Mouse Liver Homogenates Research Articles

Introducing an In Vitro Liver Stability Assay Capable of Predicting the In Vivo Pharmacodynamic Efficacy of siRNAs for IVIVC

There has been a renewed interest in therapeutic small interfering RNAs (siRNAs) over the past few years. This is particularly the result of successful and efficient delivery of N-acetylgalactosamine (GalNAc)-conjugated siRNAs to the liver. In general, the lead selection process for siRNA drugs is faster and more straightforward than traditional small molecules. Nevertheless, many siRNAs of different sequences and chemical modification patterns must still be evaluated before arriving at a final candidate. One of the major difficulties in streamlining this workflow is the well-known phenomenon that the in vitro data obtained from oligonucleotides transfected into cells are not directly predictive of their in vivo activity. Consequently, all oligonucleotides with some degree of in vitro activity are typically screened in vivo before final lead selection. Here, we demonstrate that the stability of liver-targeting GalNAc-conjugated siRNAs in a mouse liver homogenate shows an acceptable correlation to their in vivo target knockdown efficacy. Therefore, we suggest the incorporation of an in vitro liver homogenate stability assay during the lead optimization process for siRNAs. The addition of this assay to a flow scheme may decrease the need for animal studies, and it could bring cost savings and increase efficiency in siRNA drug development.

In vitro metabolism of 2'-ribose unmodified and modified phosphorothioate oligonucleotide therapeutics using liquid chromatography mass spectrometry.

Antisense oligonucleotides (ASOs) have been touted as an emerging therapeutic class to treat genetic disorders and infections. The evaluation of metabolic stability of ASOs during biotransformation is critical due to concerns regarding drug safety. Because the effects of the modifications in ASOs on their metabolic stabilities are different from unmodified ASOs, studies that afford an understanding of these effects as well as propose proper methods to determine modified and unmodified ASO metabolites are imperative. An LC-tandem mass spectrometry method offering good selectivity with a high-quality separation using 30 mm N,N-dimethylcyclohexylamine and 100 mm 1,1,1,3,3,3-hexafluoro-2-propanol was utilized to identify each oligonucleotide metabolite. Subsequently, the method was successfully applied to a variety of in vitro systems including endo/exonuclease digestion, mouse liver homogenates, and then liver microsomes, after which the metabolic stability of unmodified versus modified ASOs was compared. Typical patterns of chain-shortened metabolites generated by mainly 3'-exonucleases were observed in phosphodiester and phosphorothioate ASOs, and endonuclease activity was identically observed in gapmers that showed relatively more resistance to nuclease degradation. Overall, the degradation of each ASO occurred more slowly corresponding to the degree of chemical modifications, while 5'-exonuclease activities were only observed in gapmers incubated in mouse liver homogenates. Our findings provide further understanding of the impact of modifications on the metabolic stability of ASOs, which facilitates the development of future ASO therapeutics.

Natural Compounds Rosmarinic Acid and Carvacrol Counteract Aluminium-Induced Oxidative Stress.

Aluminum accumulation, glutathione (GSH) and malondialdehyde (MDA) concentrations as well as catalase (CAT) and superoxide dismutase (SOD) activities were determined in erythrocytes and brain and liver homogenates of BALB/c mice treated with Al3+ (7.5 mg/kg/day (0.15 LD50) as AlCl3 (37.08 mg/kg/day), whereas HCl (30.41 mg/kg/day) was used as Cl− control, the treatments were performed for 21 days, i.p., in the presence and absence of rosmarinic acid (0.2805 mg/kg/day (0.05 LD50), 21 days, i.g.) or carvacrol (0.0405 mg/kg/day (0.05 LD50), 21 days, i.g.). The treatment with AlCl3 increased GSH concentration in erythrocytes only slightly and had no effect on brain and liver homogenates. Rosmarinic acid and carvacrol strongly increased GSH concentration in erythrocytes but decreased it in brain and liver homogenates. However, AlCl3 treatment led to Al accumulation in mice blood, brain, and liver and induced oxidative stress, assessed based on MDA concentration in the brain and liver. Both rosmarinic acid and carvacrol were able to counteract the negative Al effect by decreasing its accumulation and protecting tissues from lipid peroxidation. AlCl3 treatment increased CAT activity in mice brain and liver homogenates, whereas the administration of either rosmarinic acid or carvacrol alone or in combination with AlCl3 had no significant effect on CAT activity. SOD activity remained unchanged after all the treatments in our study. We propose that natural herbal phenolic compounds rosmarinic acid and carvacrol could be used to protect brain and liver against aluminum induced oxidative stress leading to lipid peroxidation.

