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Han S, Zeng Y, Liu M, Yang L, Wang J, Song X. Regulating Sensing Patterns in Fluorescent Probes for Discriminative Detection of Biothiols. Anal Chem 2025; 97:419-426. [PMID: 39741462 DOI: 10.1021/acs.analchem.4c04523] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
Abstract
Discerning and quantifying the critical biothiols cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are vital for understanding their synergistic roles in biological systems. In this study, we synthesized a series of phenylethynylcoumarin fluorescent probes with varied structures to investigate the mechanisms underlying biothiol detection. We found that different substituents (-OCH3, -H, -CN) at the para-position of the phenylacetylene, combined with an aldehyde group at the 3-position of the coumarin, significantly affected the probes' reactivity and produced distinct response patterns toward biothiols. These insights enable the strategic development of fluorescent probes tailored to provide the personalized and discriminative detection of these biothiols. Additionally, probes CPOMe and CPCN were specifically evaluated for their efficacy in physiological environments, demonstrating their ability to accurately distinguish between Cys, Hcy, and GSH in living cells through unique fluorescent signals.
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Affiliation(s)
- Shaohui Han
- College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, China
| | - Yuyang Zeng
- College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, China
| | - Miaomiao Liu
- Shandong Provincial Key Laboratory of Detection Technology for Tumor Markers, College of Chemistry and Chemical Engineering, Linyi University, Linyi 276000, China
| | - Lei Yang
- Shandong Provincial Key Laboratory of Detection Technology for Tumor Markers, College of Chemistry and Chemical Engineering, Linyi University, Linyi 276000, China
| | - Jianxiu Wang
- College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, China
| | - Xiangzhi Song
- College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, China
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Tamargo JA, Sherman KE, Sékaly RP, Bordi R, Schlatzer D, Lai S, Khalsa JH, Mandler RN, Ehman RL, Baum MK. Cocaethylene, simultaneous alcohol and cocaine use, and liver fibrosis in people living with and without HIV. Drug Alcohol Depend 2022; 232:109273. [PMID: 35033954 PMCID: PMC8885871 DOI: 10.1016/j.drugalcdep.2022.109273] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/21/2021] [Revised: 11/29/2021] [Accepted: 12/18/2021] [Indexed: 11/03/2022]
Abstract
BACKGROUND The simultaneous consumption of cocaine and alcohol results in the production of cocaethylene (CE) in the liver, a highly toxic metabolite. Prior research suggests that cocaine use contributes to liver disease and its concomitant use with alcohol may increase its hepatotoxicity, but studies in humans are lacking. We evaluated the role of cocaine, its simultaneous use with alcohol, and CE on liver fibrosis. METHODS We performed a cross-sectional analysis of the Miami Adult Studies on HIV (MASH) cohort. Cocaine use was determined via self-report, urine screen, and blood metabolites, using liquid chromatography with tandem mass spectrometry. Hazardous drinking was determined with the AUDIT-C and liver fibrosis with the Fibrosis-4 Index (FIB-4). RESULTS Out of 649 participants included in this analysis, 281 (43.3%) used cocaine; of those, 78 (27.8%) had CE in blood. Cocaine users with CE had higher concentrations of cocaine metabolites in blood and were more likely to drink hazardously than cocaine users without CE and cocaine non-users. Overall, cocaine use was associated with liver fibrosis. CE in blood was associated with 3.17 (95% CI: 1.61, 6.23; p = 0.0008) times the odds of liver fibrosis compared to cocaine non-users, adjusting for covariates including HIV and HCV infection. The effect of CE on liver fibrosis was significantly greater than that of cocaine or alcohol alone. CONCLUSIONS CE is a reliable marker of simultaneous use of cocaine and alcohol that may help identify individuals at risk of liver disease and aid in the prevention of its development or progression.
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Affiliation(s)
| | | | - Rafick-Pierre Sékaly
- Emory University, Atlanta, GA, USA; Case Western Reserve University, Cleveland, OH, USA.
| | - Rebeka Bordi
- Emory University, Atlanta, GA, USA; Case Western Reserve University, Cleveland, OH, USA.
| | | | | | - Jag H Khalsa
- George Washington University, Washington, DC, USA.
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Wang C, Su B, Lu S, Han S, Jiang H, Li Z, Liu Y, Liu H, Yang Y. Effects of Glutathione on Growth, Intestinal Antioxidant Capacity, Histology, Gene Expression, and Microbiota of Juvenile Triploid Oncorhynchus mykiss. Front Physiol 2021; 12:784852. [PMID: 34925074 PMCID: PMC8680104 DOI: 10.3389/fphys.2021.784852] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2021] [Accepted: 10/25/2021] [Indexed: 01/27/2023] Open
Abstract
This study aimed to demonstrate the effects of dietary glutathione (GSH) on growth, intestinal antioxidant capacity, histology, gene expression, and microbiota in juvenile triploid rainbow trout (Oncorhynchus mykiss). Different diets (G0-control, G100, G200, G400, and G800) containing graded levels of GSH (0, 100, 200, 400, and 800mgkg-1) were fed to triplicate groups of 30 fish (initial mean weight 4.12±0.04g) for 56days. G400 had significantly improved weight gain and feed conversion rate. Based on the broken-line regression analysis, the optimum dietary GSH level was 447.06mgkg-1. Catalase and superoxide dismutase activities were significantly higher in G200-G800. G200 had significantly lower malondialdehyde content. The height of the intestinal muscular layer in G400 was significantly higher than that of the control group. Intestinal PepT1 and SLC1A5 gene expression was significantly increased, and the highest was observed in G400. TNF-α, IL-1β, IL-2, and IL-8 expression were significantly decreased than that of G0. Next-generation sequencing of the 16S rDNA showed a significant difference in alpha diversity whereas no differences in beta diversity. On the genus level, LefSe analysis of indicator OTUs showed Ilumatobacter, Peptoniphilus, Limnobacter, Mizugakiibacter, Chelatococcus, Stella, Filimonas, and Streptosporangium were associated with the treatment diet, whereas Arcobacter, Ferrovibrio, Buchnera, Chitinophaga, Stenotrophobacter, Solimonadaceae, Polycyclovorans, Rhodococcus, Ramlibacter, and Azohydromonas were associated with the control diet. In summary, feeding juvenile triploid O. mykiss 200-800mgkg-1 GSH improved growth and intestinal health.
