienes and conjugated trienes (KD + CT), TBA-active products and final products – Schiff bases) in the microsomal fraction of rat liver were studied under conditions of introduction into the body of sodium benzoate and ascorbic acid. Animals were divided into four groups: group I – intact animals (control); group II – rats, which were administra-tion of ascorbic acid; group III – rats, which were administration of sodium benzoate; group IV – rats, which were administration of sodium benzoate 30 min before the introduction of ascorbic acid. Sodium benzoate and ascorbic acid were administered per os daily for 21 days. Euthanasia of animals was performed under light ether anesthesia on the 21st day after the administration of sodium benzoate and ascorbic acid. It was established that the daily introduction of sodium benzoate into the body leads to the initiation of LPO in the microsomal fraction of the liver, which is evidenced by an increase in the levels of primary, secondary and final prod-ucts of LPO compared to the indicators of intact animals. Sodium benzoate exhibits a higher pro-oxidant effect when combined with ascorbic acid. It has been shown that under the conditions of administration of sodium benzoate and ascorbic acid, LPO processes are intensified, as the levels of DK, KD + ST, TBK-active products and Schiff bases significantly exceed the indicators of the group of ani-mals that were administered only sodium benzoate.

NOX2 inhibitor GSK2795039 metabolite identification towards drug optimization

Overproduction of reactive oxygen species (ROS) can lead to several disease states, such as diabetic nephropathy and amyotrophic lateral sclerosis. One of the most studied mechanisms to inhibit the over production of ROS is the inhibition of NADPH oxidase (NOX) enzymes, which catalyze the conversion of cytoplasmic NADPH to NADP+, resulting in the formation of superoxide anions. GSK2795039 has been shown to selectively inhibit the NOX2 isoform, however, clearance of the compound was high in rats and mice. Therefore, identifying metabolic soft spots would be crucial in guiding the optimization process to improve its pharmacokinetic properties. GSK2795039 (10 μM) was incubated in the presence of mouse, rat and human liver microsomal (1 mg/mL) and cytosolic (2 mg/mL) fractions and appropriate co-factors, followed by MSe fragment analysis to identify metabolic soft spots. GSK2795039 showed marked species differences in its metabolism. The alkyl side chains and indoline moiety were the most common sites of biotransformation. The compound was identified to be an aldehyde oxidase substrate. Additionally, unique human metabolites were observed in vitro. Our study sheds light on structure optimization opportunities for developing improved NOX2 inhibitors, and it will help overcome the challenges involved in preclinical species selection for its safety evaluations.

Detoxication versus Bioactivation Pathways of Lapatinib In Vitro: UGT1A1 Catalyzes the Hepatic Glucuronidation of Debenzylated Lapatinib.

O-Dealkylation of the tyrosine kinase inhibitor lapatinib by cytochrome P450 3A enzymes is implicated in the development of lapatinib-induced hepatotoxicity. Conjugative metabolism of debenzylated lapatinib (M1) via glucuronidation and sulfation is thought to be a major detoxication pathway for lapatinib in preclinical species (rat and dog), limiting formation of the quinoneimine reactive metabolite. Glucuronidation of M1 by human recombinant UDP-glucuronosyltransferases (UGTs) has been reported in vitro; however, the relative UGT enzyme contributions are unknown, and the interspecies differences in the conjugation versus bioactivation pathways of M1 have not been fully elucidated. In the present study, reaction phenotyping experiments using human recombinant UGT enzymes and enzyme-selective chemical inhibitors demonstrated that UGT1A1 was the major hepatic UGT enzyme involved in lapatinib M1 glucuronidation. Formation of the M1-glucuronide by human liver microsomes from UGT1A1-genotyped donors was significantly correlated with UGT1A1 activity as measured by 17β-estradiol 3-glucuronidation (R 2 = 0.90). Interspecies differences were found in the biotransformation of M1 in human, rat, and dog liver microsomal and 9000g supernatant (S9) fractions via glucuronidation, sulfation, aldehyde oxidase-mediated oxidation, and bioactivation to the quinoneimine trapped as a glutathione (GSH) conjugate. Moreover, we demonstrated the sequential metabolism of lapatinib in primary human hepatocytes to the M1-glucuronide, M1-sulfate, and quinoneimine-GSH conjugate. M1 glucuronidation was highly correlated with the rates of M1 formation, suggesting that O-dealkylation may be the rate-limiting step in lapatinib biotransformation. Interindividual variability in the formation and clearance pathways of lapatinib M1 likely influences the hepatic exposure to reactive metabolites and may affect the risk for hepatotoxicity. SIGNIFICANCE STATEMENT: We used an integrated approach to examine the interindividual and interspecies differences in detoxication versus bioactivation pathways of lapatinib, which is associated with idiosyncratic hepatotoxicity. In addition to cytochrome P450 (P450)-mediated bioactivation, we report that multiple non-P450 pathways are involved in the biotransformation of the primary phenolic metabolite of lapatinib in vitro, including glucuronidation, sulfation, and aldehyde oxidase mediated oxidation. UGT1A1 was identified as the major hepatic enzyme involved in debenzylated lapatinib glucuronidation, which may limit hepatic exposure to the potentially toxic quinoneimine.