Polysaccharide from Phellinus igniarius alleviates oxidative stress and hepatic fibrosis in Schistosoma japonicum-infected mice

To evaluate the role of polysaccharide from Phellinus igniarius (PPI) in the improvement of oxidative stress, hepatic granuloma and hepatic fibrosis in Schistosoma japonicum-iniected in mice. The mouse model of schistosomiasis was established by S. japonicum cercariae infection via the abdomen. Balb/c mice were randomly assigned into 5 groups, including the healthy control group (Group A), infection control group (Group B), PPI treatment group (Group C), praziquantel treatment group (Group D) and PPI-praziquantel combination group (Group E), of 10 mice in each group. Each mouse in groups B, C, D and E was infected with (30 ± 2) S. japonicum cercariae. Then, mice in groups D and E were given praziquantel by gavage at a dose of 500 mg/kg for successive two days on day 42 post-infection, while mice in groups C and E were given PPI by gavage at a dose of 400 mg/kg for successive 30 days on day 42 post-infection. Histopathological changes of hepatic tissues were observed using hematoxylin-eosin (HE) staining, and serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN) were determined, while the activities of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), glutathione reductase (GSH-R) and glutathione (GSH) were detected in Mouse liver homogenates. The expression of transforming growth factor-beta (TGF-β) and alpha-smooth muscle actin (α-SMA) was quantified in hepatic tissues using immunohistochemistry, and the Nrf2 and Gsta4 gene expression was quantified using quantitative real-time PCR (qPCR) assay. Untreated mice presented typical pathological changes of schistosomal hepatic disorders, while PPI treatment effectively alleviated hepatic egg granulomas and collagen deposition. S. japonicum infection resulted in aggravation of hepatic lipid peroxidation, induction of oxidative stress, elevated serum MDA level and a reduction in the activity of GSH and antioxidant enzymes activities in mice. As compared to infected but untreated mice, PPI treatment suppressed hepatic lipid peroxidation, increased the GSH activity and restored the activity of antioxidant enzymes. In addition, PPI treatment inhibited the TGF-β signaling pathway and up-regulated the Nrf2 and Gsta4 gene expression. PPI plays a critical role in the treatment of schistosomiasis-induced hepatic fibrosis. It may improve oxidative stress damages through up-regulating Nrf2 and Gsta4 gene expression, thereby suppressing the development of hepatic egg granulomas and hepatic fibrosis.

Metabolite Profiling of the Antisense Oligonucleotide Eluforsen Using Liquid Chromatography-Mass Spectrometry

Eluforsen (previously known as QR-010) is a 33-mer 2′-O-methyl modified phosphorothioate antisense oligonucleotide targeting the F508del mutation in the gene encoding CFTR protein of cystic fibrosis patients. In this study, eluforsen was incubated with endo- and exonucleases and mouse liver homogenates to elucidate its in vitro metabolism. Mice and monkeys were used to determine in vivo liver and lung metabolism of eluforsen following inhalation. We developed a liquid chromatography-mass spectrometry method for the identification and semi-quantitation of the metabolites of eluforsen and then applied the method for in vitro and in vivo metabolism studies. Solid-phase extraction was used following proteinase K digestion for sample preparation. Chain-shortened metabolites of eluforsen by 3′ exonuclease were observed in mouse liver in an in vitro incubation system and by either 3′ exonuclease or 5′ exonuclease in liver and lung samples from an in vivo mouse and monkey study. This study provides approaches for further metabolite characterization of 2′-ribose-modified phosphorothioate oligonucleotides in in vitro and in vivo studies to support the development of oligonucleotide therapeutics.

Simultaneous Detection of 14 Microcystin Congeners from Tissue Samples Using UPLC- ESI-MS/MS and Two Different Deuterated Synthetic Microcystins as Internal Standards.

