Liver Cytosolic Fractions Research Articles

Human Aldehyde Oxidase 1–Mediated Carbazeran Oxidation in Chimeric TK-NOG Mice Transplanted with Human Hepatocytes

Carbazeran is a potent phosphodiesterase inhibitor with species-dependent metabolic profiles in rats, dogs, and humans. In this study, we investigated the aldehyde oxidase (AOX)-mediated oxidation of carbazeran to 4-oxo derivatives in chimeric NOD/Shi-scid IL2 receptor gamma-null mice expressing a herpes simplex virus type 1 thymidine kinase transgene with humanized livers (humanized-liver mice). Liver cytosolic fractions from humanized-liver mouse effectively catalyzed carbazeran 4-oxidation with high affinity for the substrate, similar to those of the human liver cytosolic fractions and recombinant human AOX1 protein. Furthermore, hepatocytes prepared from humanized-liver mice and humans also exhibited substantial metabolism via carbazeran 4-oxidation. After a single oral administration of carbazeran (10 mg/kg), plasma levels of 4-oxo-carbazeran, N-desethyl-4-oxo-carbazeran, and 6,7-dimethoxy-1-[4-(hydroxy)-piperidino]-4-phthalazinone (three human metabolites formed via 4-oxidation) were higher in humanized-liver mice than in the control mice. In contrast, plasma levels of O-desmethylcarbazeran (a major metabolite in dogs) in control mice were higher than those in the humanized-liver mice. Relative excreted amounts of the three 4-oxidation-derived human-specific metabolites in the urine and feces were greater for humanized-liver mice than control mice, whereas the relative excreted amounts of O-desmethylcarbazeran were predominant in the urine and feces of control mice. Thus, the production of carbazeran 4-oxo derivatives was elevated in humanized-liver mice compared with control mice, in agreement with our in vitro enzyme-mediated oxidation data. These results suggest that hepatic human AOX1 functions in humanized-liver mice at the in vivo level and that humanized-liver mice may be useful for predicting drug metabolism in humans, at least with regard to human AOX1-dependent metabolism. SIGNIFICANCE STATEMENT: We found that the production of carbazeran 4-oxo derivatives was higher in humanized-liver mice than in control mice. These results were supported by the fact that carbazeran was rapidly metabolized to 4-oxo-carbazeran in humanized-liver mouse hepatocytes expressing human aldehyde oxidase 1. These results suggest that human aldehyde oxidase 1 is functional in humanized-liver mice in vivo and that chimeric NOD/Shi-scid IL2 receptor gamma-null mice expressing a herpes simplex virus type 1 thymidine kinase transgene transplanted with human hepatocytes may be a suitable model animal for predicting aldehyde oxidase-dependent biotransformation of drugs in humans.

Genetic variants of N-acetyltransferases 1 and 2 (NAT1 and NAT2) in cynomolgus and rhesus macaques

In humans, polymorphic N-acetyltransferases NAT1 and NAT2 are important enzymes that metabolize endogenous and exogenous compounds, including drugs. These enzymes exhibit considerable inter-individual variability in humans. The cynomolgus macaque is a nonhuman primate species that is widely used in drug metabolism studies. NAT1/2 in these macaques have molecular and enzymatic similarities to their human orthologs; however, genetic polymorphisms in NAT1/2 have not been fully investigated in this species. In this study, the resequencing of NAT1 and NAT2 in 114 cynomolgus macaques and 19 rhesus macaques found 15 non-synonymous variants for NAT1 and 11 non-synonymous variants and 1 insertion/deletion variant for NAT2. Nine (60%) and five (33%) NAT1 variants and seven (67%) and three (25%) NAT2 variants were unique to cynomolgus and rhesus macaques, respectively. Functional characterization of the mutant enzymes was carried out using cynomolgus NAT1 and NAT2 proteins heterologously expressed in Escherichia coli. Compared with wild-type NAT1, the D122N NAT1 variant showed substantially lower acetylation activities toward p-aminobenzoic acid but had higher acetylation activities toward isoniazid. Moreover, liver cytosolic fractions from cynomolgus macaques homozygous for T98A NAT2 showed significantly lower acetylation activities toward isoniazid than wild-type NAT2; similar results were obtained for recombinant T98A NAT2. Interestingly, all the rhesus macaques analyzed were homozygous for T98A. These findings indicate that polymorphic NAT1/2 variants in cynomolgus and rhesus macaques, especially the T98A NAT2 variant, could account for the inter-animal and/or inter-lineage variabilities of NAT-dependent drug metabolism in macaques.