Quantification of CYP2E1 in rat liver by UPLC-MS/MS-based targeted proteomics assay: a novel approach for enzyme activity assessment.

CYP2E1 is one of the most crucial isozymes of CYP450. It is responsible for metabolizing and activating a large number of toxicants and carcinogens, but the correlation between its abundance and activity has not been widely studied. With the flourishing of modern mass spectrometry technology, quantifying complex biological proteins and studying the relationship between their abundance and activity have become practicable. In our study, an accurate, sensitive, and stable LC-MS/MS-based method was developed and validated. The method can accurately quantify the abundance of CYP2E1 in the rat liver microsome and S9 fraction. The quantitative linearity of the method is between 2 and 320 ng/mL, and the run time is 16.5 minutes. Meanwhile, we used the probe substrate method (with chlorzoxazone as the substrate) as a reference to analyze the correlation between its activity and abundance. The result illustrated that the abundance of CYP2E1 by LC-MS/MS has a strong positive correlation with its activity. This is a relationship worth studying, which has not been reported before. We also explored the correlation between quantitative results by traditional methods (western blot and RT-PCR) and activity, and the positive correlation was not obvious. Therefore, when testing the correlation between metabolic enzyme abundance and activity, the LC-MS/MS-based method is confirmed to be more accurate than conventional methods. It will provide a meaningful way of researching the metabolic enzymes in drug interactions. Furthermore, we found that the S9 fraction can also be used for mass spectrometry quantitative analysis, which is helpful for promoting the practical application of targeted protein technology.

Unexpected rapid aerobic transformation of 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxyl) radical by cytochrome P450 in the presence of NADPH: Evidence against a simple reduction of the nitroxide moiety to the hydroxylamine

Aminoxyl radicals (nitroxides) are a class of compounds with important biomedical applications, serving as antioxidants, spin labels for proteins, spin probes of oximetry, pH, or redox status in electron paramagnetic resonance (EPR), or as contrast agents in magnetic resonance imaging (MRI). However, the fast reduction of the radical moiety in common tetramethyl-substituted cyclic nitroxides within cells, yielding diamagnetic hydroxylamines, limits their use in spectroscopic and imaging studies. In vivo half-lives of commonly used tetramethyl-substituted nitroxides span no more than a few minutes. Therefore, synthetic efforts have focused on enhancing the nitroxide stability towards reduction by varying the electronic and steric environment of the radical. Tetraethyl-substitution at alpha position to the aminoxyl function proved efficient in vitro against reduction by ascorbate or cytosolic extracts. Moreover, 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxyl) radical (TEEPONE) was used successfully for tridimensional EPR and MRI in vivo imaging of mouse head, with a reported half-life of over 80 min. We decided to investigate the stability of tetraethyl-substituted piperidine nitroxides in the presence of hepatic microsomal fractions, since no detailed study of their “metabolic stability” at the molecular level had been reported despite examples of the use of these nitroxides in vivo. In this context, the rapid aerobic transformation of TEEPONE observed in the presence of rat liver microsomal fractions and NADPH was unexpected. Combining EPR, HPLC-HRMS, and DFT studies on a series of piperidine nitroxides — TEEPONE, 4-oxo-2,2,6,6-tetramethyl(piperidin-1-yloxyl) (TEMPONE), and 2,2,6,6-tetraethyl-4-hydroxy(piperidin-1-yloxyl) (TEEPOL), we propose that the rapid loss in paramagnetic character of TEEPONE is not due to reduction to hydroxylamine but is a consequence of carbon backbone modification initiated by hydrogen radical abstraction in alpha position to the carbonyl by the P450-Fe(V)=O species. Besides, hydrogen radical abstraction by P450 on ethyl substituents, leading to dehydrogenation or hydroxylation products, leaves the aminoxyl function intact but could alter the linewidth of the EPR signal and thus interfere with methods relying on measurement of this parameter (EPR oximetry).

