Influence of probenecid and paracetamol (acetaminophen) on zidovudine glucuronidation in human liver in vitro.

The effects of probenecid and paracetamol on zidovudine glucuronidation were investigated, in vitro, using human liver microsomal preparations. The presence of probenecid in the incubation medium significantly reduced the maximum reaction velocity for zidovudine glucuronide formation by more than 60 per cent, and the Km was reduced by 47 per cent, suggesting an uncompetitive inhibition of zidovudine glucuronidation. In contrast, paracetamol had no significant effect on zidovudine glucuronidation. The maximum reaction velocity for zidovudine glucuronide formation and the Km were unchanged when paracetamol (5 mM) was present in the incubation medium. The effects of probenecid and paracetamol on zidovudine metabolism in vitro correlates closely with those observed in vivo. The in vitro system of human liver microsomes may have a useful role in predicting the possible interaction of other drugs with zidovudine metabolism.     

Induction of an ATPase inhibitor protein by propylthiouracil and protection against paracetamol (acetaminophen) hepatotoxicity in the rat

1. The purpose of the present study was to test the following hypothesis: propylthiouracil (PTU) treatments of rats induces an increase in the concentration and activity of the mitochondrial ATPase (m-ATPase) inhibitor protein (IF₁). The PTU-induced elevated baseline levels of this inhibitor protein inactivated m-ATPase, and prevented hepatotoxicity by a toxic dose of acetaminophen (AAP) (paracetamol), by maintaining hepatic adenosine 5′-triphosphate (ATP) levels.
2. Male Wistar rats were either gavaged with a toxic dose of AAP alone, or after pretreatment with PTU for periods of 3 and 12 days.
3. Twenty four hours after acetaminophen treatment alone, toxicity was manifested by: an approximately 10 fold increase in serum transaminase levels (serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase); depletion of hepatic reduced glutathione (GSH) and ATP levels; loss of inhibitor protein activity, and extensive pericentral necrosis of the hepatocytes. Propylthiouracil pretreatment for 12 days enhanced the concentration of the following metabolites in the liver: ATP (1.5 fold), ATPase inhibitor protein (IF₁) (4.5 fold), and reduced glutathione (1.3 fold), while the activity of the inhibitor protein increased 2 fold. When the PTU treated rats were challenged with AAP, transaminases were not elevated, and only sporadic areas of necrosis were detected by histological examination of the liver tissue. In contrast to the 12 day treatment with PTU the 3 day treatment had no protection against AAP. No histological evidence of protection was manifested and the transaminases were not different from AAP treated controls. Most of the protective metabolites were depleted.
4. Our findings suggest that PTU-induced increased concentration of inhibitor protein and GSH, are contributing factors in the prevention of hepatotoxicity by maintaining hepatic m-ATP levels and reducing the harmful effect of the toxic metabolite of AAP.

     

Hepatoprotective effect of Taraxacum officinale leaf extract on sodium dichromate‐induced liver injury in rats

Taraxacum officinale (L.) Weber, commonly known as Dandelion, has been widely used as a folkloric medicine for the treatment of liver and kidney disorders and some women diseases such as breast and uterus cancers. The main objective of the present study was to assess the efficiency of T. officinale leaf extract (TOE) in treating sodium dichromate hazards; it is a major environmental pollutant known for its wide toxic manifestations witch induced liver injury. TOE at a dose of 500 mg/kg b.w was orally administered once per day for 30 days consecutively, followed by 10 mg/kg b.w sodium dichromate was injected (intraperitoneal) for 10 days. Our results using Wistar rats showed that sodium dichromate significantly increased serum biochemical parameters. In the liver, it was found to induce an oxidative stress, evidenced from increase in lipid peroxidation and changes in antioxidative activities. In addition, histopathological observation revealed that sodium dichromate causes acute liver damage, necrosis of hepatocytes, as well as DNA fragmentation. Interestingly, animals that were pretreated with TOE, prior to sodium dichromate administration, showed a significant hepatoprotection, revealed by a significant reduction of sodium dichromate‐induced oxidative damage for all tested markers. These finding powerfully supports that TOE was effective in the protection against sodium dichromate‐induced hepatotoxicity and genotoxicity and, therefore, suggest a potential therapeutic use of this plant as an alternative medicine for patients with acute liver diseases. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 339–349, 2016.     