Modulation of Superoxide Dismutase Activity by Mercury, Lead, and Arsenic.

Arsenic, mercury and lead are the environmental toxicants which exert their toxic effects through binding with certain proteins including their structures and functions. The toxicity of these heavy metal results is associated to its interaction with the metalloenzymes. They replace the essential metals required for normal biochemical functions of enzymes. The superoxide dismutase (SOD), a metalloenzyme, requires certain cofactors such as Cu2+ and Zn2+ for their optimal activity. However, the studies on the in vitro kinetic characterization of SOD from the rat liver cytosolic fraction have not been reported. The main objective of this study concerns the determination of the effect of three heavy metals such as arsenic, mercury, and lead on the activity of cytosolic SOD isolated from post nuclear supernatant (PNS) of rat liver. The activity of SOD was calculated using pyrogallol as a substrate. The stability and the sensitivity of enzyme activity were measured by assaying the enzyme activity at different temperature conditions. In order to determine the IC50 of the heavy metals, the enzyme activity was monitored in the presence of different concentrations of heavy metals. The values of all kinetic parameters including Km, Vmax, and Kcat were calculated by assaying SOD in the presence and absence of heavy metals. The results indicated that these heavy metals were able to significantly modulate the kinetic behavior of hepatic SOD. The data from present study could be utilized to develop suitable antidotes to mitigate the adverse effects of these heavy metals. Graphical Abstract.

Spectrophotometric Assessment of Heme Oxygenase-1 Activity in Leishmania-infected Macrophages.

Heme oxygenase-1 (HO-1) is a stress responsive enzyme that metabolizes heme and releases free iron, carbon monoxide (CO), and biliverdin (BV), which rapidly undergoes conversion to bilirubin (BL). Estimation of bilirubin is the basis of HO-1 assay. HO-1 activity is widely employed to determine antioxidant response of cells under different physiological stress environment. Intra-macrophage infection often acts as such a stress inducer and measurement of HO-1 activity in infected cells indicates the ability of pathogens towards modulating oxidative response of host. The present protocol describes analysis of HO-1 activity in infected macrophages by spectrophotometric method, which is much less complex and therefore advantageous over other methods like high-performance liquid chromatography, radiochemical methods and detection of CO by gas chromatography. The main steps include: (1) Preparation of macrophage microsomal fraction containing HO-1 (2) Isolation of rat liver cytosolic fraction containing biliverdin reductase and (3) Assessment of heme oxygenase-1 activity by spectrophotometric detection of bilirubin. This method provides a simple and sensitive approach to measure cellular antioxidant response under infected condition.

The impact of bilberry leaves' polyphenols on the anxiety level, spatial learning and memory of db/db mice

Numerous experimental and clinical studies have shown high efficiency of plant polyphenolic compounds in restoring age-related memory and learning disorders. In the present study a functional food ingredient (FFI) was obtained by sorption of an aqueous solution of bilberry leaves extract on buckwheat flour, which allowed to concentrate polyphenols and increase their storage stability. The aim of this study was to evaluate the impact of a developed FFI, enriched with bilberry leaves` polyphenols, on the anxiety level, locomotor activity, memory and spatial learning of db/db mice with genetical type 2 diabetes. Material and methods. The experiment was conducted using 10 heterozygote male db/db mice and 10 homozygote male db/+ mice as the comparison control group (7 weeks of age). According to body weight, blood glucose level, the results of insulin resistance test and elevated plus-maze (EPM) test, animals were randomized into three groups: control group C1 - db/+ animals, control group C2 and experimental group G3 - obese db/db mice. Buckwheat flour was included into the diet of C2 group in a dose 22.5 g/100 g; FFI was included into the diet of G3 group in a dose 2.5 g/100 g (that was equal to 59.2± 1.4 mg-eq gallic acid per 100 g of the diet). The anxiety level and general locomotor activity were evaluated in the EPM test. The evaluation of behavior, memory and spatial learning was performed using passive avoidance test (PAT). Glycated hemoglobin level was determined in blood, insulin and leptin levels were determined in blood plasma, general antioxidant activity was determined in liver cytosolic fraction. Results and discussion. The obtained data on biochemical parameters and insulin resistance tests showed the absence of normalizing effects of developed FFI. However, the inclusion of polyphenol-containing FFI into the diet led to beneficial changes in physiological parameters. Animals of G3 group, provided with FFI, were significantly less anxious compared to both control groups. During PAT testing of short-term memory, no animals in G3 group entered to the dark compartment (0%), what demonstrated increased learning ability and well-established memory of these animals in comparison with C1 (50%) and C2 groups (80%). Conclusion. The results prove the effectiveness of bilberry leaves` polyphenols, sorbed on the brown buckwheat flour, in the correction of central nervous system disorders in db/ db mice with genetically altered type 2 diabetes, what points at possible prospect of FFI inclusion in therapeutic products for patients with type 2 diabetes mellitus.