Lycopene increases metabolic activity of rat liver CYP2B, CYP2D and CYP3A.

Lycopene as a naturally occurring carotenoid is a common part of the human diet. Several beneficial properties of lycopene have been identified, with the most studied being anti-cancer and antioxidant activity. However, no evidence of possible drug-drug or drug-food supplement interactions has been found. We studied the in vivo effect of lycopene on the selected rat liver cytochromes P450 (CYPs): CYP1A2, CYP2B, CYP2C11, CYP2C6, CYP2D, and CYP3A. Lycopene was administered to rats intragastrically at doses of 4, 20, and 100 mg/kg/day for 10 consecutive days. Total protein content, P450 Content, and metabolic activity of selected CYPs were evaluated in the rat liver microsomal fraction. Increased CYP2B, CYP2D, and CYP3A metabolic activities were observed in animals treated with the lycopene dose of 100 mg/kg/day. The content of CYP3A1 protein was increased by the dose of 100 mg/kg/day and CYP3A2 protein was increased by all administered doses of lycopene. The results of our study indicate that lycopene increased the metabolic activity of enzymes that are orthologues to the most clinically important human enzymes involved in xenobiotic metabolism. The risk of pharmacokinetic interactions between lycopene dietary supplements and co-administered drugs should be evaluated.

Effect of Silver Nanoparticles on Protein Composition of Rat Liver Microsomal Fraction.

We studied the effect of oral administration of metallic silver nanoparticles to rats on the proteome of the liver microsomal fraction. Nanoparticles (5-80 nm) were administered daily to growing Wistar male rats over 92 days. Controls received pure water. To control the effect of the carrier, the rats were administered aqueous solution of a stabilizer polyvinylpyrrolidone. The protein composition (proteome) of the liver microsomal fraction was analyzed by 2D-electrophoresis with identification of variable protein spots using the high-resolution nanoHPLC-MS/MS. Eight, 6, and 8 proteins absent in the control groups appeared in the microsomal fraction under the action of nanoparticles in doses of 0.1, 1, and 10 mg/kg body weight, among these, proteasome activator complex subunit 1 (Psme1 gene), and the heat shock protein HSP60 (Hspd1 gene) were reliably identified. The consumption of silver nanoparticles led to disappearance of protein of β2a tubulin chain (Tuba1b gene) from the microsomal fraction. The expression of catalase, present in the proteome of the liver microsomal fraction in animals of all groups was significantly decreased after consumption of silver nanoparticles in doses of 0.1 and 10 mg/kg. The observed changes in the proteome are considered as manifestations of hepatotoxicity of silver nanoparticles and can be related to the antagonistic effect of silver on the status of the essential trace element selenium.

Lack of Mutagenic Activity of Sulfur Nanoparticles in Micronucleus Test on L5178Y Cell Culture

The nature and physicochemical characteristics of nanomaterials affect their biological activity. Sulfur microparticles and sulphur nanoparticles have different levels of activity against bacterial, fungal, and animal cells. The cytotoxicity and genotoxicity of sulfur nanoparticles (SNPs) of about 70 nm in size were examined on the L5178Y mouse lymphoma cell culture. The concentration of SNPs causing 50% cell death is 0.078–0.312 mg/mL. The micronucleus assay revealed no mutagenic properties of SNPs. Metabolic activation of SNPs by the rat liver microsomal fraction does not affect the toxicity. It is assumed that the mechanism of cytotoxic action is associated with the interaction of elemental sulfur with sulfhydryl groups of molecules inside the cell.

Oral pharmacokinetics and in-vitro metabolism of metyrapone in male rats.