Cyanobacterial microcystins (MCs), potent serine/threonine-phosphatase inhibitors, pose an increasing threat to humans. Current detection methods are optimised for water matrices with only a few MC congeners simultaneously detected. However, as MC congeners are known to differ in their toxicity, methods are needed that simultaneously quantify the congeners present, thus allowing for summary hazard and risk assessment. Moreover, detection of MCs should be expanded to complex matrices, e.g., blood and tissue samples, to verify in situ MC concentrations, thus providing for improved exposure assessment and hazard interpretation. To achieve this, we applied two synthetic deuterated MC standards and optimised the tissue extraction protocol for the simultaneous detection of 14 MC congeners in a single ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run. This procedure was validated using plasma and liver homogenates of mice (male and female) spiked with deuterated MC standards. For proof of concept, tissue and plasma samples from mice i.p. injected with MC-LR and MC-LF were analysed. While MC-LF was detected in all tissue samples of both sexes, detection of MC-LR was restricted to liver samples of male mice, suggesting different toxicokinetics in males, e.g., transport, conjugation or protein binding. Thus, deconjugation/-proteinisation steps should be employed to improve detection of bound MC.

Abstract 2799: Targeting CXCR2-mediated neutrophil recruitment to lung cancer

Abstract With recent advances in immunotherapy, it is evident that targeting the tumor microenvironment (TME) is an effective strategy to treat lung cancer (LC), however, more than half LC patients are still resistant to therapy. Limited attention was given to the relevance of the innate immune system despite its critical role in triggering adaptive responses. Neutrophils (PMNs) are the predominant circulating leukocyte in humans. PMNs are associated with developing lesions and are the main immune component of primary non-small cell LC (NSCLC). Multiple studies support the notion that PMNs promote tumor progression, however, the exact mechanisms in which these PMNs are recruited to the primary and metastatic lung TMEs remain unclear. To this end, we examined available genomic databases of > 1,000 NSCLC primary adenocarcinoma (ADC) patients and observed that high expression of all CXCR2 ligands (CXCL1-8 and MIF) correlate with poor survival in lung ADC. Lung ADC patients display one of the highest fold increases of these ligands as compared to all other cancers. We then performed shRNA knock down (KD) of CXCL1 and MIF in A549 and tested the migration of PMNs towards treated and control cell lines using the novel microfluidic device. We observe 3-fold increase of PMN migration towards A549 compared to control. This increase was significantly inhibited in MIF and CXCL1 KDs as well as using MIF and CXCL1 neutralizing antibodies (NA) as compared to controls. PMN migration was higher to A549 then to PC9EN, and treatment of PMNs with a CXCR2 NA led to a decrease in their migration to A549 while unaffecting their migration to PC9EN. Due to the lack of similar genomic databases on LC metastasis, we profiled liver homogenates of mice intrasplenically injected with liver-metastatic Lewis lung carcinoma (LLC) and observed that Cxcl1 was the most overexpressed gene as compared to non-tumor bearing mice (non-TBM). We then KD CXCL1 from the liver metastatic LLC cell line and compared its capacity to recruit PMNs in live mice using intravital microscopy. We observe a decrease in the number of PMNs around developing CXCL1 KD LLC tumors compared to control LLC. We also observe a decrease in PMN migration toward the CXCL1 KD LLC tumors as compared the control LLC. This resulted in a significant decrease in liver metastasis of the CXCL1 KD LLC as compared to control LLC injected mice. Altogether, our data highlight the importance of CXCR2-mediated PMN migration in primary LC and the establishment of liver metastasis from LC. Thus, inhibiting CXCR2 represents a promising strategy to impede primary tumor growth and metastatic dissemination of LC. Citation Format: Roni F. Rayes, Jack G. Mouhanna, Claire Wang, Simon Milette, Carson Wong, Mariana Usatii, Betty Giannias, France Bourdeau, Rachel Mot, Arvind Chandrasekaran, Christopher Moraes, Sidong Huang, Daniela Quail, Logan Walsh, Veena Sangwan, Nicholas Bertos, Pierre-Olivier Fiset, Jonathan Cools-Lartigue, Lorenzo E. Ferri, Jonathan D. Spicer. Targeting CXCR2-mediated neutrophil recruitment to lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2799.

The pharmacological properties of 3-arm or 4-arm DOTA constructs for conjugation to α-melanocyte-stimulating hormone analogues for melanoma imaging.