Application of in vitro Drug Metabolism Studies in Chemical Structure Optimization for the Treatment of Fibrodysplasia Ossificans Progressiva (FOP).

Currently no approved treatment exists for fibrodysplasia ossificans progressiva (FOP) patients, and disease progression results in severe restriction of joint function and premature mortality. LDN-193189 has been demonstrated to be efficacious in a mouse FOP disease model after oral administration. To support species selection for drug safety evaluation and to guide structure optimization for back-up compounds, in vitro metabolism of LDN-193189 was investigated in liver microsome and cytosol fractions of mouse, rat, dog, rabbit, monkey and human. Metabolism studies included analysis of reactive intermediate formation using glutathione and potassium cyanide (KCN) and analysis of non-P450 mediated metabolites in cytosol fractions of various species. Metabolite profiles and metabolic soft spots of LDN-193189 were elucidated using LC/UV and mass spectral techniques. The in vitro metabolism of LDN-193189 was significantly dependent on aldehyde oxidase, with formation of the major NIH-Q55 metabolite. The piperazinyl moiety of LDN-193189 was liable to NADPH-dependent metabolism which generated reactive iminium intermediates, as confirmed through KCN trapping experiments, and aniline metabolites (M337 and M380), which brought up potential drug safety concerns. Subsequently, strategies were employed to avoid metabolic liabilities leading to the synthesis of Compounds 1, 2, and 3. This study demonstrated the importance of metabolite identification for the discovery of novel and safe drug candidates for the treatment of FOP and helped medicinal chemists steer away from potential metabolic liabilities.

In Vitro Approach to Addressing Violative Drug Residues in Cattle

Violative residues of the veterinary drug flunixin (FNX) are sometimes found in cattle that were presumably slaughtered in accordance with the withdrawal period prescribed by federal guidelines. A possible cause for violative residues may be slow rates of FNX metabolism associated with sex, age or illness of animals. Experiments were designed to investigate the effects of sex and age on rates of flunixin metabolism using liver microsomal, S9, cytosol, and homogenate fractions from heifers (n=5) and steers (n=6) less than 30 months of age, and cows (n=8) older than 48 months of age. Validation of subcellular fractions was achieved through enzyme analyses: cytochrome P450 (P450), NADPH‐P450 reductase and glucose‐6‐phosphate dehydrogenase (G6PDH) activities under optimized conditions. Similarly, conditions for flunixin metabolism were optimized and fractional activity and metabolite formation was determined. Cows had lower (P < 0.05) concentrations of P450, NADPH‐P450 reductase activity, and FNX metabolism than their younger counterparts (heifers or steers). Using 15 min incubations, 5‐hydroxy FNX was the major metabolite detected by LC MS‐MS. Variables including exact animal age, cattle breed, diet, health and physiological status of animals at slaughter could have influenced results but were not assessed because of limitations associated with tissue collection.Support or Funding InformationAll funding of work was provided by USDA‐ARS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hepatic GSTZ1 Expression in Pregnant Ewes and Their Offspring: Influence of Treatment with Dichloroacetate