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Affiliation(s)
- Chang’an Wang
- Key Open Laboratory of Cold Water Fish Germplasm Resources and Breeding of Heilongjiang Province, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin, China
- College of Animal Science, Northeast Agricultural University, Harbin, China
| | - Baohui Su
- College of Animal Science, Northeast Agricultural University, Harbin, China
| | - Shaoxia Lu
- Key Open Laboratory of Cold Water Fish Germplasm Resources and Breeding of Heilongjiang Province, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin, China
| | - Shicheng Han
- Key Open Laboratory of Cold Water Fish Germplasm Resources and Breeding of Heilongjiang Province, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin, China
| | - Haibo Jiang
- College of Animal Science, Guizhou University, Guiyang, China
| | - Zhuang Li
- Fishery Technical Extension Station of Jilin Province, Changchun, China
| | - Yang Liu
- Key Open Laboratory of Cold Water Fish Germplasm Resources and Breeding of Heilongjiang Province, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin, China
| | - Hongbai Liu
- Key Open Laboratory of Cold Water Fish Germplasm Resources and Breeding of Heilongjiang Province, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin, China
| | - Yuhong Yang
- College of Animal Science, Northeast Agricultural University, Harbin, China
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4
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Zhang Y, Wen L, Zhang W, Yue Y, Chao J, Huo F, Yin C. Sulphide activity-dependent multicolor emission dye and its applications in in vivo imaging. Analyst 2021; 146:5517-5527. [PMID: 34515714 DOI: 10.1039/d1an01345a] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Reactive sulfur species (RSS) play pivotal roles in various pathological and physiological processes. There exists an intricate relevance in generation and metabolism among these substances. Although they are nucleophilic, there are still some differences in their reactivity. There are many methods to detect them by using reactive fluorescent probes, but the systematic study of their reactivity is still lacking. In our study, we designed a multiple reaction site fluorescent probe based on benzene conjugated benzopyrylium and NBD. The study revealed that besides both biothiols and hydrogen sulfide, sulfur dioxide (SO2) can cleave the ether bond. There are two reaction forms for GSH with low reactivity: cutting the ether bond and adding the conjugated double bond of benzopyrylium. Nevertheless, Cys/Hcy with higher activity can further rearrange with NBD after cutting the ether bond. In addition, SO2 can not only cleave the ether bond, but also continue to add the conjugated double bond of benzopyrylium. The above processes lead to multicolor emission of the probe, thus realizing the characteristic analysis of different sulfides. Thus the probe can be used for the detection of sulfide in mitochondria, and further for the imaging of sulfide in cells and zebrafish. This effective analysis method will provide a broad application prospect for practical applications.
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Affiliation(s)
- Yongbin Zhang
- Shanxi Key Laboratory of Functional Molecules, Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China
| | - Le Wen
- Shanxi Key Laboratory of Functional Molecules, Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China.,School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China.
| | - Weijie Zhang
- Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China
| | - Yongkang Yue
- Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China
| | - Jianbin Chao
- Scientific Instrument Center, Shanxi University, Taiyuan 030006, China
| | - Fangjun Huo
- Shanxi Key Laboratory of Functional Molecules, Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China
| | - Caixia Yin
- School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China. .,Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China
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Abstract
Drug-induced liver injury (DILI) is a leading cause of attrition during the early and late stages of drug development and after a drug is marketed. DILI is generally classified as either intrinsic or idiosyncratic. Intrinsic DILI is dose dependent and predictable (e.g., acetaminophen toxicity). However, predicting the occurrence of idiosyncratic DILI, which has a very low incidence and is associated with severe liver damage, is difficult because of its complex nature and the poor understanding of its mechanism. Considering drug metabolism and pharmacokinetics, we established experimental animal models of DILI for 14 clinical drugs that cause idiosyncratic DILI in humans, which is characterized by the formation of reactive metabolites and the involvement of both innate and adaptive immunity. On the basis of the biomarker data obtained from the animal models, we developed a cell-based assay system that predicts the potential risks of drugs for inducing DILI. These findings increase our understanding of the mechanisms of DILI and may help predict and prevent idiosyncratic DILI due to certain drugs.
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Affiliation(s)
- Tsuyoshi Yokoi
- Department of Drug Safety Sciences, Division of Clinical Pharmacology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan;
| | - Shingo Oda
- Department of Drug Safety Sciences, Division of Clinical Pharmacology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan;
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Nemcova M, Pikula J, Zukal J, Seidlova V. Diclofenac-induced cytotoxicity in cultured carp leukocytes. Physiol Res 2020; 69:S607-S618. [PMID: 33646004 DOI: 10.33549/physiolres.934609] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
Diclofenac is a drug commonly used in human and veterinary medicine for the treatment of diseases associated with inflammation and pain. Medicinal products enter waste and surface waters on an everyday basis and contaminate the aquatic environment. Fish are therefore permanently exposed to these chemicals dissolved in their aquatic environment. To simulate variable environmental conditions, the aim of our study was to examine adverse effects of diclofenac under different temperatures of cell incubation (18, 21, 24, 27 and 30 °C). Cyto-toxic and -static effects of diclofenac in concentrations of 0.001 mcg/ml, 0.01 microg/ml, 0.1 mcg/ml, 1 mcg/ml, 10 mcg/ml and 100 mcg/ml for the carp (Cyprinuscarpio) cultured leukocytes were quantified using detection of lactate dehydrogenase released from damaged cells. Overall DCF cytotoxicity was relatively low and its impact was pronounced at higher temperature and DCF concentration. Cells growth inhibition is changing more rapidly but it is high mainly at the highest concentration from low temperature. DNA fragmentation was not detected in tested leukocyte cell line. CYP450 increased diclofenac cytotoxicity only at the highest concentration but at incubation temperatures 18 and 27 °C. Leukocyte viability is essential for immune functions and any change can lead to reduction of resistance against pathogens, mainly in cold year seasons, when the immune system is naturally suppressed.
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Affiliation(s)
- M Nemcova
- Department of Ecology and Diseases of Zoo Animals, Game, Fish and Bees, University of Veterinary and Pharmaceutical Sciences Brno,Czech Republic.