BackgroundAlthough a 3-arm DOTA construct, which has three carboxylic acids, h has been applied for conjugation to many peptides, we investigated if a 4-arm DOTA construct conjugated to peptides improves chemical properties for melanoma imaging of the melanocortin 1 receptor compared to 3-arm DOTA-conjugated peptides.MethodsSpecific activities, radiolabeling efficiencies, and partition coefficients were evaluated using 111In-labeled 3-arm and 4-arm DOTA-α-melanocyte-stimulating hormone (MSH). For assessment of MC1-R affinity and accumulation in tumor cells in vitro, B16-F1 melanoma and/or 4T1 breast cancer cells were incubated with 111In-labeled 3-arm and 4-arm DOTA-α-MSH with and without α-MSH as a substrate. The stability was evaluated using mouse liver homogenates and plasma. Biological distribution and whole-body single photon emission computed tomography imaging of 111In-labeled 3-arm and 4-arm DOTA-α-MSH were obtained using B16-F1 melanoma-bearing mice.ResultsSpecific activities and radiolabeling efficiencies of both radiotracers were about 1.2 MBq/nM and 90–95%, respectively. The partition coefficients were −0.28 ± 0.03 for 111In-labeled 3-arm DOTA-α-MSH and −0.13 ± 0.04 for 111In-labeled 4-arm DOTA-α-MSH. Although accumulation was significantly inhibited by α-MSH in B16-F1 cells, the inhibition rate of 111In-labeled 4-arm DOTA-α-MSH was lower than that of 111In-labeled 3-arm DOTA-α-MSH. 111In-labeled 4-arm DOTA-α-MSH was taken up early into B16-F1 cells and showed higher accumulation than 111In-labeled 3-arm DOTA-α-MSH after 10 min of incubation. Although these stabilities were relatively high, the stability of 111In-labeled 4-arm DOTA-α-MSH was higher than that of 111In-labeled 3-arm DOTA-α-MSH. Regarding biological distribution, 111In-labeled 4-arm DOTA-α-MSH showed significantly lower average renal accumulation (1.38-fold) and significantly higher average melanoma accumulation (1.32-fold) than 111In-labeled 3-arm DOTA-α-MSH at all acquisition times. 111In-labeled 4-arm DOTA-α-MSH showed significantly higher melanoma-to-kidney, melanoma-to-blood, and melanoma-to-muscle ratios than 111In-labeled 3-arm DOTA-α-MSH.ConclusionsThe 4-arm DOTA construct has better chemical properties for peptide radiotracers than the 3-arm DOTA construct.

PROTECTIVE EFFECT OF SYNTHETIC ANTIOXIDANTS AND ETHANOLIC EXTRACT OF BACOPA MONNIERI AGAINST LEAD TOXICITY INDUCED METABOLIC DYSFUNCTIONS IN MICE BRAIN AND LIVER HOMOGENATES: AN IN VITRO APPROACH

Objective: The main objective of the present study was to investigate the ameliorative potential of synthetic antioxidants mixture comprising N-acetyl cysteine, ascorbic acid, tocopheryl acetate, and thiamine as micronutrient combinational therapy and ethanolic extract of Bacopa monnieri as herbal antioxidant therapy approach against lead toxicity-induced metabolic dysfunctions in vitro.
 Methods: Experimental study involved in vitro exposure of mice brain and liver homogenates to different doses (100 μM, 250 μM, and 500 μM) of lead acetate. The study also involved coadministration of high-dose lead acetate (500 μM) and specific dosage of synthetic antioxidants or ethanolic extract of B. monnieri separately to homogenate cultures. Alterations in metabolic parameters of protein levels and lipid peroxidation were analyzed for evaluating the protective effect of synthetic antioxidants and B. monnieri against lead intoxication.
 Results: Results revealed dose-dependent statistically significant (p<0.001) reduction in protein levels and elevation in lipid peroxidation in lead acetate exposed mice brain and liver homogenates as compared to their respective control groups. Coadministration of lead acetate and synthetic antioxidants mixture or B. monnieri in the brain and liver homogenates conferred protection and manifested maintenance of studied biochemical parameters nearest to control groups. Ameliorative efficacy of B. monnieri against lead-induced neurotoxicity and hepatotoxicity was found to be more pronounced than that of a mixture of synthetic antioxidants.
 Conclusion: Synthetic antioxidants mixture (N-acetyl cysteine, ascorbic acid, tocopheryl acetate, and thiamine) and B. monnieri exhibited remarkable therapeutic efficacy against lead toxicity-induced metabolic dysfunctions in mice brain and liver homogenates by virtue of their antioxidant, neuroprotective, and hepatoprotective abilities.