Dichloroacetate (DCA) is an investigational drug that can stimulate mitochondrial energy metabolism by inhibiting pyruvate dehydrogenase kinase. We hypothesize that DCA could be used in high‐risk pregnancy to reduce perinatal mitochondrial deficiency and resulting cardiac dysfunction in at‐risk fetuses and are testing this postulate in a pregnant sheep model. DCA is metabolized by the enzyme glutathione transferase zeta1 (GSTZ1) to glyoxylate. GSTZ1 is not expressed in human fetal liver, but expression rises after birth. Furthermore, DCA inactivates GSTZ1, resulting in altered pharmacokinetics with repeated dosing. The objectives of this research were (1) to investigate the relative expression of GSTZ1 in maternal and fetal sheep liver from untreated controls close to full term (140 days gestation) and (2) to determine if treatment of sheep with DCA resulted in reduced expression of GSTZ1. We administered DCA intravenously to either the ewe or the fetus at daily divided doses of 25 mg/kg for three to five days prior to sacrifice 24 h after the last dose. We used samples of maternal and fetal liver to prepare cytosol and mitochondria and employed a custom‐made polyclonal antibody to rat GSTZ1 to measure expression relative to a single rat hepatic cytosol standard by Western blot. The sheep liver samples exhibited good cross‐reactivity to the rat antibody. Surprisingly, expression of GSTZ1 in the full‐term fetal liver cytosol fractions from controls was similar to that in the control maternal liver. Treatment with DCA resulted in reduced expression in both maternal and fetal liver cytosol fractions to less than 10% of the control values. In control fetuses, mitochondrial expression of GSTZ1 was 17.6 ± 7.6% of the cytosolic expression. In the DCA‐treated group, mitochondrial expression decreased and levels were undetectable in one‐half of the fetal sheep. These studies demonstrate that (1) GSTZ1 expression in liver cytosol and mitochondria in the late fetal period varies with animal species and (2) repeated treatment with DCA results in reduced GSTZ1 expression, which is likely to alter DCA pharmacokinetics.Support or Funding InformationSupported in part by the US Public Health Service, grant R21HD91599This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibitory effects of ficus deltoidea extracts on UDP-glucuronosyltransferase and glutathione s-transferase drug-metabolizing enzymes

Background: Mas cotek or Ficus deltoidea is conventionally used to treat various diseases and often consumed with other medication and this may give rise to herb–drug interaction. The potential of F. deltoidea for interactions with drug-metabolizing enzymes of UDP-gucuronosytranserase (UGT) and glutathione S-transferase (GST) have not been investigated. Objective: We had evaluated the potential of methanol, ethanol, and aqueous extracts of F. deltoidea to cause UGT- and GST-mediated herb–drug interaction in vitro. Materials and Methods: The total phenolic content and total phenolic content were determined using modified colorimetric method. In the UGT study, para -nitrophenol (p -NP) was employed as a substrate to determine the UGT enzymes activity in rat liver microsomes (RLM) and human liver microsomes (HLM). For the GST study, 1-chloro-2,4-dinitrobenzene was employed as a substrate to determine GST activity in rat liver cytosolic fraction. Results: The total phenolic content in F. deltoidea extracts can be ranked as follows: Methanol extract > aqueous extract > ethanol extract, whereas the content of flavonoid compounds in F. deltoidea extracts can be ranked as: Methanol extract > ethanol extract > aqueous extract. Assessment using the UGT enzymes of RLM (IC50 [Half-maximal inhibitory concentration] = 881.40 ± 1.14 μg/mL) and HLM (IC50 =63.44 ± 1.20 μg/mL) showed that the methanol extract of F. deltoidea significantly inhibited p -NP glucuronidation compared with ethanol and aqueous extracts. For GST inhibition study, methanol extract strongly inhibited GST activity (IC50 =70.73 ± 1.07 μg/mL), whereas no IC50values were determined for ethanol and aqueous extracts. Conclusion: The methanol extract of F. deltoidea containing the highest flavonoid content highlights the possibility of herb–drug interaction through the modulation of p -NP UGT and GST activity.

Sulfate conjugates are the major metabolites in rats administrated with sesamin

Sesamin is known to have various biological effects. Although several metabolites of sesamin have been identified, its metabolism by phase II enzymes remains unclear, because usually its sulfo- and glucurono-conjugates in plasma and urine are analyzed after sulfatase/β-glucuronidase treatment. In this study, the metabolites of sesamin in rats administrated with sesamin (100 mg/kg b.w.) were analyzed without sulfatase/β-glucuronidase treatment. Two sulfate conjugates of sesamin monocatechol (SC-1) were detected in the liver and plasma. Their Cmax values were 5- and 10-times higher than that of sesamin itself. The Vmax/Km values for sulfate conjugation in the cytosol fraction of human liver were 1.7-times larger than that in the cytosol fraction of rat liver, suggesting that sulfate conjugation also occurs in human liver. The recombinant human proteins SULT1A1, 1A3, 1B1, and 1E1 expressed in Saccharomyces cerevisiae cells produced sulfate conjugates effectively. Our results could help revealing the mechanism of physiological effects of sesamin.