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Liu D, Lin L, Lin Y, Zhong Y, Zhang S, Liu W, Zou B, Liao Q, Xie Z. Zengye decoction induces alterations to metabolically active gut microbiota in aged constipated rats. Biomed Pharmacother 2019; 109:1361-1371. [PMID: 30551387 DOI: 10.1016/j.biopha.2018.11.013] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2018] [Revised: 11/02/2018] [Accepted: 11/02/2018] [Indexed: 12/19/2022] Open
Abstract
Zengye decoction (ZYD), a traditional Chinese medicinal formula, has been used in the treatment of various chronic diseases, such as constipation and skin dryness syndrome. Clinically, the specific mechanisms and targets of ZYD for treating disease remain unclear. The present study was undertaken to investigate the effects of ZYD on the gut microbiota and host metabolites in aged constipated rats and the relationship between the intestinal microbiota and the host. Rats were divided randomly into three groups, the control group (n = 10), recovery group (n = 10) and ZYD group (n = 10). First, the aged constipation model was established for the ZYD group and recovery group. Then, rats in the ZYD group were treated with ZYD. Urinary and faecal samples of each animal were collected in microcentrifuge tubes. Next, 16s rRNA gene sequencing was employed to analyse the composition of the gut microbiome in faecal samples and afterwards the metabolic function of the altered gut microbiota was predicted. Additionally, 1H NMR profiling was used to detect the alterations of host metabolites in urine and faecal samples to verify the metabolic function results obtained from sequencing. As a result, ZYD reduced the level of harmful bacteria, such as Desulfovibrio, Ruminococcus, Prevotella and Dorea, and increased the abundance of Oxalobacter, Clostridium and Roseburia. The functional prediction of changes in the gut microbiota induced by ZYD revealed that ZYD promoted energy storage, regulated amino acid metabolism, inhibited methane metabolism, strengthened the physiological function of glutathione and reduced bacterial toxin. The 1H NMR profiles revealed that ZYD regulated the carbohydrates, short chain fatty acids, amino acids and amines in the aged constipated rats. In addition, most metabolic changes observed were related to the function of intestinal microbiota. These results suggest that ZYD can regulate the intestinal microbiota of constipated rats to normal levels and change the endogenous metabolites of the host through the intestinal microbiota to achieve therapeutic effects.
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Affiliation(s)
- Deliang Liu
- School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou, 510006, PR China
| | - Lei Lin
- School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, PR China
| | - Yixuan Lin
- School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou, 510006, PR China
| | - Yuping Zhong
- School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou, 510006, PR China
| | - Shaobao Zhang
- School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, PR China
| | - Wen Liu
- School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou, 510006, PR China
| | - Baorong Zou
- School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou, 510006, PR China
| | - Qiongfeng Liao
- School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou, 510006, PR China.
| | - Zhiyong Xie
- School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou, 510006, PR China; School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, PR China.
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The Role of Acetyl Cysteine in Cocaethylene (Non-Acetaminophen) Acute Liver Failure. Case Rep Emerg Med 2018; 2018:4393064. [PMID: 30356434 PMCID: PMC6178182 DOI: 10.1155/2018/4393064] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2018] [Accepted: 06/19/2018] [Indexed: 11/17/2022] Open
Abstract
Background Acute liver failure can result from acetaminophen overdose, viral infection, toxins, and other disease conditions. Liver transplant is available in limited fashion and the criteria are strict as to who should get an available liver. N- Acetyl Cysteine (NAC) has been used in non-acetaminophen induced liver failure with success. Here we report a case of acute liver failure from cocaethylene that was reversed with NAC along with other medical therapy. Case Presentation A 50-year-old female patient presented to the Emergency Department (ED) with a two-day history of coffee ground vomiting and hematemesis. She reported occasional substance abuse and heavy alcoholism. She reported shortness of breath and chest pain from the recurrent forceful vomiting. The rest of the review of systems was unremarkable except a fall from intoxication. Physical examination revealed anicteric conjunctiva and nontender abdomen and her vital signs were within normal limits. Initial blood work revealed acute liver and renal failure. The patient was started with general medical management and liver transplant service rejected the case due to active substance abuse. She underwent brief hemodialysis and was started on NAC. Over the course of her hospital stay her liver function and kidney function improved significantly and patient was discharged to home. Conclusion In cases where liver transplant is not an option for various reasons including active substance abuse, a trial of N-Acetyl Cysteine may be beneficial and should be considered in the Emergency Department.
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Mai HN, Lee SH, Sharma G, Kim DJ, Sharma N, Shin EJ, Pham DT, Trinh QD, Jang CG, Nah SY, Jeong JH, Kim HC. Protein kinase Cδ knockout mice are protected from cocaine-induced hepatotoxicity. Chem Biol Interact 2018; 297:95-108. [PMID: 30393195 DOI: 10.1016/j.cbi.2018.10.017] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2018] [Revised: 09/09/2018] [Accepted: 10/22/2018] [Indexed: 12/25/2022]
Abstract
We investigated whether protein kinase Cδ (PKCδ) mediates cocaine-induced hepatotoxicity in mice. Cocaine treatment (60 mg/kg, i.p.) significantly increased cleaved PKCδ expression in the liver of wild-type (WT) mice, and led to significant increases in oxidative parameters (i.e., reactive oxygen species, 4-hydroxylnonenal and protein carbonyl). These cocaine-induced oxidative burdens were attenuated by pharmacological (i.e., rottlerin) or genetic depletion of PKCδ. We also demonstrated that treatment with cocaine resulted in significant increases in nuclear factor erythroid-2-related factor 2 (Nrf-2) nuclear translocation and increased Nrf-2 DNA-binding activity in wild-type (WT) mice. These increases were more pronounced in the rottlerin-treated WT or PKCδ knockout mice than in the saline-treated WT mice. Although cocaine treatment increased Nrf-2 nuclear translocation, DNA binding activity, and γ-glutamyl cysteine ligases (i.e., GCLc and GCLm) mRNA expressions, while it reduced the glutathione level and GSH/GSSG ratio. These decreases were attenuated by PKCδ depletion. Cocaine treatment significantly increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum of WT mice signifying the hepatic damage. These increases were also attenuated by PKCδ depletion. In addition, cocaine-induced hepatic degeneration in WT mice was evident 1 d post-cocaine. At that time, cocaine treatment decreased Bcl-2 and Bcl-xL levels, and increased Bax, cytosolic cytochrome c, and cleaved caspase-3 levels. Pharmacological or genetic depletion of PKCδ significantly ameliorated the pro-apoptotic properties and hepatic degeneration. Therefore, our results suggest that inhibition of PKCδ, as well as activation of Nrf-2, is important for protecting against hepatotoxicity induced by cocaine.
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Affiliation(s)
- Huynh Nhu Mai
- Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon, 24341, Republic of Korea
| | - Sung Hoon Lee
- Department of Pharmacology, College of Pharmacy, Chung-Ang University, Seoul, 06974, Republic of Korea
| | - Garima Sharma
- Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon, 24341, Republic of Korea
| | - Dae-Joong Kim
- Department of Anatomy and Cell Biology, Medical School, Kangwon National University, Chunchon, 24341, Republic of Korea.
| | - Naveen Sharma
- Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon, 24341, Republic of Korea
| | - Eun-Joo Shin
- Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon, 24341, Republic of Korea
| | - Duc Toan Pham
- Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon, 24341, Republic of Korea
| | - Quynh Dieu Trinh
- Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon, 24341, Republic of Korea
| | - Choon-Gon Jang
- Department of Pharmacology, School of Pharmacy, Sungkyunkwan University, Suwon, 16419, Republic of Korea
| | - Seung-Yeol Nah
- Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul, 05029, Republic of Korea
| | - Ji Hoon Jeong
- Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul, 06974, Republic of Korea
| | - Hyoung-Chun Kim
- Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon, 24341, Republic of Korea.