Protective effects and action mechanism of extract from Tibetan medicine Yajima(Chrysosplenium nudicaule) on mice with intrahepatic cholestasis induced by ANIT

Chrysosplenium nudicaule,Tibetan name " Yajima",is recorded as an effective medicine for the treatment of liver and gallbladder diseases by Tibetan Pharmacopoeia published in the past dynasties,but its traditional efficacy has not yet been investigated by means of modern pharmacological research methods. In this paper,the protective effect of extract of C. nudicaule(ECN) on liver injury in mice was observed by using the mice model of intrahepatic cholestasis(IC) induced by α-naphthyl isothiocyanate(ANIT) and the possible mechanism by which ECN work as the therapeutic agent was discussed. The results showed that the serum levels of AST,ALT,ALP,DBIL,TBIL and TBA of the model mice were notably reduced in dose-dependent manner(P<0. 01,P<0. 05). The activity of SOD and GSH-Px in the liver homogenate of mice was increased,while the content of MDA was decreased(P<0. 01,P<0. 05).Pathological examination of liver in mice showed that ECN could improve the pathological changes of liver tissue in mice. The mRNA expression level of genes related to bile acid metabolism were detected by RT-PCR and the results suggested that ECN could significantly increase the expression of genes such as BSEP,FXR and MRP2(P<0. 01,P<0. 05),meanwhile significantly reduce the expression of CYP7 A1(P<0. 01,P<0. 05). These results confirmed the protective effect of ECN on intrahepatic cholestasis-induced liver injury in mice,and indicated that the mechanism may be related to activating FXR and its target genes,reducing bile acid synthesis and increasing bile acid excretion. This study provides a modern pharmacological basis for the clinical application of Yajima in Tibetan medicine.

The impact of ionizing irradiation on liver detoxifying enzymes. A re-investigation

By looking at many studies describing the impact of ionizing irradiations in living mice on a few key detoxifying enzymes like catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione transferase, we noted conflicting evidences: almost all papers finalized to demonstrate the protective effects of natural or synthetic drugs against the damage by irradiations, described also a relevant inactivation of these enzymes in the absence of these compounds. Conversely, no inactivation and even enhanced activity has been noted under similar irradiation modality in all studies supporting the “adaptive response”. Motivated by these curious discrepancies, we performed irradiation experiments on living mice, explanted mouse livers and liver homogenates observing that, in all conditions the activity of all these enzymes remained almost unchanged except for a slight increase found in explanted livers. Our results put a question about many previous scientific reports in this field.

Mitigation of radiation-induced oxidative stress by methanolic extract of Tragia involucrata in swiss albino mice

Background: Tragia involucrata L. has been used in Indian traditional medicine since centuries to treat various ailments. The plant remains unexplored for its radioprotective properties and hence the present study. Objective: The main objective of the study is to ascertain the radioprotective effects of T. involucrata methanolic extract (TME) in Swiss albino mice against whole-body gamma radiation. Materials and Methods: The acute toxicity of TME was evaluated. The optimal protective dose of TME against radiation-induced mortality in mice was determined by survival analysis using the Kaplan–Meier curves. To understand the mechanism of radioprotection, TME was tested for hematological changes and antioxidant levels in mice by injecting100 mg/kg body weight intraperitoneally for 5 days and irradiated with a sublethal dose of 6 Gy gamma radiation. Results: The lethal dose 50%/of TME was about 645.65 mg/kgbw. The pretreatment of mice with 100 mg/kgbw of TME increased its survivability to 30% compared with the radiation control group and hence is considered as a radioprotective dose. The lethal dose 50%/30% of the irradiation alone group was found to be 9 Gy and 10.35 Gy for the TME + irradiation group. Hence, the dose reduction factor was about 1.15. The radiation decreased the hematological parameters in the blood, but on pretreatment with TME (100 mg/kgbw), increase in levels was noted. The antioxidant enzyme levels in the mice liver homogenate were found to be replenished in the test group compared with the radiation control. Conclusion: The present study indicates the protective role of TME against gamma radiation-induced mortality and oxidative stress. Hence, it is proposed as a candidate for radioprotection.