In vitro subcellular characterization of flunixin liver metabolism in heifers, steers, and cows

The majority of cattle found to have violative liver residues of flunixin (FNX) in the United States are dairy cows. It has been hypothesized that illness of cows decreases the rate of FNX metabolism, resulting in violative residues at slaughter. Another contributing factor might be an age-related decrease in FNX metabolism, as dairy cull cows are typically older at slaughter than cattle raised for beef, rather than milk production. In order to investigate this possibility, subcellular fractions were prepared from liver slices from steers (n = 6) and heifers (n = 5) <30 months of age, and cows (n = 8) >48 mos of age. Cytochrome P450 (P450), NADPH-P450 reductase and glucose-6-phosphate dehydrogenase (G6PDH) activity and rate of 5-hydroxy FNX (5-OH FNX) formation were measured in liver homogenate, cytosolic, microsomal, and S9 fractions. Cows had lower concentrations of P450, NADPH-P450 reductase activity, and 5-OH FNX formation (P ≤ 0. 02), supporting the theory that advanced age may contribute to the higher incidence of violative FNX residues in dairy cows.

Functionally relevant genetic variants of glutathione S-transferase GSTM5 in cynomolgus and rhesus macaques

Glutathione S-transferase (GST) is a family of enzymes important for conjugation with glutathione of endogenous and exogenous compounds. Human GSTM1 null allele is associated with toxicity and cancers. Cynomolgus and rhesus macaques have molecular and enzymatic similarities of GSTs to humans; however, genetic variants have not been investigated. In macaques, instead of pseudogenized GSTM1, GSTM5 is a predominant GSTM isoform.In this study, re-sequencing of GSTM5 in 64 cynomolgus and 31 rhesus macaques found 6 non-synonymous variants, and 1 variant (IVS5 + 1) causing exon skip. Of these 7 variants, 3 and 1 were found only in Indochinese and Indonesian cynomolgus macaques, respectively.Cynomolgus GSTM5-mediated styrene 7,8-oxide and trans-stilbene oxide conjugation activities correlated with GSTM protein levels immunochemically quantified in cynomolgus liver samples. Using recombinant GSTM5 proteins, 4 of the 6 non-synonymous variants including E29Q, L96R, M166V and S201N showed substantially lower metabolic activities. Moreover, a homozygote for E29Q and heterozygotes for S201N or IVS5 + 1 showed significantly lower conjugation activities in liver cytosolic fractions as compared with wild-type animals.Therefore, the present results suggest that inter-animal variability of GST-dependent drug metabolism is at least partly accounted for by GSTM5 variants in cynomolgus and rhesus macaques as pre-clinical animal models.

Identification of Blood–Brain Barrier-Permeable Proteins Derived from a Peripheral Organ: In Vivo and in Vitro Evidence of Blood-to-Brain Transport of Creatine Kinase

Certain proteins, such as inflammatory cytokines, that are released from injured or diseased organs are transported from the circulating blood through the blood-brain barrier (BBB) into the brain and contribute to the pathogenesis of related central nervous system dysfunctions. However, little is known about the protein transport mechanisms involved in the central nervous system dysfunctions. The aims of the present study were to identify BBB-permeable protein(s) derived from liver and to clarify their transport characteristics at the BBB. After administration of biotin-labeled liver cytosolic protein fraction to mice in vivo, we identified 9 biotin-labeled proteins in the brain. Among them, we focused here on creatine kinase (CK). In vitro uptake studies with human brain microvessel endothelial cells (hCMEC/D3 cells) showed preferential uptake of muscle-type CK (CK-MM) compared with brain-type CK (CK-BB) at the BBB. Integration plot analysis revealed that CK-MM readily penetrated into brain parenchyma from the circulating blood across the BBB. The uptake of CK-MM by hCMEC/D3 cells was decreased at 4 °C and in the presence of clathrin- and caveolin-dependent endocytosis inhibitors. These results indicate that entry of CK into the brain is mediated by a transport system(s) at the BBB.