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Mai HN, Jung TW, Kim DJ, Sharma G, Sharma N, Shin EJ, Jang CG, Nah SY, Lee SH, Chung YH, Lei XG, Jeong JH, Kim HC. Protective potential of glutathione peroxidase-1 gene against cocaine-induced acute hepatotoxic consequences in mice. J Appl Toxicol 2018; 38:1502-1520. [DOI: 10.1002/jat.3666] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2018] [Revised: 05/29/2018] [Accepted: 06/10/2018] [Indexed: 01/22/2023]
Affiliation(s)
- Huynh Nhu Mai
- Neuropsychopharmacology and Toxicology Program, College of Pharmacy; Kangwon National University; Chunchon 24341 Republic of Korea
| | - Tae Woo Jung
- Research Administration Team; Seoul National University Bundang Hospital; Seongnam 13620 Republic of Korea
| | - Dae-Joong Kim
- Department of Anatomy and Cell Biology, Medical School; Kangwon National University; Chunchon 24341 Republic of Korea
| | - Garima Sharma
- Neuropsychopharmacology and Toxicology Program, College of Pharmacy; Kangwon National University; Chunchon 24341 Republic of Korea
| | - Naveen Sharma
- Neuropsychopharmacology and Toxicology Program, College of Pharmacy; Kangwon National University; Chunchon 24341 Republic of Korea
| | - Eun-Joo Shin
- Neuropsychopharmacology and Toxicology Program, College of Pharmacy; Kangwon National University; Chunchon 24341 Republic of Korea
| | - Choon-Gon Jang
- Department of Pharmacology, School of Pharmacy; Sungkyunkwan University; Suwon 16419 Republic of Korea
| | - Seung-Yeol Nah
- Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine; Konkuk University; Seoul 05029 Republic of Korea
| | - Sung Hoon Lee
- Department of Pharmacology, College of Pharmacy; Chung-Ang University; Seoul 06974 Republic of Korea
| | - Yoon Hee Chung
- Department of Anatomy, College of Medicine; Chung-Ang University; Seoul 06974 Republic of Korea
| | - Xin Gen Lei
- Department of Animal Science; Cornell University; Ithaca New York 14853 USA
| | - Ji Hoon Jeong
- Department of Pharmacology, College of Medicine; Chung-Ang University; Seoul 06974 Republic of Korea
| | - Hyoung-Chun Kim
- Neuropsychopharmacology and Toxicology Program, College of Pharmacy; Kangwon National University; Chunchon 24341 Republic of Korea
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Xu J, Oda S, Yokoi T. Cell-based assay using glutathione-depleted HepaRG and HepG2 human liver cells for predicting drug-induced liver injury. Toxicol In Vitro 2018; 48:286-301. [PMID: 29407385 DOI: 10.1016/j.tiv.2018.01.019] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2017] [Revised: 12/14/2017] [Accepted: 01/22/2018] [Indexed: 02/06/2023]
Abstract
Immortalized liver cells have been used for evaluating the toxicity of compounds; however, excessive glutathione is considered to lessen cytotoxicity. In this study, we compared the effects of glutathione depletion on cytotoxicities of drugs using HepaRG and HepG2 cells, which express and lack drug-metabolizing enzymes, respectively, for predicting drug-induced liver injury (DILI) risks. These cells were pre-incubated with L-buthionine-S,R-sulfoximine (BSO) and then exposed to 34 test compounds with various DILI risks for 24 h. ATP level exhibited the highest predictability of DILI among tested parameters. BSO treatment rendered cells susceptible to drug-induced cytotoxicity when evaluated by cell viability and caspase 3/7 activity with the sensitivity of cell viability from 50% in non-treated HepaRG cells to 71% in BSO-treated HepaRG cells. These results indicate that cytotoxicity assays using GSH-depleted HepaRG cells improve the predictability of DILI risks. However, HepaRG cells were not always superior to HepG2 cells when assessed by ATP level. The combination of HepG2 and HepaRG cells index produced the best prediction in the cases of caspase 3/7 acitivity and ATP level. In conclusions, the developed highly sensitive cell-based assay using GSH-reduced cells would be useful for predicting potential DILI risks at an early stage of drug development.
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Affiliation(s)
- Jieyu Xu
- Department of Drug Safety Sciences, Division of Clinical Pharmacology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
| | - Shingo Oda
- Department of Drug Safety Sciences, Division of Clinical Pharmacology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
| | - Tsuyoshi Yokoi
- Department of Drug Safety Sciences, Division of Clinical Pharmacology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
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Mai HN, Chung YH, Shin EJ, Kim DJ, Jeong JH, Nguyen TTL, Nam Y, Lee YJ, Nah SY, Yu DY, Jang CG, Ho YS, Lei XG, Kim HC. Genetic depletion of glutathione peroxidase-1 potentiates nephrotoxicity induced by multiple doses of cocaine via activation of angiotensin II AT1 receptor. Free Radic Res 2016; 50:467-83. [DOI: 10.3109/10715762.2016.1143097] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
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A novel cell-based assay for the evaluation of immune- and inflammatory-related gene expression as biomarkers for the risk assessment of drug-induced liver injury. Toxicol Lett 2015; 241:60-70. [PMID: 26546780 DOI: 10.1016/j.toxlet.2015.10.029] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2015] [Revised: 10/13/2015] [Accepted: 10/30/2015] [Indexed: 01/20/2023]
Abstract
Drug-induced liver injury (DILI) is a major problem in drug development. Although some in vitro methods assessing DILI risk that utilize hepatic cell death or cellular stress as markers have been developed, the predictive ability of these tests is low. In this study, we sought to develop a novel cell-based assay for the risk assessment of DILI that considers drug metabolism as well as immune- and inflammatory-related gene expression. To accomplish this goal, human hepatoma HepaRG or HepG2 cells were treated with 96 drugs with different clinical DILI risks. The conditioned media were subsequently used to treat human promyelocytic leukemia HL-60 cells, and the mRNA expression levels of immune- and inflammatory-related genes in the cells were measured. An area under the receiver operating characteristic curve (ROC-AUC) was calculated to evaluate the predictive performance of the mRNA levels as markers to discriminate DILI risk. The expression of interleukin-8 (IL-8) in HL-60 cells treated with conditioned media from HepaRG cells (HL-60/HepaRG) exhibited the highest ROC-AUC value of 0.758, followed by the expression of IL-1β in HL-60/HepaRG (ROC-AUC: 0.726). Notably, the ROC-AUC values of these genes were higher in HL-60/HepaRG than in HL-60/HepG2, which suggests that HL-60/HepaRG has a higher potential for detecting the metabolic activation of drugs. An integrated score calculated from the levels of S100 calcium-binding protein A9 (S100A9), IL-1β, and IL-8 more precisely determined the DILI risks than individual gene expression did. The developed cell-based assay that utilizes immune-related gene expression would aid in the assessment of potential DILI risks.