Antioxidant and prooxidant effects of Piptadeniastrum africanum as the possible rationale behind its broad scale application in African ethnomedicine

Ethnopharmacological relevancePiptadeniastrum africanum is widely used in treating oxidative stress related diseases. Oxidative stress, defined as the disturbance in the balance between the production of free radicals and antioxidant defenses, is the root cause of many pathophysiological conditions. Based on the dual properties of prooxidants as toxic and beneficial compounds, both prooxidants and antioxidants may be effective in the treatment of these conditions when the right dose is given to the right subject at the right time for the right duration. Aim of the studyThis study was aimed at investigating the in vitro and ex vivo anti- and pro-oxidative effects of P. africanum. Materials and methodsTotal phenolic and flavonoid contents of methanol and aqueous extracts of P. africanum stem back were quantified spectrophotometrically. The methanol extract, ascorbate radicals and reactive oxygen species in brain and liver homogenates of mice treated with the methanol stem bark extract were analyzed by electron paramagnetic resonance (EPR) spectroscopy. Free radical scavenging of DPPH was determined by spectrophotometric and EPR assays. ResultsThe methanol extract was richer in both phenolic and flavonoid contents compared to the aqueous extracts and also showed better DPPH radical scavenging capacity. The EPR spectroscopy in vitro analysis exhibited high DPPH scavenging capacity before and after UV irradiation (99.5% and 98.76%) at 40 μg/ml extract. The ex vivo EPR spectroscopy studies demonstrated increased levels of ascorbate radicals (•Asc) in liver and brain homogenates of healthy mice treated with P. africanum in comparison with those of the non treated controls (0.6141 ± 0.026 vs 0.1800 ± 0.0073 arb. units for liver homogenates and 0.9605 ± 0.0492 vs 0.3375 ± 0.0062 arb. units for brain homogenates, correspondingly). Considerably, higher levels of reactive oxygen species (ROS) were measured in mice liver and brain homogenates after treatment with P. africanum extract compared to the control group, as well (1.9402 ± 0.1200 vs 0.6699 ± 0.062 arb. units for liver homogenates and 1.7325 ± 01503 vs 0.3167 ± 0.0403 arb.units, respectively). ConclusionTherefore, P. africanum exhibited antioxidant and pro-oxidant properties which may explain its broad spectrum use in a wide variety of ailments.

Antioxidant activities of aqueous extracts from nine different rose cultivars

Rose petals have been applied as food additives in teas, cakes and flavor extracts. The aim of this research study was to explore and reveal the antioxidant potential of aqueous extracts of rose petals belonging to nine genotypes of rose (wild as well as hybrid). The in vitro antioxidant activities of roses were studied by lipid peroxidation assay, DPPH radical scavenging assay, iron chelation assay, phosphomolybdenum reduction assay and total phenolic and flavonoid contents. The aqueous extract showed inhibition against lipid peroxidation (TBARS), induced by prooxidants (10 µM FeSO 4 ) in mice liver homogenate. The free radical scavenging activities of the extracts were determined by scavenging of the DPPH radical. Extracts also showed metal chelating activities and high antioxidant activity in the phosphomolybdenum assay. The high content of phenolics and flavonoids detected in aqueous extracts may be responsible for the antioxidant activity. Amongst the different rose genotypes, screened, Rosa moschata (musk rose) was found to carry slightly higher antioxidant potential, owing to its higher phytochemical content.

Antioxidant activities of aqueous extracts from nine different rose cultivars

Rose petals have been applied as food additives in teas, cakes and flavor extracts. The aim of this research study was to explore and reveal the antioxidant potential of aqueous extracts of rose petals belonging to nine genotypes of rose (wild as well as hybrid). The in vitro antioxidant activities of roses were studied by lipid peroxidation assay, DPPH radical scavenging assay, iron chelation assay, phosphomolybdenum reduction assay and total phenolic and flavonoid contents. The aqueous extract showed inhibition against lipid peroxidation (TBARS), induced by prooxidants (10 µM FeSO4) in mice liver homogenate. The free radical scavenging activities of the extracts were determined by scavenging of the DPPH radical. Extracts also showed metal chelating activities and high antioxidant activity in the phosphomolybdenum assay. The high content of phenolics and flavonoids detected in aqueous extracts may be responsible for the antioxidant activity. Amongst the different rose genotypes, screened, Rosa moschata (musk rose) was found to carry slightly higher antioxidant potential, owing to its higher phytochemical content.