Evaluation of Carbazeran 4-Oxidation and O 6-Benzylguanine 8-Oxidation as Catalytic Markers of Human Aldehyde Oxidase: Impact of Cytosolic Contamination of Liver Microsomes.

The present study investigated the contribution of microsomal cytochrome P450 and cytosolic aldehyde oxidase-1 (AOX-1) to carbazeran 4-oxidation and O 6-benzylguanine 8-oxidation in human liver microsomal, cytosolic, and S9 fractions. Incubations containing carbazeran and human liver microsomes with or without exogenously added NADPH yielded comparable levels of 4-oxo-carbazeran. O 6-Benzylguanine 8-oxidation occurred in microsomal incubations, and the extent was increased by NADPH. Human recombinant CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 did not catalyze carbazeran 4-oxidation, whereas CYP1A2 was highly active in O 6-benzylguanine 8-oxidation. 1-Aminobenzotriazole, a pan-cytochrome P450 inhibitor, decreased O 6-benzylguanine 8-oxidation, but not carbazeran 4-oxidation, in microsomal incubations, whereas 1-aminobenzotriazole and furafylline (a CYP1A2-selective inhibitor) did not inhibit carbazeran 4-oxidation or O 6-benzylguanine 8-oxidation in human liver S9 fraction. Carbazeran 4-oxidation in incubations containing human liver microsomes (from multiple donors and commercial suppliers) was attributed to microsomal preparations contaminated with AOX-1, as suggested by liver microsomal experiments indicating a decrease in carbazeran 4-oxidation by an AOX-1 inhibitor (hydralazine), and to detection of AOX-1 protein (at one-third the level of that in liver cytosol). Cytosolic contamination of liver microsomes was further demonstrated by the formation of dehydroepiandrosterone sulfate (catalyzed by cytosolic sulfotransferases) in liver microsomal incubations containing dehydroepiandrosterone. In conclusion, carbazeran 4-oxidation and O 6-benzylguanine 8-oxidation are enzyme-selective catalytic markers of human AOX-1, as shown in human liver S9 fraction expressing cytochrome P450 and AOX-1. This study highlights the negative impact of cytosolic contamination of liver microsomes on the interpretation of reaction phenotyping data collected in an in vitro study performed in microsomal fractions.

Genetic Variants of Glutathione S-Transferase GSTT1 and GSTT2 in Cynomolgus Macaques: Identification of GSTT Substrates and Functionally Relevant Alleles

Glutathione S-transferase (GST) is a family of important drug-metabolizing enzymes, conjugating endogenous and exogenous compounds. Genetic polymorphisms result in the inter-individual variability of GST activity in humans. Especially, human GSTT1 and GSTT2 null alleles are associated with toxicity and various cancers derived from chemicals. Cynomolgus macaque, a nonhuman primate species widely used in drug metabolism studies, has molecular and enzymatic similarities of GSTs to the human orthologs; however, genetic polymorphisms have not been investigated in this species. In this study, resequencing of GSTT1 and GSTT2 in 64 cynomolgus and 32 rhesus macaques found 15 nonsynonymous variants and 1 nonsense variant for GSTT1 and 15 nonsynonymous variants for GSTT2. Some of these GSTT variants were distributed differently in Indochinese and Indonesian cynomolgus macaques and rhesus macaques. For analysis of functional relevance of the GSTT variants, 1-iodohexane and dibromomethane were determined to be suitable substrates for cynomolgus GSTT1 and GSTT2. However, the conjugation activities were roughly correlated with GSTT protein levels immunochemically quantified in cynomolgus liver samples with no statistical significances, implying the contributions of the GST genetic variants. Among the GSTT1 variants identified, the animals carrying R76C and D125G mutations showed lower conjugation activities toward dibromomethane than those of the wild-type in liver cytosolic fractions. Moreover, the recombinant R76C/D125G and D125G GSTT variant proteins showed significantly lower 1-iodohexane or dibromomethane conjugation activities than those of the wild-type protein. Therefore, inter-animal variability of GSTT-dependent drug metabolism is at least partly accounted for by GSTT1 and possibly GSTT2 variants in cynomolgus and rhesus macaques.

New glutathione conjugate of pyrrolizidine alkaloids produced by human cytosolic enzyme-dependent reactions in vitro.

