Journal of Biochemical and Molecular Toxicology

Short‐, medium‐, and long‐chain fatty acid:CoA ligases from human liver were tested for their sensitivity to inhibition by triacsin C. The short‐chain fatty acid:CoA ligase was inhibited less than 10% by concentrations of triacsin C as high as 80 μM. The two mitochondrial xenobiotic/medium‐chain fatty acid:CoA ligases (XM‐ligases), HXM‐A and HXM‐B, were partially inhibited by triacsin C, and the inhibitions were characterized by low affinity for triacsin C (K_I values > 100 μM). These inhibitions were found to be the result of triacsin C competing with medium‐chain fatty acid for binding at the active site. The microsomal and mitochondrial forms of long‐chain fatty acid:CoA ligase (also termed long‐chain fatty acyl‐CoA synthetase, or long‐chain acyl‐CoA synthetase LACS) were potently inhibited by triacsin C, and the inhibition had identical characteristics for both LACS forms. Dixon plots of this inhibition were biphasic. There is a high‐affinity site with a K_I of 0.1 μM that accounts for a maximum of 70% of the inhibition. There is also a low affinity site with a K_I of 6 μM that accounts for a maximum of 30% inhibition. Kinetic analysis revealed that the high‐affinity inhibition of the mitochondrial and microsomal LACS forms is the result of triacsin C binding at the palmitate substrate site.

The high‐affinity triacsin C inhibition of both the mitochondrial and microsomal LACS forms was found to require a high concentration of free Mg²⁺, with the EC₅₀ for inhibition being 3 mM free Mg²⁺. The low affinity triacsin C inhibition was also enhanced by Mg²⁺. The data suggests that Mg²⁺ promotes triacsin C inhibition of LACS by enhancing binding at the palmitate binding site. In contrast, the partial inhibition of the XM‐ligases by triacsin C, which showed only a low‐affinity component, did not require Mg²⁺. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:100–106, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20009

This study evaluates the protective effects of 7‐hydroxycoumarin (7‐HC) on dyslipidemia and cardiac hypertrophy in isoproterenol (ISO) induced myocardial infarction (MI) in rats. Rats were pre‐ and co treated with 7‐HC (16 mg/kg) daily for 8 days. ISO (100 mg/kg) was subcutaneously injected into rats on seventh and eighth days to induce MI. Increased activity/levels of serum creatine kinase‐MB (CK‐MB), troponin‐T, plasma lipid peroxidation products, and altered levels of lipids in the serum and heart and serum lipoproteins were noted in ISO‐induced rats. ISO‐induced myocardial infarcted rats revealed increased hypertrophy (cardiac and left ventricular) and hepatic 3‐hydroxyl 3‐methylglutaryl‐coenzyme‐A reductase (HMG‐CoA reductase) activity. Pre and cotreatment with 7‐HC revealed significant protective effects on all the biochemical parameters evaluated. The in vitro study demonstrated its free radical scavenging property. Thus, 7‐HC protects ISO‐induced MI in rats by its free radical scavenging and antihyperlipidaemic and antihypertrophic properties.

The purpose of this study was to investigate the antioxidant role of coumarin on streptozotocin–nicotinamide‐induced Type 2 diabetic rats. In experimental rats, the levels of plasma glucose, insulin, and the levels of thiobarbituric acid reactive substances, lipid hydroperoxides, conjugated dienes, and the activities of superoxide dismutase, catalase, glutathione‐S‐transferase, and glutathione peroxidase were assayed in liver and kidney. Diabetic rats showed elevated levels of plasma glucose and lipid peroxidation markers and reduced plasma insulin and antioxidant enzymes. Oral administration of coumarin resulted in a significant reduction in the plasma glucose and lipid peroxides and a significant increase in the plasma insulin and antioxidant enzymes. Chronic treatment of coumarin remarkably restored the normal status of the histopathological changes observed in the selected tissues. It can be concluded that coumarin has antioxidant effect in Type 2 diabetic rats. © 2011 Wiley Periodicals, Inc. J Biochem Mol Toxicol 25:355–361 2011; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.20395