The purpose of this study was to investigate the pharmacokinetics of a single oral administration of metyrapone (MP) and metabolites produced from it in male Wistar rats, and the major tissues and enzymes involved in the production of the MP metabolites. Furthermore, the MP metabolism in human liver subcellular fractions was compared with that in rats. High-performance liquid chromatography with ultraviolet detection (HPLC-UV) was used to determine the concentrations of MP and its metabolites in plasma and urine after administration, and the production activity of MP metabolites in subcellular fractions of various tissues. Plasma concentration of MP was rapidly increased and decreased, and the primary metabolite, metyrapol (MPOL), was immediately produced. The production activity of MPOL was substantially inhibited by an 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitor in the rat and human liver microsomal and mitochondrial fractions. In the liver cytosolic fraction, the activity was inhibited by a carbonyl reductase inhibitor in the humans but not rats. In this study, we elucidated the plasma pharmacokinetics of MP and its metabolites in male rats after an oral administration. MPOL is most likely to be produced by 11β-HSD1 in the male rats and humans.

Abstract 826: DNA adducts induced by in vitro activation of diesel and biodiesel exhaust extracts

Abstract Petroleum diesel exhaust is a complex mixture containing many probable and known carcinogens. The development of biodiesel and biodiesel blends offers a renewable alternative to petroleum diesel, but few data are available concerning the carcinogenic potential of biodiesel exhausts. We compared the formation of covalent DNA adducts by the in vitro metabolic activation of organic extracts of diesel exhaust particles (DEP)from petroleum diesel and biodiesel. Two different petroleum diesel DEPs were examined (C-DEP and B0), as well as one biodiesel DEP (B100), and DEP resulting from combustion of a blend of 20% B100 and 80% B0 (B20), which is representative of commercially available biodiesel. Oxidative activation was performed in the presence of calf thymus DNA (ctDNA) using rat liver microsomal fractions with required cofactors (S9), and nitroreductive activation was performed using xanthine oxidase (XO) and hypoxanthine in the presence of ctDNA. The modified DNA was hydrolyzed and analyzed by 32P-postlabeling using either butanol extraction or nuclease P1 pre-enrichment. Multiple DNA adducts were produced with chromatographic mobilities consistent with polycyclic aromatic hydrocarbon (PAH) and nitro-PAH adducts. The types and quantities of DNA adducts produced from two independent petroleum diesel DEPs were similar, with evidence of formation of both PAH- and nitroPAH- derived adducts. In contrast, the biodiesel DEP B100 induced higher total levels of DNA adducts in both activation systems. The lowest levels of DNA adduct formation were observed with B20 DEP. These results suggest that the DEP from available biodiesel blends (B20) pose lower risk for induction of DNA damage than petroleum diesel. This is an abstract or a proposed presentation and does not necessarily reflect EPA policy. Citation Format: Jeffrey A. Ross, Garret B. Nelson, Esrta Mutlu, Sarah H. Warren, Matthews P. Peggy, M. Ian Gilmour, David M. DeMarini. DNA adducts induced by in vitro activation of diesel and biodiesel exhaust extracts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 826. doi:10.1158/1538-7445.AM2015-826

Influence of human serum albumin on the bile acid-mediated inhibition of liver microsomal type 1 11β-hydroxysteroid dehydrogenase.

The influence of human serum albumin (HSA) on the bile acid-mediated inhibition of liver microsomal type 1 11β-hydroxysteroid dehydrogenase (11β-HSD1) was studied in vitro. A rat liver microsomal fraction was prepared, and the 11β-HSD1 enzyme activity in the presence of various concentrations of bile acids and HSA was determined using hydrocortisone as the substrate. The products of the reaction were extracted and analyzed using high-performance liquid chromatography. The magnitude of the inhibition decreased with the addition of HSA in a dose-dependent manner. Four percent human albumin decreased the inhibitory effects of 100 μM chenodeoxycholic acid and lithocholic acid from 89.9 ± 5.6 to 54.5 ± 6.1% and from 83.8 ± 4.8 to 20.8 ± 4.2%, respectively. In contrast, ursodeoxycholic acid and deoxycholic acid showed no inhibitory effect on the enzyme activity in the presence of 4% human serum albumin, and the addition of 1% γ-globulin to the assay mixture in the presence of bile acids did not affect the enzyme activity. Our in vitro study showed that the addition of HSA ameliorated the inhibition of 11β-HSD1 and that the magnitude of the change is dependent on the species of bile acid, presumably based on the numbers of hydroxyl groups. These results suggest that HSA seems to protect the bile acid-mediated inhibition of 11β-HSD1 in the healthy subject. On the other hand, in the patients with obstructive biliary diseases, not only elevated serum bile acid but also the accompanying hypoalbuminemia is important to evaluate the pathophysiology of the bile acid-mediated inhibition of 11β-HSD1 of the disease.