The toxic metabolites of pyrrolizidine alkaloids (PAs) are initially formed by cytochrome P450-mediated oxidation reactions and primarily eliminated as glutathione (GSH) conjugates. Although the reaction between the reactive metabolites and GSH can occur spontaneously, the role of the cytosolic enzymes in the process has not been studied. The toxic metabolites of selected PAs (retrorsine, monocrotaline, senecionine, lasiocarpine, heliotrine or senkirkine) were generated by incubating them in 100mM phosphate buffer (pH7.4) containing liver microsomes of human, pig, rat or sheep, NADPH and reduced GSH in the absence or presence of human, pig, rat or sheep liver cytosolic fraction. The supernatants were analyzed using liquid chromatography connected to Finnigan LTQ ion-trap, Agilent QTOF or Thermo Scientific Q Exactive Focus quadrupole-orbitrap mass spectrometers. Retrorsine, senecionine and lasiocarpine yielded three GSH conjugates producing [M-H]- ions at m/z 439 (7-GSH-DHP (CHO)), m/z 441 (7-GSH-DHP (OH)) and m/z 730 (7,9-diGSH-DHP) in the presence of human liver cytosolic fraction. 7-GSH-DHP (CHO) was a novel metabolite. Monocrotaline, heliotrine and senkirkine did not produce this novel 7-GSH-DHP (CHO) conjugate. 7-GSH-DHP (CHO) disappeared when incubated with hydroxylamine, and a new oxime derivative was formed. This metabolite was formed only by the human liver cytosolic enzymes but not in the presence of rat or sheep liver cytosolic fractions under otherwise identical reaction conditions. 7-GSH-DHP (CHO) has not been reported before, and thus it was considered as a novel metabolite of PAs. This may clarify the mechanisms involved in PA detoxification and widely observed but less understood species differences in response to PA exposure.

Suspect and untargeted screening of bisphenol S metabolites produced by in vitro human liver metabolism

Bisphenol S (BPS) is increasingly used as substitute for bisphenol A, resulting in higher potential of human exposure to this compound. Yet, information on the human metabolism of BPS is limited. Hence, current biomonitoring studies rely only on the measurement of BPS itself, leading to a potential underestimation of assessing human exposure to this emerging contaminant. The aims of this study were to investigate the in vitro metabolic pathways of BPS using human liver microsomes and cytosol fractions and propose in vitro metabolites for evaluation in pharmacokinetics studies. Liquid chromatography coupled to quadrupole time-of-flight high-resolution mass spectrometry was used for the screening, identification, and structural elucidation of Phase I and II metabolites of BPS for the first time. Metabolite identification was performed using two complementary workflows: suspect and untargeted screening. Two Phase I metabolites were formed through hydroxylation of the phenolic rings. Four Phase II metabolites were formed through conjugation with glucuronic acid or sulfate. Three of these metabolites, namely dihydroxy-BPS, hydroxy-BPS-glucuronide and hydroxy-BPS-sulfate were identified and structurally elucidated for the first time. As such, we provide an expanded set of in vitro biotransformation products of BPS, which can potentially support a reliable assessment of BPS exposure in future biomonitoring studies.