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Womersley JS, Uys JD. S-Glutathionylation and Redox Protein Signaling in Drug Addiction. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2015; 137:87-121. [PMID: 26809999 DOI: 10.1016/bs.pmbts.2015.10.001] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Drug addiction is a chronic relapsing disorder that comes at a high cost to individuals and society. Therefore understanding the mechanisms by which drugs exert their effects is of prime importance. Drugs of abuse increase the production of reactive oxygen and nitrogen species resulting in oxidative stress. This change in redox homeostasis increases the conjugation of glutathione to protein cysteine residues; a process called S-glutathionylation. Although traditionally regarded as a protective mechanism against irreversible protein oxidation, accumulated evidence suggests a more nuanced role for S-glutathionylation, namely as a mediator in redox-sensitive protein signaling. The reversible modification of protein thiols leading to alteration in function under different physiologic/pathologic conditions provides a mechanism whereby change in redox status can be translated into a functional response. As such, S-glutathionylation represents an understudied means of post-translational protein modification that may be important in the mechanisms underlying drug addiction. This review will discuss the evidence for S-glutathionylation as a redox-sensing mechanism and how this may be involved in the response to drug-induced oxidative stress. The function of S-glutathionylated proteins involved in neurotransmission, dendritic spine structure, and drug-induced behavioral outputs will be reviewed with specific reference to alcohol, cocaine, and heroin.
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Affiliation(s)
- Jacqueline S Womersley
- Department of Cellular and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Joachim D Uys
- Department of Cellular and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina, USA.
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Valente MJ, Henrique R, Vilas-Boas V, Silva R, Bastos MDL, Carvalho F, Guedes de Pinho P, Carvalho M. Cocaine-induced kidney toxicity: an in vitro study using primary cultured human proximal tubular epithelial cells. Arch Toxicol 2012; 86:249-261. [PMID: 21983858 DOI: 10.1007/s00204-011-0749-3] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2011] [Accepted: 09/14/2011] [Indexed: 12/13/2022]
Abstract
Renal failure resulting from cocaine abuse has been well documented, although the underlying mechanisms remain to be investigated. In the present study, primary cultured human proximal tubular epithelial cells (HPTECs) of the kidney were used to investigate its ability to metabolize cocaine, as well as the cytotoxicity induced by cocaine and its metabolites benzoylecgonine (BE), ecgonine methyl ester (EME) and norcocaine (NCOC). Gas chromatography/ion trap-mass spectrometry (GC/IT-MS) analysis of HPTECs exposed to cocaine (1 mM) for 72 h confirmed its metabolism into EME and NCOC, but not BE. EME levels increased along the exposure time to cocaine, while NCOC concentration diminished after reaching a maximum at 6 h, indicating a possible secondary metabolism for this metabolite. Cocaine promoted a concentration-dependent loss of cell viability, whereas BE and EME were found to be non-toxic to HPTECs at the tested conditions. In contrast, NCOC revealed to have higher intrinsic nephrotoxicity than the parent compound. Moreover, cocaine-induced cell death was partially reversed in the presence of ketoconazole (KTZ), a potent CYP3A inhibitor, supporting the hypothesis that NCOC may play a role in cocaine-induced nephrotoxicity. Cocaine-induced cytotoxicity was found to involve intracellular glutathione depletion at low concentrations and to induce mitochondrial damage at higher concentrations. Under the present experimental conditions, HPTECs death pathway followed an apoptotic pattern, which was evident for concentrations as low as 0.1 mM.
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Affiliation(s)
- Maria João Valente
- REQUIMTE-Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, rua Aníbal Cunha, 164, 4099-030, Porto, Portugal.
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16
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Gould NS, Min E, Gauthier S, Martin RJ, Day BJ. Lung glutathione adaptive responses to cigarette smoke exposure. Respir Res 2011; 12:133. [PMID: 21982222 PMCID: PMC3215650 DOI: 10.1186/1465-9921-12-133] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2011] [Accepted: 10/07/2011] [Indexed: 01/21/2023] Open
Abstract
Background Smoking tobacco is a leading cause of chronic obstructive pulmonary disease (COPD), but although the majority of COPD cases can be directly related to smoking, only a quarter of smokers actually develop the disease. A potential reason for the disparity between smoking and COPD may involve an individual's ability to mount a protective adaptive response to cigarette smoke (CS). Glutathione (GSH) is highly concentrated in the lung epithelial lining fluid (ELF) and protects against many inhaled oxidants. The changes in GSH that occur with CS are not well investigated; therefore the GSH adaptive response that occurs with a commonly utilized CS exposure was examined in mice. Methods Mice were exposed to CS for 5 h after which they were rested in filtered air for up to 16 h. GSH levels were measured in the ELF, bronchoalveolar lavage cells, plasma, and tissues. GSH synthesis was assessed by measuring γ-glutamylcysteine ligase (GCL) activity in lung and liver tissue. Results GSH levels in the ELF, plasma, and liver were decreased by as much as 50% during the 5 h CS exposure period whereas the lung GSH levels were unchanged. Next, the time course of rebound in GSH levels after the CS exposure was examined. CS exposure initially decreased ELF GSH levels by 50% but within 2 h GSH levels rebound to about 3 times basal levels and peaked at 16 h with a 6-fold increase and over repeat exposures were maintained at a 3-fold elevation for up to 2 months. Similar changes were observed in tissue GCL activity which is the rate limiting step in GSH synthesis. Furthermore, elevation in ELF GSH levels was not arbitrary since the CS induced GSH adaptive response after a 3d exposure period prevented GSH levels from dropping below basal levels. Conclusions CS exposures evoke a powerful GSH adaptive response in the lung and systemically. These data suggests there may be a sensor that sets the ELF GSH adaptive response to prevent GSH levels from dipping below basal levels. Factors that disrupt GSH adaptive responses may contribute to the pathophysiology of COPD.