Інтенсивність окисних процесів у мікросомній фракції печінки щурів за умов різного забезпечення поліненасиченими жирними кислотами

The effect of fat compositions with the varying ratio of polyunsaturated fatty acids (PUFAs) of families ω-3 and ω-6 on oxidation process intensity in microsomal fraction of rat liver has been investigated. The aim of the study was to investigate the level of markers of oxidative modification of lipids and proteins in microsomal fraction of rat liver. Fat components in the experiment diets were presented by sunflower oil, soybean oil and fish oil. Rats were fed using one of the fillowing 5 diets for the period of 4 weeks: 1) AIN-93 diet with 7% sunflower oil and fish oil, with the inclusion of linoleic acid, eicosapentaenoic acid and docosahexaenoic acid in the ratio of ω-6:ω-3 – 7:1 (control diet); 2) AIN-93 diet with 7% soybean oil, with the inclusion of linoleic acid and α-linolenic acid in the ratio of 7:1; 3) the diet containing only ω-6 PUFAs; 4) the diet containing only ω-3 PUFAs; 5) the diet without PUFAs. The fatty acid compositions of the diets were analysed by gas chromatography. We measured the primary and secondary lipoperoxidation products, proteins carbonyl derivatives and SH-groups of proteins. It was shown that inclusion of linoleic acid and α-linolenic acid in the ratio of 7:1 or ω-6 PUFAs into the animal diet increased lipid peroxidation in microsomal fraction of the rat liver as compared with the control group. Only ω-6 PUFAs increased the oxidative modification of proteins in microsomal fraction of the rat liver as compared with the control rat group. High dose of ω-3 PUFAs – eicosapentaenoic acid and docosahexaenoic acid had no influence on free radical oxidation of lipids and proteins. Using the diet without PUFAs increased oxidation process intensity in microsomal fraction of rat liver. According to our study, ω-6 PUFAs increased the oxidative modification of lipids and proteins in microsomal fraction of the rat liver. ω-3 PUFAs, in particular, eicosapentaenoic acid and docosahexaenoic acid, increased lipid and protein resistance to oxidative modification in microsomal fraction of the rat liver.

Study of the fullerene C60 stability in biological substrates using in vitro model system

This paper presents the results of an in vitro study of the unmodified fullerene C60 stability in samples of biological substrates obtained from Wistar rats. The known amounts of fullerene C60 at various concentrations are added to the freshly prepared samples of rat whole blood, homogenates, and liver microsomal fractions; the decrease in its concentration over a day is studied using HPLC. The data testify to the instability of fullerene C60 in biological media in situations reproducing with some degree of approximation the processes of biodegradation of the substance taking place in a living organism (nitrogen-oxygen atmosphere, +37°C, in the dark).

Nucleotide Excision Repair Is Not Induced in Human Embryonic Lung Fibroblasts Treated with Environmental Pollutants

The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells) and tested their response to treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 µm (PM2.5) collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs) including the levels of bulky DNA adducts and the nucleotide excision repair (NER) response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS)]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9) and absence (–S9) of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (–S9); the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced.

Analytical power of LLE–HPLC–PDA–MS/MS in drug metabolism studies: Identification of new nabumetone metabolites