Identification of biologically active δ-lactone eicosanoids as paraoxonase substrates

The mammalian paraoxonases (PONs 1, 2 and 3) are a family of esterases that are highly conserved within and between species. They exhibit antioxidant and anti-inflammatory activities. However, their physiological function(s) and native substrates are uncertain. Previous structure-activity relationship studies demonstrate that PONs have a high specificity for lipophilic lactones, suggesting that such compounds may be representative of native substrates. This report describes the ability of PONs to hydrolyze two bioactive δ-lactones derived from arachidonic acid, 5,6-dihydroxy-eicosatrienoic acid lactone (5,6-DHTL) and cyclo-epoxycyclopentenone (cyclo-EC). Both lactones were very efficiently hydrolyzed by purified PON3. PON1 efficiently hydrolyzed 5,6-DHTL, but with a specific activity about 15-fold lower than PON3. 5,6-DHTL was a poor substrate for PON2. Cyclo-EC was a poor substrate for PON1 and not hydrolyzed by PON2. Studies with the PON inhibitor EDTA and a serine esterase inhibitor indicated that the PONs are the main contributors to hydrolysis of the lactones in human and mouse liver homogenates. Studies with homogenates from PON3 knockout mouse livers indicated that >80% of the 5,6-DHTL and cyclo-EC lactonase activities were attributed to PON3. The findings provide further insight into the structural requirements for PONs substrates and support the hypothesis that PONs, particularly PON1 and PON3, evolved to hydrolyze and regulate a class of lactone lipid mediators derived from polyunsaturated fatty acids.

A Synthetic Peptide AWRK6 Alleviates Lipopolysaccharide-Induced Liver Injury.

During lipopolysaccharide (LPS)-induced sepsis, the liver plays central roles in toxins phagocytosis and clearance to protect the whole body. The liver cells were constantly irritated by LPS which leads to liver injury. While most anti-LPS agents showed little clinical activity against LPS-induced liver injury. Here, the protective effects of the synthetic peptide AWRK6 against LPS-induced liver injury have been investigated in vivo and in vitro. In mice liver homogenate, LPS administration elevated ALT (alanine aminotransferase), iNOS (inducible nitric oxide synthase) and repressed SOD (superoxide dismutase) activities and these changes were remarkably reversed by AWRK6. Histologically, AWRK6 effectively alleviated the histological changes and repressed LPS-induced neutrophils infiltration. By TUNEL assay on liver sections, AWRK6 was proven to inhibit apoptosis induced by LPS in mice livers, which was also verified by the protein levels of cleaved-caspase 9, Bax and Bcl-2. In addition, by in vitro study using HepG2 cells, AWRK6 was found to recover the LPS-reduced cell viability and reduce LPS-induced apoptosis. For mechanisms, AWRK6 was demonstrated to alleviate the LPS-induced phosphorylation of ERK, JNK and p38 MAPK, indicating the involvement of MAPKs in the protection of AWRK6 against liver injury. In summary, we have found the synthetic peptide AWRK6 as a promising novel agent for LPS-induced liver injury, by inhibiting cell apoptosis through MAPK signaling pathways, which might bring new strategies for the treatment of acute and chronic liver injuries.

A reliable LC-MS/MS method for the quantification of N-acetyl-p-benzoquinoneimine, acetaminophen glutathione and acetaminophen glucuronide in mouse plasma, liver and kidney: Method validation and application to a pharmacokinetic study.