Ibuprofen cases Proteasome dysfunction in mice liver

Introduction and objectivesPrevious clinical studies have suggested that nonsteroidal anti‐inflammatory drugs (NSAIDs) are associated with increased risk of stroke and cardiovascular disease. Acetaminophen, the active ingredient in Tylenol, is not an NSAID and is associated with increased risk of liver toxicity. The common use of NSAIDs for pain and fever relief is a concern but it is unclear if NSAIDs is also causing adverse effects on other tissues such as the liver, kidney and brain. Previous research in our laboratory has suggested that NSAIDs cause proteasome dysfunction in cardiac tissue. To investigate the possibility that NSAIDs also influence liver function, ibuprofen treated mice were used to determine if this NSAID affects the ubiquitin proteasome system (UPS). The UPS is critical for cell function as it is responsible for degradation of 60–80% of intracellular proteins.Method/ResultsEight‐week‐old male and female C57BL/6J mice were randomly assigned to one of 4 groups: (A) control (B) Group treated with ibuprofen (C) Group treated with ibuprofen + vitamin C (D) Group treated with vitamin C alone. Mice were treated with water or ibuprofen (100 mg/kg) and vitamin C (150 mg/kg) dissolved in drinking water for 6 weeks. Liver from 6 week treated animals were homogenized and cytosol fractions obtained. The three independent proteolytic proteasome activities, β1 caspase‐like activity, β2 trypsin‐like activity, and β5 chymotrypsin‐like activity were measured in the different cytosolic liver fractions. The proteasome inhibitor bortezomib was utilized to ensure the activity measured was due to the proteasome.Semi‐quantitative western blotting suggests that the 20S proteasome amount was similar in all 4 groups but the immunoproteasome subunit β5i was increased in both male and female ibuprofen treated and ibuprofen with vitamin C groups. The β1 and β2 proteasome activities were significantly decreased in both male and female mice treated with ibuprofen. Although inclusion of vitamin C with the ibuprofen did not fully restore proteasome activity, it resulted in an improvement in the β1 proteasome activity compared to ibuprofen treated mice. The β5 proteasome activity was also significantly decreased in both male and female mice treated with ibuprofen. However, this decrease was significantly more pronounced in female mice than in male mice suggesting gender‐specific effects of ibuprofen on proteasome function.ConclusionThese results suggest that ibuprofen significantly disrupts the UPS and causes proteasome dysfunction in liver tissue. The experimental data also suggests that proteasome dysfunction in livers from ibuprofen treated mice differs in female and male mice.Support or Funding InformationAmerican Heart Association Award #16GRNT31350040This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vitro Phase I and Phase II metabolism of the new designer benzodiazepine cloniprazepam using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

Designer benzodiazepines have recently emerged as a class of new psychoactive substances. These substances are used in recreational settings and as alternatives to prescription benzodiazepines as self-medication for patients suffering from anxiety or other mental disorders. Due to the limited information available on the metabolic fate of these new substances, it is challenging to reliably detect their usage in bioanalytical (e.g. clinical and forensic) settings. The objective of this study was to investigate the in vitro Phase I and Phase II metabolism of the new designer benzodiazepine cloniprazepam and identify potential biomarkers for its detection in human biological fluids. Cloniprazepam was incubated with human liver microsomes and cytosolic fractions to generate both Phase I and II metabolites. The extracts were analysed using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Identification of the metabolites was performed using two complementary workflows, including a suspect screening based on in silico predictions and a non-targeted screening. A total of nine metabolites were identified, eight Phase I metabolites and one Phase II metabolite, of which five were specific for cloniprazepam. Clonazepam was the major metabolite of cloniprazepam. Hydroxy-cloniprazepam, dihydroxy-cloniprazepam, 3-keto-cloniprazepam, 7-amino-cloniprazepam, hydroxy-clonazepam, 7-amino-clonazepam and 3-hydroxy-7-amino-clonazepam were formed through oxidation, hydroxylation, and/or reduction of the nitro-group. Glucuronidated hydroxy-cloniprazepam was the only Phase II metabolite detected. Five metabolites were specific for cloniprazepam. This study provided a set of human in vitro biotransformation products which can assist specific detection of cloniprazepam consumption in future studies.

Identification of Peroxisomal Protein Complexes with PTS Receptors, Pex5 and Pex7, in Mammalian Cells.

Pex5 and Pex7 are cytosolic receptors for peroxisome targeting signal type-1 (PTS1) and type-2 (PTS2), respectively, and play a pivotal role in import of peroxisomal matrix proteins. Recent advance in mass spectrometry analysis has facilitated comprehensive analysis of protein-protein interaction network by a combination with immunoprecipitation or biochemical purification. In this chapter, we introduce several findings obtained by these methods applied to mammalian cells. Exploring Pex5-binding partners in mammalian cells revealed core components comprising the import machinery complex of matrix proteins and a number of PTS1-type cargo proteins. Biochemical purification of the Pex5-export stimulating factor from rat liver cytosol fraction identified Awp1, providing further insight into molecular mechanisms of the export step of mono-ubiquitinated Pex5. Identification of DDB1 (damage-specific DNA-binding protein 1), a component of CRL4 (Cullin4A-RING ubiquitin ligase) E3 complex, as a Pex7-interacting protein revealed that quality control of Pex7 by CRL4A is important for PTS2 protein import by preventing the accumulation of dysfunctional Pex7. Furthermore, analysis of binding partners of an intraperoxisomal processing enzyme, trypsin-domain containing 1 (Tysnd1), showed a protein network regulating peroxisomal fatty acid β-oxidation.