Nabumetone is a non-acidic, nonsteroidal anti-inflammatory prodrug. Following oral administration, the prodrug is converted in the liver to 6-methoxy-2-naphthylacetic acid (6-MNA), which was found to be the principal metabolite responsible for the NSAID effect. The pathway of nabumetone transformation to 6-MNA has not been clarified, with no intermediates between nabumetone and 6-MNA having been identified to date.In this study, a new, as yet unreported phase I metabolite was discovered within the evaluation of nabumetone metabolism by human and rat liver microsomal fractions. Extracts from the biomatrices were subjected to chiral LLE–HPLC–PDA and achiral LLE–UHPLC–MS/MS analyses to elucidate the chemical structure of this metabolite. UHPLC–MS/MS experiments detected the presence of a structure corresponding to elemental composition C15H16O3, which was tentatively assigned as a hydroxylated nabumetone. Identical nabumetone and HO-nabumetone UV spectra obtained from the PDA detector ruled out the presence of the hydroxy group in the aromatic moiety of nabumetone. Hence, the most likely structure of the new metabolite was 4-(6-methoxy-2-naphthyl)-3-hydroxybutan-2-one (3-hydroxy nabumetone). To confirm this structure, the standard of this nabumetone metabolite was synthesized, its spectral (UV, CD, NMR, MS/MS) and retention properties on chiral and achiral chromatographic columns were evaluated and compared with those of the authentic nabumetone metabolite.To elucidate the subsequent biotransformation of 3-hydroxy nabumetone, the compound was used as a substrate in incubation with human and rat liver microsomal fraction. A number of 3-hydroxy nabumetone metabolites (products of conjugation with glucuronic acid, O-desmethylation, carbonyl reduction and their combination) were discovered in the extracts from the incubated microsomes using LLE–HPLC–PDA–MS/MS experiments. On the other hand, when 3-hydroxy nabumetone was incubated with isolated rat hepatocytes, 6-MNA was detected as the principal metabolite of 3-hydroxy nabumetone. Hence, 3-hydroxy nabumetone could be the missing link in nabumetone biotransformation to 6-MNA (i.e. nabumetone→3-hydroxy nabumetone→6-MNA).

Ultrafine particles are not major carriers of carcinogenic PAHs and their genotoxicity in size-segregated aerosols

Some studies suggest that genotoxic effects of combustion-related aerosols are induced by carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) and their derivatives, which are part of the organic fraction of the particulate matter (PM) in ambient air. The proportion of the organic fraction in PM is known to vary with particle size. The ultrafine fraction is hypothesized to be the most important carrier of c-PAHs, since it possesses the highest specific surface area of PM. To test this hypothesis, the distribution of c-PAHs in organic extracts (EOMs) was compared for four size fractions of ambient-air aerosols: coarse (1<dae<10μm), upper (0.5<dae<1μm), and lower (0.17<dae<0.5μm) accumulation aerosol particles and ultrafine particles (dae<0.17). High-volume aerosol samples were collected consecutively in four localities that differed in the level of environmental pollution. The genotoxicity of EOMs was measured by analysis of DNA adducts induced in an a cellular assay consisting of calf thymus DNA with/without rat liver microsomal S9 fraction coupled with 32P-postlabelling. The upper accumulation fraction was the major size fraction in all four localities, forming 37–46% of the total PM mass. Per m3 of sampled air, this fraction also bound the largest amount of c-PAHs. Correspondingly, the upper accumulation fraction induced the highest DNA-adduct levels. Per PM mass itself, the lower accumulation fraction is seen to be the most efficient in binding DNA-reactive organic compounds. Interestingly, the results suggest that the fraction of ultrafine particles of various ambient-air samples is neither a major carrier of c-PAHs, nor a major inducer of their genotoxicity, which is an important finding that is relevant to the toxicity and health effects of ultrafine particles, which are so extensively discussed these days.

Combination of hollow‐fiber liquid‐phase microextraction and capillary electrophoresis for pioglitazone and its main metabolites determination in rat liver microsomal fraction

Pioglitazone (PGZ), a thiazolidinedione antidiabetic agent, is reported as a potent and selective activator of peroxisome proliferator-activated receptor γ (PPAR γ). This drug has been widely prescribed for the treatment of Type 2 diabetes mellitus. In this regard, this manuscript presents, for the first time, an alternative electrophoretic method for PGZ and its main metabolites determination in rat liver microsomal fraction. The electrophoretic analyses were performed using an uncoated fused-silica capillary of 50 μm id, 48 cm in total length and 40 cm in effective length, and 50 mmol/L sodium phosphate buffer solution (pH 2.5). All experiments were carried out under the normal mode. The capillary temperature was set at 35°C and a constant voltage of +30 kV was applied during the analyses. Samples were introduced into the capillary by hydrodynamic injection (50 mbar, 15 s) and detection was performed at 190 nm. The sample preparation procedure, based on hollow-fiber liquid-phase microextraction, was optimized using multifactorial experiments. Next, the following optimal condition was established: sample agitation at 1500 rpm, extraction for 15 min, 0.01 mol/L hydrochloric acid as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 6.0. The method demonstrated LOQs of 200 ng/mL. Additionally, it was linear over the concentration range of 200-25,000 ng/mL for PGZ and 200-2000 ng/mL for the metabolites. Finally, the validated method was employed to study the in vitro metabolism of PGZ using rat liver microsomal fraction.