A rapid, specific, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated to simultaneously quantify N-acetyl-p-benzoquinoneimine (NAPQI), acetaminophen-glutathione (acetaminophen-glut) and acetaminophen-glucuronide (acetaminophen-gluc) in mouse plasma, liver and kidney homogenates. Analytes were eluted by a binary gradient mobile phase composed of water (phase A) and methanol containing 0.1% formic acid (phase B) at a flow rate of 0.3 mL/min, which was performed on a CAPCELL PAK C18 MG II column. It took 3.2 min to detect three analytes in a single run. Quantification was carried out in positive mode combined with multiple reaction monitoring. The validation of the LC-MS/MS method consisted of specificity, linearity, precision, accuracy, protein precipitation recovery, matrix effect, dilution integrity and stability. The plasma and tissue homogenate calibration curves were linear over concentration ranges of 0.050-5.00, 0.050-5.00 and 0.100-40.0 μg/mL, with a lower limit of quantification of 0.050, 0.050, and 0.100 μg/mL for NAPQI, acetaminophen-glut and acetaminophen-gluc, respectively. The intra- and inter-run precision values were within 12.47% for NAPQI, 12.11% for acetaminophen-glut and 11.86% for acetaminophen-gluc at their lower limit of quantitation levels. The samples were stable under all tested conditions. This method was successfully applied to study the pharmacokinetics of NAPQI, acetaminophen-glut and acetaminophen-gluc in ICR mice following oral administration of 200 mg/kg of acetaminophen suspension.

123I-iomazenil whole-body imaging to detect hepatic carboxylesterase drug-metabolizing enzyme activity.

Drugs are mainly metabolized by hepatic enzymes, the activity of which can differ between individuals. Although it is ideal to measure the hepatic clearance of liver-targeted drugs in individualized medicine, blood enzyme tests typically measure metabolic drug clearance in the entire body, and not just in the liver. We investigated whether I-iomazenil imaging can directly assess and quantify the activity of hepatic drug-metabolizing enzymes. Hepatic enzymes that metabolize I-iomazenil were identified by thin-layer chromatography in mouse liver homogenates with bis(4-nitrophenyl) phosphate (BNPP) inhibitor for carboxylesterase enzymes and nicotinamide adenine dinucleotide phosphate (NADPH) generator for cytochrome P450 enzymes. Whole-body images of mice were acquired using I-iomazenil with and without BNPP, and the distribution was also obtained. The metabolism of I-iomazenil in the blood, liver, gall bladder, and bladder was investigated by thin-layer chromatography. From the in-vitro metabolism of I-iomazenil using BNPP, the enzyme converting I-iomazenil to I-R-COOH was identified as carboxylesterase, and that converting I-iomazenil to M2 was identified as cytochrome P450 in experiments with and without an NADPH generator. The biological distribution and whole-body imaging showed increased accumulation in the liver of mice administered BNPP compared with normal mice, but decreased levels in the gall bladder and small intestine. The main fraction in bile and urine was I-R-COOH, with two unknown metabolites (M1 and M2), I, and I-iomazenil also being present. I-iomazenil whole-body imaging has good possibility of direct measurement of hepatic carboxylesterase activity as accumulation of I-R-COOH in the gall bladder through bile and in the bladder through urine.

Activated charcoal significantly improved the reliability of methods for quantitative analysis of endogenous substances in biological specimens: Glutathione and cysteine as cases

When developing a quantitative assay for exogenous or endogenous compounds, guidelines for method validation normally recommend that the biological specimens should be prepared in corresponding authentic matrices, yet “analyte-free authentic matrices” is in general not available. It is generally known that GSH and CYS are endogenous compounds and present in both prokaryotes and eukaryotes. Herein, an efficient approach for the quantitative analysis of endogenous substances in biological specimens was developed, and glutathione (GSH) & cysteine (CYS) were chosen as model endogenous substances. Activated carbon (AC), a common adsorbent for the adsorption of environmental pollutants, was used to remove the endogenous GSH and CYS and prepare “GSH&CYS-free biological matrix”. The endogenous GSH and CYS in mouse plasma, blood and liver homogenate were found can be almost removed via incubating with 100 mg of AC for 2 h. After optimizing the derivatization reagents, internal standard and analytical parameters, a reliable quantitative assay of GSH and CYS in mouse plasma, blood and liver homogenate was developed and validated on LC-ESI-MS/MS using corresponding AC-adsorbed mouse biological matrices. The validation results indicated that the developed method provided suitable accuracy, sensitivity, specificity and high throughput for the analysis of GSH and CYS. Finally, the developed LC-ESI-MS/MS assay was successfully applied to measure the concentrations of GSH and CYS in liver injury mice. The presently developed methodology could be widely applied in the quantitative analysis of endogenous compounds in various complex mixtures such as biological, herbal and environmental samples.