Hepatoprotective effect of ginsenoside Rg1 from Panax ginseng on carbon tetrachloride‐induced acute liver injury by activating Nrf2 signaling pathway in mice

Oxidative stress and inflammatory response are well known to be involved in the pathogenesis of acute liver injury. This study was performed to examine the hepatoprotective effect of ginsenoside Rg1 (Rg1) against CCl₄‐induced acute liver injury, and further to elucidate the involvement of Nrf2 signaling pathway in vivo and in vitro. Mice were orally administered Rg1 (15, 30, and 60 mg/kg) or sulforaphane (SFN) once daily for 1 week prior to 750 μL/kg CCl₄ injection. The results showed that Rg1 markedly altered relative liver weights, promoted liver repair, increased the serum level of TP and decreased the serum levels of ALT, AST and ALP. Hepatic oxidative stress was inhibited by Rg1, as evidenced by the decrease in MDA, and increases in GSH, SOD, and CAT in the liver. Further research demonstrated that Rg1 suppressed liver inflammation response through repressing the expression levels of inflammation‐related genes including TNF‐α, IL‐1β, IL‐6, COX‐2, and iNOS. In addition, Rg1 enhanced antioxidative stress and liver detoxification abilities by up‐regulating Nrf2 and its target‐genes such as GCLC, GCLM, HO‐1, NQO1, Besp, Mrp2, Mrp3, Mrp4, and down‐regulating Cyp2e1. However, the changes in Nrf2 target‐genes, as well as ameliorative liver histology induced by Rg1 were abrogated by Nrf2 antagonist all‐transretinoic acid in vivo and Nrf2 siRNA in vitro. Overall, the findings indicated that Rg1 might be an effective approach for the prevention against acute liver injury by activating Nrf2 signaling pathway.     

Proteomic profiling in incubation medium of mouse,rat and human precision‐cut liver slices for biomarker detection regarding acute drug‐induced liver injury

Drug‐induced liver injury is one of the leading causes of drug withdrawal from the market. In this study, we investigated the applicability of protein profiling of the incubation medium of human, mouse and rat precision‐cut liver slices (PCLS) exposed to liver injury‐inducing drugs for biomarker identification, using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. PCLS were incubated with acetaminophen (APAP), 3‐acetamidophenol, diclofenac and lipopolysaccharide for 24–48 h. PCLS medium from all species treated with APAP demonstrated similar changes in protein profiles, as previously found in mouse urine after APAP‐induced liver injury, including the same key proteins: superoxide dismutase 1, carbonic anhydrase 3 and calmodulin. Further analysis showed that the concentration of hepcidin, a hepatic iron‐regulating hormone peptide, was reduced in PCLS medium after APAP treatment, resembling the decreased mouse plasma concentrations of hepcidin observed after APAP treatment. Interestingly, comparable results were obtained after 3‐acetamidophenol incubation in rat and human, but not mouse PCLS. Incubation with diclofenac, but not with lipopolysaccharide, resulted in the same toxicity parameters as observed for APAP, albeit to a lesser extent. In conclusion, proteomics can be applied to identify potential translational biomarkers using the PCLS system. Copyright © 2013 John Wiley & Sons, Ltd.     

Mechanism of the vasorelaxant effect induced by trans‐4‐methyl‐β‐nitrostyrene,a synthetic nitroderivative,in rat thoracic aorta

Mechanisms underlying the vasorelaxant effects of trans‐4‐methyl‐β‐nitrostyrene (T4MeN) were studied in rat aortic rings. In endothelium‐intact preparations, T4MeN fully and similarly relaxed contractions induced by phenylephrine (PHE) (IC₅₀ = 61.41 [35.40‐87.42] μmol/L) and KCl (IC₅₀ = 83.50 [56.63‐110.50] μmol/L). The vasorelaxant effect of T4MeN was unchanged by endothelium removal, pretreatment with L‐NAME, indomethacin, tetraethylammonium, ODQ or MDL‐12,330A. Under Ca²⁺‐free conditions, T4MeN significantly reduced with a similar potency: (i) phasic contractions induced by PHE, but not by caffeine; (ii) contractions due to CaCl₂ in aortic preparations stimulated with PHE (in the presence of verapamil) or high KCl; (iii) contractions evoked by the restoration of external Ca²⁺ levels after depletion of intracellular Ca²⁺ stores in the presence of thapsigargin. In contrast, T4MeN was more potent at inhibiting contractions evoked by the tyrosine phosphatase inhibitor, sodium orthovanadate, than those induced by the activator of PKC, phorbol‐12,13‐dibutyrate. These results suggest that T4MeN induces an endothelium‐ independent vasorelaxation that appears to occur intracellularly through the inhibition of contractions that are independent of Ca²⁺ influx from the extracellular milieu but involve phosphorylation of tyrosine residues.     