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Affiliation(s)
- Neal S Gould
- Department of Pharmaceutical Sciences, University of Colorado, Denver, CO, USA
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Guindalini C, O'Gara C, Laranjeira R, Collier D, Castelo A, Vallada H, Breen G. A GSTP1 functional variant associated with cocaine dependence in a Brazilian population. Pharmacogenet Genomics 2006; 15:891-3. [PMID: 16272961 DOI: 10.1097/01213011-200512000-00007] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Cocaine dependence aetiology is complex and genetically influenced. We hypothesize that, for many users, efficient metabolism of cocaine and its toxic byproducts aids persistent cocaine use, such as that leading to dependence. The glutathione-S-transferases - in particular, GST-Pi - may be important in preventing cocaine and alcohol-induced oxidative damage. We genotyped a GST-Pi functional polymorphism (Ile105Val) in 654 male cocaine users and 572 controls from Brazil. Genotype and allele frequencies of Ile105Val differed significantly (chi = 6.74; P=0.03 and chi = 6.54; P = 0.01, respectively). Ile/Ile cocaine dependents had an OR = 1.31 (95%CI: 1.04-1.65), and Ile/Ile dependents consuming >50 units alcohol weekly an OR of 1.44 (95% CI:1.06-1.96). Population stratification was assessed and did not affect the results. These data require replication but do suggest that the high activity Ile105 GST-Pi allele may influence the aetiology and development of cocaine dependence.
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Affiliation(s)
- Camila Guindalini
- MRC Social Genetic and Developmental Psychiatry Research Centre, Institute of Psychiatry, King's College London, UK
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18
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Liu LG, Yan H, Yao P, Zhang W, Zou LJ, Song FF, Li K, Sun XF. CYP2E1-dependent hepatotoxicity and oxidative damage after ethanol administration in human primary hepatocytes. World J Gastroenterol 2005; 11:4530-5. [PMID: 16052683 PMCID: PMC4398703 DOI: 10.3748/wjg.v11.i29.4530] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To observe the relationship between ethanol-induced oxidative damage in human primary cultured hepatocytes and cytochrome P450 2E1 (CYP2E1) activity, in order to address if inhibition of CYP2E1 could attenuate ethanol-induced cellular damage.
METHODS: The dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) exposures of primary human cultured hepatocytes to ethanol were carried out. CYP2E1 activity and protein expression were detected by spectrophotometer and Western blot analysis respectively. Hepatotoxicity was investigated by determination of lactate dehydrogenase (LDH) and aspartate transaminase (AST) level in hepatocyte culture supernatants, as well as the intracellular formation of malondialdehyde (MDA).
RESULTS: A dose-and time-dependent response between ethanol exposure and CYP2E1 activity in human hepatocytes was demonstrated. Moreover, there was a time-dependent increase of CYP2E1 protein after 100 mmol/L ethanol exposure. Meanwhile, ethanol exposure of hepatocytes caused a time-dependent increase of cellular MDA level, LDH, and AST activities in supernatants. Furthermore, the inhibitor of CYP2E1, diallyl sulfide (DAS) could partly attenuate the increases of MDA, LDH, and AST in human hepatocytes.
CONCLUSION: A positive relationship between ethanol-induced oxidative damage in human primary cultured hepatocytes and CYP2E1 activity was exhibited, and the inhibition of CYP2E1 could partly attenuate ethanol-induced oxidative damage.
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Affiliation(s)
- Lie-Gang Liu
- Department of Nutrition and Food Hygiene, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
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Ceballos NA, Houston RJ, Smith ND, Bauer LO, Taylor RE. N400 as an index of semantic expectancies: differential effects of alcohol and cocaine dependence. Prog Neuropsychopharmacol Biol Psychiatry 2005; 29:936-43. [PMID: 15967560 DOI: 10.1016/j.pnpbp.2005.04.036] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 04/18/2005] [Indexed: 11/27/2022]
Abstract
BACKGROUND Chronic substance abuse has been associated with decrements in the processing and expression of language. The present study utilized the N400 event-related electroencephalographic potential to index semantic processing in 133 adults with (n=49) or without (n=84) a history of alcohol and/or cocaine dependence. The contributions of age, gender, and comorbid marijuana and nicotine dependence, and antisocial symptomology to N400 decrements were either covaried or controlled. METHODS A continuous series of 300 stimuli was presented for 150 ms each (interstimulus interval=1475 ms) on a computer screen. The series was arranged such that a word (approximately 17% of stimuli) immediately preceded presentations of its antonym (primed condition; approximately 17% of stimuli), or a semantically unrelated word (unprimed condition; approximately 17% of stimuli). The remaining 50% of stimuli consisted of unpronounceable letter combinations (non-word condition). EEG responses to the antonyms, unrelated words, and letter jumbles were retained for analysis. Throughout the task, the subject pressed response keys to discriminate words from non-words. RESULTS Analyses revealed a detrimental effect of alcohol dependence on N400 amplitude and no significant main or interactive effects of cocaine dependence. CONCLUSION The present findings suggest that alcohol-dependent individuals may exhibit verbal processing decrements. These findings also challenge hypotheses suggesting that the combined use of cocaine and alcohol is more deleterious to brain function than alcohol use alone.
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Affiliation(s)
- Natalie A Ceballos
- Department of Behavioral Sciences, 1035 University Drive, 236 Medical School Building, University of Minnesota School of Medicine, Duluth, MN 55812-3031, United States.
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20
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Liguori MJ, Anderson MG, Bukofzer S, McKim J, Pregenzer JF, Retief J, Spear BB, Waring JF. Microarray analysis in human hepatocytes suggests a mechanism for hepatotoxicity induced by trovafloxacin. Hepatology 2005; 41:177-86. [PMID: 15619227 DOI: 10.1002/hep.20514] [Citation(s) in RCA: 77] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/07/2022]
Abstract
Idiosyncratic drug toxicity, defined as toxicity that is dose independent, host dependent, and usually cannot be predicted during preclinical or early phases of clinical trials, is a particularly confounding complication of drug development. An understanding of the mechanisms that lead to idiosyncratic liver toxicity would be extremely beneficial for the development of new compounds. We used microarray analysis on isolated human hepatocytes to understand the mechanisms underlying the idiosyncratic toxicity induced by trovafloxacin. Our results clearly distinguish trovafloxacin from other marketed quinolone agents and identify unique gene changes induced by trovafloxacin that are involved in mitochondrial damage, RNA processing, transcription, and inflammation that may suggest a mechanism for the hepatotoxicity induced by this agent. In conclusion, this work establishes the basis for future microarray analysis of new compounds to determine the presence of these expression changes and their usefulness in predicting idiosyncratic hepatotoxicity. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience. Wiley.com/jpages/0270-9139/suppmat/index.htnd).