Method for Detecting NADPH-Cytochrome P450 Reductase in Liver Microsomal Fractions by Using Native Polyacrylamide Gel Electrophoresis and NADPH-Diaphorase Staining

By combining native polyacrylamide gel electrophoresis (PAGE) and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining, a simple method for detecting NADPH-cytochrome P450 reductase in tissue samples was established. When rat liver microsomal fractions were examined by this method, several bands with different mobility were visualized. Western blot analysis indicated that the band which appeared in the most anodal position among them represented NADPH-cytochrome P450 reductase. SDS-PAGE/Western blot analysis revealed that the molecular weight of the protein forming the band was around 80 kDa, which was identical to that of rat NADPH-cytochrome P450 reductase. The intensity level of NADPH-diaphorase staining assigned to this enzyme estimated by this method increased four times in microsomal fractions prepared from rat fed ethanol chronically compared to that from controls. When a dilution series of a rat liver microsomal fraction was examined by this method and SDS-PAGE/Western blot analysis, their staining intensities representing this enzyme were significantly correlated with each other. Using the naive PAGE/NADPH-diaphorase staining method, NADPH-cytochrome P450 reductase is detected in rat liver microsomes. This method is beneficial because compared with the conventional SDS-PAGE/Western blot analysis, the quantification of NADPH-cytochrome P450 reductase in tissue samples is allowed to be more easily done.

Genotoxic potential of organic extracts from particle emissions of diesel and rapeseed oil powered engines

The present study was performed to identify possible genotoxicity induced by organic extracts from particulate matter in the exhaust of two typical diesel engines run on diesel fuel and neat heated fuel-grade rapeseed oil: a Cummins ISBe4 engine tested using the World Harmonized Steady State Test Cycle (WHSC) and modified Engine Steady Cycle (ESC) and a Zetor 1505 engine tested using the Non-Road Steady State Cycle (NRSC). In addition, biodiesel B-100 (neat methylester of rapeseed oil) was tested in the Cummins engine run on the modified ESC. Diluted exhaust was sampled with high-volume samplers on Teflon coated filters. Filters were extracted with dichlormethane (DCM) and DNA adduct levels induced by extractable organic matter (EOM) in an acellular assay of calf thymus DNA coupled with 32P-postlabeling in the presence and absence of rat liver microsomal S9 fraction were employed. Simultaneously, the chemical analysis of 12 priority PAHs in EOM, including 7 carcinogenic PAHs (c-PAHs) was performed. The results suggest that diesel emissions contain substantially more total PAHs than rapeseed oil emissions (for the ESC) or that these concentrations were comparable (for the WHSC and NRSC), while c-PAHs levels were comparable (for the ESC) or significantly higher (for the WHSC and NRSC) for rapeseed oil emissions. DNA adduct levels induced by diesel and rapeseed oil derived EOM were comparable, but consistently slightly higher for diesel than for rapeseed oil. Highly significant correlations were found between 12 priority PAHs concentrations and DNA adduct levels (0.980; p<0.001) and these correlations were even stronger for c-PAHs (0.990; p<0.001). Metabolic activation by the microsomal S9 fraction resulted in several fold higher genotoxicity, suggesting a major contribution of PAHs to genotoxicity. Directly acting compounds, other than c-PAHs, and not requiring S9 to exhibit DNA reactivity were also significant. Generally, DNA adduct levels were more dependent on the type of engine and the test cycle than on the fuel. Our findings suggest that the genotoxicity of particulate emissions from the combustion of rapeseed oil is significant and is comparable to that from the combustion of diesel fuel. A more detailed study is ongoing to verify and extent these preliminary findings.