Establishment of a mouse model for amiodarone‐induced liver injury and analyses of its hepatotoxic mechanism

Drug‐induced liver injury (DILI) is the most frequent cause of post‐marketing warnings and withdrawals. Amiodarone (AMD), an antiarrhythmic, presents a risk of liver injury in humans, and its metabolites, formed by cytochrome P450 3A4, are likely more toxic to hepatocytes than AMD is. However, it remains to be clarified whether the metabolic activation of AMD is involved in liver injury in vivo. In this study, to elucidate the underlying mechanisms of AMD‐induced liver injury, mice were administered AMD [1000 mg kg^–1, per os (p.o.)] after pretreatment with dexamethasone [DEX, 60 mg kg^–1, intraperitoneal (i.p.)], which induces P450 expression, once daily for 3 days. The plasma alanine aminotransferase (ALT) levels were significantly increased by AMD administration in the DEX‐pretreated mice, and the liver concentrations of desethylamiodarone (DEA), a major metabolite of AMD, were correlated with the changes in the plasma ALT levels. Cytochrome c release into the hepatic cytosol and triglyceride levels in the plasma were increased in DEX plus AMD‐administered mice. Furthermore, the ratio of reduced glutathione to oxidized glutathione disulfide in the liver significantly decreased in the DEX plus AMD‐administered mice. The increase of ALT levels was suppressed by treatment with gadolinium chloride (GdCl₃), which is an inhibitor of Kupffer cell function. From these results, it is suggested that AMD and/or DEA contribute to the pathogenesis of AMD‐induced liver injury by producing mitochondrial and oxidative stress and Kupffer cell activation. This study proposes the mechanisms of AMD‐induced liver injury using an in vivo mouse model. Copyright © 2015 John Wiley & Sons, Ltd.     

(E)‐2‐Benzylidene‐4‐phenyl‐1,3‐diselenole ameliorates signals of renal injury induced by cisplatin in rats

The present study investigated the protective role of antioxidant (E)‐2‐benzylidene‐4‐phenyl‐1,3‐diselenole (BPD), an organoselenium compound, against the renal injury induced by cisplatin in rats. Canola oil or BPD (50 mg kg^?1) was administered orally by gavage once a day for 6 days to rats. The first dose of BPD was given 24 h before a single intraperitoneal injection of saline or cisplatin (7 mg kg^?1). At day 7, animals were killed and parameters related to renal injury were determined. The histological analysis showed that cisplatin caused renal injury in rats, which was accompanied by an increase in urea and creatinine levels in plasma. The increase of plasma creatinine levels negatively correlated with renal antioxidant defenses including ascorbic acid (AA) and reduced glutathione (GSH) content as well as glutathione S‐transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) activities. As revealed by histological analysis, BPD ameliorated tubular injury in rat kidney and reduced plasma markers altered by cisplatin. The administration of BPD to rats attenuated the reduction of renal AA and GSH content in animals exposed to cisplatin. The decrease of GST activity, but not GPx and CAT activities, in rats exposed to cisplatin was totally reversed by BPD administration. BPD was also effective in attenuating the inhibition of a sulfhydryl enzyme sensitive to oxidative stress, δ‐aminolevulinic dehydratase, in kidneys of rats exposed to cisplatin. The present study demonstrated that BPD reduced renal injury induced by cisplatin in rats and this effect seems to be related to antioxidant mechanisms. Copyright © 2012 John Wiley & Sons, Ltd.     