Acrylamide (ACR) is a known industrial neurotoxic chemical. Evidence suggests that ACR neurotoxic effect is related to brain neurotransmission disturbances. Since nitric oxide (NO) acts as a neurotransmission modulator and is produced by nitric oxide synthase (NOS), the neuronal NOS (nNOS) and inducible NOS (iNOS) expression pattern were determined in rat cerebral cortex and striatum after subchronic exposure to ACR. Using immunocytochemistry, the neuronal count of nNOS or optical density of iNOS from sections at three coronal levels, bregma 1.0, −0.4, and −2.3 mm, were compared between ACR‐treated and control rats. At all three levels, nNOS expressions were uniformly decreased in most of the neocortical subregions following the treatment of ACR. At bregma level 1.0 mm, total numbers of nNOS expressing neurons were significantly decreased to 58.7% and 64.7% of the control in the cortex and striatum of ACR‐treated rats, respectively. However, at the bregma level −2.3 mm, ACR treatment did not produce a significant difference in the numbers of nNOS expressing neurons both in the cortex and striatum. Contrary to nNOS, iNOS expressions were consistently increased to approximately 32% in the neocortex and 25% in the striatum, following the subchronic ACR treatment. These data suggest that subchronic ACR exposure involves compensatory mechanism on nNOS and iNOS expression to maintain the homeostasis of NO at the rostral part of the neocortex and the striatum. However, in the caudal brain, increased iNOS expression did not suppress nNOS expression. Therefore, the present study is consistent with the hypothesis that ACR toxicity is mediated through the disturbance to the NO signaling pathway and exhibits a rostrocaudal difference through the differential expressions of nNOS and iNOS in the neocortex and the striatum. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:162–168, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20069

The effect of dysfunctional mitochondria in several cell pathologies has been reported in renal diseases, including diabetic nephropathy and acute kidney injury. Previous studies have reported that mitochondrial transplantation provided surprising results in myocardial and liver ischemia, as well as in Parkinson's disease. We aimed to investigate the beneficial effects of isolated mitochondria transplantation from mesenchymal stem cells (MSCs) in vivo, to mitigate renal damage that arises from doxorubicin‐mediated nephrotoxicity and its action mechanism. In this study, a kidney model of doxorubicin‐mediated nephrotoxicity was used and isolated mitochondria from MSCs were transferred to the renal cortex of rats. The findings showed that the rate of isolated mitochondria from MSCs maintains sufficient membrane integrity, and was associated with a beneficial renal therapeutic effect. Following doxorubicin‐mediated renal injury, isolated mitochondria or vehicle infused into the renal cortex and rats were monitored for five days. This study found that mitochondrial transplantation decreased cellular oxidative stress and promoted regeneration of tubular cells after renal injury (P < .001, P = .009). Moreover, mitochondrial transplantation reduced protein accumulation of tubular cells and reversed renal deficits (P = .01, P < .001). Mitochondrial transplantation increased Bcl‐2 levels, and caspase‐3 levels decreased in injured renal cells (P < .015, P < .001). Our results provide a direct link between mitochondria dysfunction and doxorubicin‐mediated nephrotoxicity and suggest a therapeutic effect of transferring isolated mitochondria obtained from MSCs against renal injury. To our knowledge, this study is the first study in the literature that showed good therapeutic effects of mitochondrial transplantation in a nephrotoxicity model, which is under‐researched.

Despite the extensive use of cisplatin (CP) as a chemotherapeutic agent, its clinical use is often restricted by undesirable side effects, such as toxicity to normal tissues. The aim of this study was to probe the effect of a combinatorial treatment of low multiple doses of antioxidants on CP‐induced toxicity and the mitochondrial apoptotic pathway in hepatocytes. Animals received a single toxic dose of CP (7.5 mg/kg body weight) with or without combined multiple doses of epigallocatechin gallate (EGCG) and coenzyme Q10 (CoQ10) (15 and 5 mg/kg body weight, respectively). CP‐treated animals showed altered biochemical parameters, denoting hepatotoxicity, which was markedly improved by the multidose treatment with EGCG + CoQ10. The increased levels of oxidants found in the cytosolic and mitochondrial fractions isolated from the liver of CP‐administered rats were significantly attenuated by the combinatorial doses of antioxidants. EGCG + CoQ10 ameliorated the CP‐induced compromised antioxidant defenses, oxidative modification of macromolecules, decreased activities of respiratory chain enzymes, altered membrane depolarization, and swelling of liver mitochondria. Furthermore, EGCG + CoQ10 treatment inhibited CP‐induced apoptosis by suppressing the activation and mitochondrial accumulation of proapoptotic proteins and preventing the inhibition of antiapoptotic protein expression, cytochrome c efflux, caspase‐3 activation, and DNA fragmentation. Histological findings further confirmed the protective effects of EGCG + CoQ10 against CP‐induced cellular injury. Our findings revealed that the combination of EGCG and CoQ10, owing to their individual antioxidant properties, can be an effective remedy, which by maintaining redox hemostasis attenuate the mitochondrial stress‐mediated molecular and cellular processes involved in CP‐induced liver toxicity and cell death.