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Affiliation(s)
- Michael J Liguori
- Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064-6104, USA
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21
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Labib R, Turkall R, Abdel-Rahman MS. Endotoxin potentiates cocaine-mediated hepatotoxicity by nitric oxide and reactive oxygen species. Int J Toxicol 2003; 22:305-16. [PMID: 12933325 DOI: 10.1080/10915810305117] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Exposure to small, noninjurious doses of the inflammagen, bacterial endotoxin (lipopolysaccharide, LPS) augments the toxicity of certain hepatotoxicants, including cocaine. The mechanism of this interaction has not been clearly elucidated, but it seems that aspects of the inflammatory response initiated by exposure to LPS may be responsible. In particular, this study examined the role of Kupffer cells and the modulating effects of nitric oxide (NO) and reactive oxygen species (ROS) on the LPS potentiation of cocaine-mediated hepatotoxicity (CMH). Mice were administered oral cocaine hydrochloride for 5 consecutive days at a dose of 20 mg/kg with and without 12 x 10(6) EU LPS/kg given intraperitoneally (IP) 4 hours after the last cocaine injection. Pretreatment regimens consisted of administration of 300 mg/kg, IP, of aminoguanidine (AM) or 1,3-dimethylthiourea (DMU) at 1 hour or 15 minutes, respectively, before each cocaine administration. In another group, mice were pretreated with saline using the same cocaine and LPS treatment protocol, but received a single pretreatment of 7 mg gadolinium chloride (GdCl(3))/kg intravenously (IV), or sterile saline 24 hours prior to the LPS administration. The GdCl(3) (Kupffer cell inhibitor) pretreatment inhibited the LPS potentiation of CMH, but did not reverse the effects of cocaine alone. On the other hand, AM (NO synthase inhibitor), decreased the synthesis of NO as observed by the decrease in the plasma nitrate/nitrite level and completely reversed the hepatotoxic effects of cocaine and LPS alone and in combination. Moreover, DMU (hydroxyl free radical scavenger) ameliorated the effects of cocaine and significantly reduced the hepatotoxicity observed with the cocaine and LPS administration. These data suggest that cocaine sensitizes the liver and subsequent activation of Kupffer cells by LPS leads to the formation of increased levels of NO, which can promote oxidant stress and thus provide an environment favoring the generation of more reactive species such as the hydroxyl free radical.
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Affiliation(s)
- Ramez Labib
- Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA
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22
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Sinbandhit-Tricot S, Cillard J, Chevanne M, Morel I, Cillard P, Sergent O. Glutathione depletion increases nitric oxide-induced oxidative stress in primary rat hepatocyte cultures: involvement of low-molecular-weight iron. Free Radic Biol Med 2003; 34:1283-94. [PMID: 12726916 DOI: 10.1016/s0891-5849(03)00108-4] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Various drugs and chemicals can cause a glutathione (GSH) depletion in the liver. Moreover, nitric oxide (NO) can be generated in response to physiological and pathological situations such as inflammation. The aim of this study was to estimate oxidative stress when primary rat hepatocytes were exposed to GSH depletion after NO production. For this purpose, cells were preincubated with lipopolysaccharide (LPS) and gamma-interferon (IFN) for 18 h in order to induce NO production by NO synthase and then L-buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, was added for 5 h. In hepatocyte cultures preincubated with LPS and IFN before BSO addition, an increase in lipid peroxidation was noted. In those cells, an elevation of iron-bound NO and a decrease in free NO led us to suggest the involvement of low-molecular-weight iron (LMW iron) in the enhancement of oxidative stress. Indeed, addition of deferiprone, a chelator of LMW iron, reduced iron-bound NO levels and the extent of oxidative stress. Moreover, an important elevation of LMW iron levels was also observed. As both, N-acetylcysteine, a GSH precursor, and N(G)-monomethyl-L-arginine, a NO synthase inhibitor, totally inhibited the elevation of LMW iron and oxidative stress, a cooperative role could be attributed to NO production and GSH depletion.
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Labib R, Turkall R, Abdel-Rahman MS. Endotoxin potentiates the hepatotoxicity of cocaine in male mice. JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH. PART A 2002; 65:977-993. [PMID: 12133232 DOI: 10.1080/00984100290071252] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
Cocaine produces hepatotoxicity by a mechanism that remains undefined but that has been linked to its oxidative metabolism. Endotoxin (lipopolysaccharide, LPS) is also a well-known cause of hepatic damage, where exposure to non-injurious doses of LPS increases the toxicity of certain hepatotoxins. This study was conducted to investigate the possible potentiation of cocaine-mediated hepatotoxicity (CMH) by LPS. Male CF-1 mice were administered oral cocaine hydrochloride for 5 consecutive days at a dose of 20 mg/kg with and without 12 x 10(6) EU LPS/kg given intraperitoneally 4 h after the last cocaine injection. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured as markers of liver injury. Blood and liver glutathione (GSH) levels were determined, as well as the activities of glutathione peroxidase (GPx) and catalase (CAT). In addition, the activity of liver glutathione reductase (GRx) was measured. The results demonstrate that endotoxin potentiated the hepatotoxicity of cocaine. Serum ALT and AST were significantly elevated with the combined cocaine and LPS treatment versus all other treatments. While cocaine alone resulted in centrilobular necrosis, the cocaine and LPS combination produced submassive necrosis. The increased hepatic GSH content and GRx activity observed with cocaine alone were not observed with the combination treatment, rendering the liver more susceptible to oxidative stress. Moreover, there was a significant decrease in the activities of hepatic GPx and CAT, particularly with the combination treatment. In conclusion, this study demonstrates that LPS potentiates the hepatotoxicity of cocaine as revealed by an array of biochemical and morphological markers.
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Affiliation(s)
- Ramez Labib
- Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103-2714, USA
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Lee YW, Hennig B, Fiala M, Kim KS, Toborek M. Cocaine activates redox-regulated transcription factors and induces TNF-alpha expression in human brain endothelial cells. Brain Res 2001; 920:125-33. [PMID: 11716818 DOI: 10.1016/s0006-8993(01)03047-5] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Cocaine abuse is frequently associated with cerebrovascular pathology. Although the cellular and molecular mechanisms of these alterations are not fully understood, they may involve oxidative injury or dysfunction of brain microvascular endothelial cells. To test this hypothesis, total glutathione levels, activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), as well as induction of the TNF-alpha gene expression were determined in human brain microvascular endothelial cells (HBMEC) exposed to cocaine. Exposure of HBMEC to cocaine resulted in a dose-dependent depletion of total glutathione levels. In addition, cocaine markedly activated redox-regulated transcription factors, NF-kappaB and AP-1. Activation of these transcription factors was accompanied by induction of AP-1- or NF-kappaB-dependent transcription, as measured by dual luciferase assay in HBMEC transfected with the AP-1- or NF-kappaB-responsive reporter constructs. Furthermore, HBMEC treatment with cocaine induced a dose-dependent expression of the tumor necrosis factor-alpha (TNF-alpha) gene. These results indicate that exposure to cocaine can trigger inflammatory pathways via activation of redox-sensitive transcription factors and induction of expression of the inflammatory genes in HBMEC. These events may contribute to the cerebrovascular insults observed in cocaine-abused patients.