一种中药提取物的制备及对东莨菪碱模型小鼠学习记忆能力的影响

目的 优选自制中药的最佳提取工艺,并考察采用最佳提取工艺得到的提取物对东莨菪碱致记忆障碍模型小鼠学习记忆的影响.方法 以出膏率为评价指标,采用L9(34) 正交试验,对提取溶剂、溶剂倍数、浸泡时间及提取时间4个因素进行考察;将昆明种小鼠随机分为正常对照组、东莨菪碱模型组、脑复康组(阳性对照药,400 mg·kg-1)、中药提取物高剂量组(2.0 g·kg-1)、中剂量组(1.0 g·kg-1)、低剂量组(0.5 g·kg-1)共6组,灌胃给药4周后,除正常对照组腹腔注射生理盐水外,其他各组腹腔注射东莨菪碱3 mg·kg-1,20 min后进行Morris水迷宫测试.结果 自制中药最佳提取工艺为加入12倍量的50% 乙醇溶液,浸泡90 min后,加热回流提取3次,每次提取2 h;最佳提取工艺得到的提取物灌胃给药能够提高东莨菪碱模型小鼠学习记忆能力,缩短Morris水迷宫游泳时间(P<0.05).结论 优选出的提取工艺设计合理、结果可靠,该工艺得到的自制中药提取物可以改善东莨菪碱模型小鼠的记忆障碍.     

Preparation of [11C]methyl nona‐fluorobutyl‐1‐sulfonate ([11C]MeONf) and its use in the synthesis of [11C]‐6‐OH‐BTA‐1

The rapid, simple and high‐yield synthesis of the extraordinarily reactive ¹¹C‐methylating agent, [¹¹C]methyl nona‐fluorobutyl‐1‐sulfonate ([¹¹C]MeONf), and its use in the synthesis of the promising β‐amyloid imaging agent, [¹¹C]‐6‐OH‐BTA‐1, is reported. In terms of radioactive methylation yields, [¹¹C]MeONf seems to surpass [¹¹C]methyl trifluoromethansulfonate ([¹¹C]MeOTf) as a methylating agent in this particular case giving the ¹¹C‐labelled compound in high‐preparative radiochemical yields between 27 and 29% EOS with a minimum formation of radioactive by‐products. Copyright © 2007 John Wiley & Sons, Ltd.     

Cr(VI)‐induced genotoxicity and cell cycle arrest in human osteoblast cell line MG‐63

Occupational environments are major exposure routes to Cr(VI). However, Cr(VI) may also establish in bone tissues by ingestion or through Cr containing orthopaedic prostheses that, due to wear and corrosion, may release metal particles and ions potentially affecting bone tissue. The aim of this work was to evaluate the effects of clinically relevant concentrations of Cr(VI) in human osteoblasts, by integrating genotoxic effects, evaluated by the comet assay and cytokinesis‐blocked micronucleus assay (scoring the presence of micronucleus, nucleoplasmic bridges and nuclear division index), with the effects on cell cycle and cell viability. Human osteoblasts MG‐63 were in vitro exposed to Cr(VI) at concentrations ranging from 0.1 to 5 μm , for 24 and 48 hours. Results pointed out to a decrease of cell viability for both time exposures in a time‐ and dose‐dependent manner, which was related to cell cycle arrest and DNA damage. Chromosome abnormalities were also observed. Hence, these data suggest that cells arrested in the cell division with DNA damage may have followed cell death pathways, while some surviving ones still revealed DNA damage at chromosome level indicating abnormal cell division progression. In conclusion, Cr(VI) induced cytotoxic and genotoxic effects in human bone cells at concentrations that could be found in patients with metal‐on‐metal prostheses. In addition, the early onset of genotoxic damage induced by Cr(VI) at low concentrations after 24 hours of cell exposure alert to the relevance of periodic monitoring of patients for genotoxicity diagnosis after implantation of prostheses before clinical symptoms appear.     

Involvement of c-jun N-terminal kinase in G2/M arrest and caspase-mediated apoptosis induced by cardiotoxin III (Naja naja atra) in K562 leukemia cells.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, may have a potentiality as a structural template for rational drug design in killing cancer cells. Treatment of K562 cells with 0.3 microM of CTX III resulted in G2/M phase cell cycle arrest that was associated with a marked decline in protein levels of G2/M regulatory proteins including cyclin A, cyclin B1, Cdk2 and Cdc25C. In contrast to no effect on the phosphorylation of ERK, p38 MAPK and Akt, an activation of JNK was noted when K562 cells were exposed to CTX III. CTX III-mediated G2/M phase arrest and apoptosis were reduced by treatment with the JNK-specific inhibitor SP600125, but not by ERK and p38MAPK inhibitors. Further investigation showed that the specific JNK inhibitor, SP600125, reduced the activation of caspase-3, caspase-9, and reversed the decline in the expression of cyclin B1. Taken together, our data show for the first time that JNK, but not ERK, p38MAPK or Akt signaling, plays an important role in CTX III-mediated G2/M arrest and apoptosis in K562 cancer cells.