Aminoglycoside antibiotics are widely used against Gram‐negative infections. On the other hand, nephrotoxicity is a deleterious side effect associated with aminoglycoside therapy. Gentamicin is the most nephrotoxic aminoglycoside. Because of serious health complications ensue the nephrotoxicity induced by aminoglycosides, finding new therapeutic strategies against this problem has a great clinical value. This study has attempted to compare the nephrotoxic properties of gentamicin and a new nanosized formulation of this drug in a mice model. Animals were treated with gentamicin (100 mg/kg, i.p. for eight consecutive days) and nanogentamicin (100 mg/kg, i.p. for eight consecutive days). Blood urea nitrogen (BUN), plasma creatinine levels, and histopathological changes of kidney proximal tubule were monitored. It was found that gentamicin caused severe degeneration of kidney proximal tubule cells and an increase in serum creatinine and BUN. No severe injury was observed after nanogentamicin administration. This study proved that nanosized gentamicin is less nephrotoxic.

The oxysterol 7beta‐hydroxycholesterol (7β‐OH) has been shown to induce apoptosis in a number of cell lines. Though not fully elucidated, the mechanism through which this oxysterol induces cell death is thought to involve the generation of an oxidative stress leading to perturbation of the mitochondrion and release of cytochrome c into the cytosol. Cytochrome c together with Apaf‐1 causes activation of the initiator caspase, caspase‐9, which in turn activates caspase‐3 ultimately leading to the degradation of poly(ADP‐ribose) polymerase (PARP). The objective of the present study was to investigate the signalling pathway in 7β‐OH‐induced apoptosis in U937 cells, a human monocytic blood cell line known to undergo apoptosis upon treatment with 7β‐OH, over a time course of 48 h. Apoptosis was evident after 24 h incubation. Glutathione levels were decreased after 6 h and this was coupled with an increase in SOD activity. Through western blot analysis we examined expression of caspase‐3, ‐8, and ‐9 and cleavage of the caspase‐3 substrate PARP. The sequence proceeded with activation of caspase‐9 after 9 h, caspase‐3 at the 12 h timepoint, and cleavage of PARP after 24 h treatment with 7β‐OH. Caspase‐8 did not appear to play a major role in this particular apoptotic pathway. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:50–59, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20007

Oxysterols, such as 7β‐hydroxy‐cholesterol (7β‐OH) and cholesterol‐5β,6β‐epoxide (β‐epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca²⁺) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca²⁺ changes on apoptosis induced by 7β‐OH and β‐epoxide. Ca²⁺ responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura‐2. Over 15‐min exposure of differentiated U937 cells to 30 μM of 7β‐OH induced a slow but significant rise in fura‐2 ratio. The Ca²⁺ channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca²⁺. Moreover, dihydropyridine (DHP) binding sites identified with BODIPY‐FLX‐DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca²⁺ occurred through L‐type channels. However, following long‐term incubation with 7β‐OH, elevated levels of cytoplasmic Ca²⁺ were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca²⁺ may be an initial trigger of 7β‐OH–induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca²⁺ on apoptotic cell death appears to be less significant. In contrast, Ca²⁺ did not appear to be involved in β‐epoxide–induced apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:324–332, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20295

Eupatorium adenophorum leaves cause hepatotoxicity and cholestasis in rats. The hepatotoxicant has been characterized as 9‐oxo‐10,11‐dehydroageraphorone (ODA), a cadinene sesquiterpene. Oral administration of ODA, mixed in feed to rats, caused jaundice in 24 h. The liver of the intoxicated animals had focal areas of hepatocellular necrosis, proliferation, and dilation of bile ducts with degenerative changes in the lining epithelium. There was marked increase in the conjugated form of plasma bilirubin and in the activities of the enzymes glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, alkaline phosphatase, lactate dehydrogenase, γ‐glutamyltranspeptidase, glutamate dehydrogenase, and 5′‐nucleotidase. The histopathological lesions in liver and biochemical profile of marker enzymes show that ODA induced hepatotoxicity and cholestasis in rats. This is the first report on the toxicity of a cadinene sesquiterpene in rats. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:279–286, 2001