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Affiliation(s)
- Y W Lee
- Department of Surgery, Division of Neurosurgery, University of Kentucky Medical Center, 800 Rose Street, Lexington, KY 40536, USA
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Abstract
BACKGROUND About 10 percent of patients who undergo liver transplantation have cryptogenic liver disease. In animal models, the absence of heteropolymeric keratins 8 and 18 or the presence of mutant keratins in hepatocytes causes or promotes liver disease. We have previously described a mutation in the keratin 18 gene in a patient with cryptogenic cirrhosis, but the importance of mutations in the keratin 8 and keratin 18 genes in such patients is unclear. METHODS We tested for mutations in the keratin 8 and keratin 18 genes in purified genomic DNA isolated from 150 explanted livers and 89 peripheral-blood specimens from three groups of patients: 55 patients with cryptogenic liver disease; 98 patients with noncryptogenic liver disease, with causes that included alcohol use, autoimmunity, drug use, and viral infections; and 86 randomly selected inpatients and outpatients who provided blood to the hematology laboratory. RESULTS Of the 55 patients with cryptogenic liver disease, 3 had glycine-to-cysteine mutations at position 61 (a highly conserved glycine) of keratin 8, and 2 had tyrosine-to-histidine mutations at position 53 of keratin 8. These mutations were not detected in the patients with other liver diseases or in the randomly selected patients. We verified the presence of the mutations in specimens of explanted livers by protein analysis and by the detection of unique restriction-enzyme cleavage sites. In transfected cells, the glycine-to-cysteine mutation limited keratin-filament reorganization when the cells were exposed to oxidative stress. In contrast, the tyrosine-to-histidine mutation destabilized keratin filaments when transfected cells were exposed to heat or okadaic acid stress. CONCLUSIONS Mutations in the keratin 8 gene may predispose people to liver disease and may account for cryptogenic liver disease in some patients.
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Affiliation(s)
- N O Ku
- Gastroenterology Section, Palo Alto Veterans Affairs Medical Center and Stanford University School of Medicine, Calif 94304, USA
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26
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Reid MJ, Bornheim LM. The effects of phencyclidine pretreatment on cocaine-mediated hepatotoxicity in mice. Toxicol Appl Pharmacol 2001; 172:194-202. [PMID: 11312647 DOI: 10.1006/taap.2001.9146] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Cocaine-mediated hepatotoxicity (CMH) requires cocaine (CCN) bioactivation by microsomal monooxygenase enzymes that results in cell death. Proposed mechanisms of toxicity involve reactive metabolites that covalently bind to hepatocellular proteins, depletion of cellular reducing equivalents through redox cycling, and/or the generation of reactive oxygen and nitrogen species that alter lipids and proteins. We have previously shown that phencyclidine (PCP) pretreatment potentiated CMH in CF-1 mice without increasing in vitro N-demethylation or N-hydroxylation of CCN. We have now further characterized PCP-potentiated CMH and determined that it is a dose- and time-dependent process, with PCP doses as low as 2.5 mg/kg for 3 days significantly increasing CMH. Immunohistochemistry and histology of livers from mice pretreated with PCP before CCN administration revealed a marked correlation between the regions of CCN metabolite binding and that of necrosis, whereas there was little binding or necrosis in vehicle-pretreated mice. Although hepatic GSH levels were not altered after repetitive PCP treatment alone, a sustained decrease (at least 6 h) in these levels was observed following CCN administration. Inhibitors of inducible nitric oxide synthase (NOS) abrogated PCP-potentiated CMH, although repetitive PCP treatment alone did not increase nitric oxide synthesis systemically or locally in hepatic tissue nor did lipopolysaccharide induction of NOS (without PCP) directly potentiate CMH. The precise mechanisms of PCP potentiation of CMH and involvement of NOS in CMH remain unclear, however, sustained depletion of GSH levels and increased hepatocellular binding of reactive cocaine metabolites have been demonstrated.
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Affiliation(s)
- M J Reid
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, 94143, USA
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Zaragoza A, Díez-Fernández C, Alvarez AM, Andrés D, Cascales M. Mitochondrial involvement in cocaine-treated rat hepatocytes: effect of N-acetylcysteine and deferoxamine. Br J Pharmacol 2001; 132:1063-70. [PMID: 11226137 PMCID: PMC1572648 DOI: 10.1038/sj.bjp.0703909] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
The cytotoxicity of cocaine (0 - 1000 microM), was studied on parameters related to the mitochondrial role and the cascade of events that lead to apoptosis in hepatocyte cultures from phenobarbitone (PB) pretreated rats. Cytotoxicity was dose-dependent and LDH leakage was significantly enhanced above 100 microM cocaine. Apoptosis was visualized by DNA fragmentation on agarose gel, and appeared at 50 and 100 microM cocaine. Cocaine induced biphasic changes in mitochondrial transmembrane potential and significantly increased the mitochondrial release of cytochrome c, the caspase-3 like DEVDase activity and the level of 20 kDa subunit, a product of pro-caspase-3 cleavage. The protective effect of N-acetylcysteine (NAC) and deferoxamine (DFO) on all these parameters confirmed the involvement of oxygen radicals in cocaine-induced necrosis/apoptosis. We conclude: first, that the biphasic changes recorded in mitochondrial inner membrane potential by the effect of cocaine, were parallel to apoptosis; second, that caspase-3 activity and cleavage to it p20 subunit increased sharply in parallel to the translocation of cytochrome c from mitochondria to cytosol; and third, that the antioxidants, NAC or DFO exerted a noticeable protective role in counteracting the cytotoxicity of cocaine, these effects being more pronounced in the case of DFO than NAC. These findings demonstrate that cocaine cytotoxicity involves mitochondrial damage.
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Affiliation(s)
- Asunción Zaragoza
- Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
| | - Carmen Díez-Fernández
- Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
| | - Alberto M Alvarez
- Centro de Citometría de Flujo, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
| | - David Andrés
- Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
| | - María Cascales
- Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
- Author for correspondence:
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