Diclofenac induced in vivo nephrotoxicity may involve oxidative stress-mediated massive genomic DNA fragmentation and apoptotic cell death.

Diclofenac (DCLF) is a nonsteroidal anti-inflammatory drug that is widely used for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and acute muscle pain conditions. Toxic doses of DCLF can cause nephrotoxicity in humans and experimental animals. However, whether this DCLF-induced nephrotoxicity involves apoptotic cell death in addition to necrosis is unknown. The goals of this investigation were to determine whether DCLF-induced nephrotoxicity involves oxidative stress and apoptotic type genomic DNA fragmentation, and if so, whether DCLF-induced oxidative stress and DNA fragmentation cause apoptotic cell death in mouse kidneys. Male ICR mice (CD-1; 25-45 g), fed ad libitum, were administered nephrotoxic doses of DCLF (100, 200, 300 mg/Kg, po) and sacrificed 24 h later. Blood was collected to evaluate renal injury (BUN), lipid peroxidation (MDA: malondialdehyde levels), and superoxide dismutase (SOD) activity (a marker of oxidative stress). Kidney tissues were analyzed both quantitatively and qualitatively to determine the degree and type of DNA damage, and evaluated histopathologically for the presence of apoptotic characteristics in the nucleus of diverse types of kidney cells. Results show that diclofenac is a powerful nephrotoxicant (at 100, 200, and 300 mg/kg: 4.7-, 4.9-, and 5.0-fold increases in BUN compared to the control, respectively) and a strong inducer of oxidative stress (significant increase in MDA levels). Oxidative stress induced by DCLF was also coupled with massive kidney DNA fragmentation (100, 200, and 300 mg/kg: 3-, 8-, and 10-fold increases compared to control, respectively). A dose-dependent increase in MDA levels and SOD activity was also observed, which indicated a link between oxidative stress and nephrotoxicity. Qualitative analysis of DNA fragmentation by gel electrophoresis showed a DNA ladder indicative of Ca2+-Mg2+-endonuclease activation. Histopathological examination of kidney sections revealed numerous apoptotic nuclei across proximal and distal tubular cell linings. Collectively, these data for the first time suggest that DCLF-induced nephrotoxicity may involve production of reactive oxygen species leading to oxidative stress and massive genomic DNA fragmentation, and these two free radical mediated events may ultimately translate into apoptotic cell death of kidney cells in vivo, and reveal a DNA-active role for DCLF.

Unique organoprotective properties of a novel IH636 grape seed proanthocyanidin extract on cadmium chloride-induced nephrotoxicity, dimethylnitrosamine (DMN)-induced splenotoxicity and mocap-induced neurotoxicity in mice.

Several observations, both in humans and laboratory animals, have suggested that proanthocyanidins exhibit a broad spectrum of pharmacological, therapeutic and chemoprotective properties. Specifically, some of our earlier studies have shown that IH636 grape seed proanthocyanidin extract (GSPE, commercially known as ActiVin) provides excellent concentration- and dose-dependent protection against toxicities induced by diverse agents, such as acetaminophen, hydrogen peroxide, 12-O-tetradecanoylphorbol-13-acetate (TPA), smokeless-tobacco extract, idarubicin and 4-hydroxyperoxycyclophosphamide in both in vitro and in vivo models. In some models, GSPE proved to be a better cytoprotectant than vitamins C, E and beta-carotene. The purpose of this investigation was three fold: (i) to indirectly assess the bioavailability of GSPE in multiple target organs, (ii) quantify GSPE's capacity to avert cadmium chloride (CdCl2)-induced nephrotoxicity, dimethylnitrosamine (DMN)-induced splenotoxicity and O-ethyl-S,S-dipropyl phosphorodithioate (MOCAP)-induced neurotoxicity, and lastly (iii) to evaluate possible mechanisms of protection in mice. In order to determine all these, three separate experiments were designed and each experiment consisted of four groups, such as vehicle control, GSPE alone, toxicant alone and GSPE + toxicant. GSPE was administered orally (100 mg/Kg) for 7-8 days prior to the toxicant exposure. Parameters of the analyses included evaluation of serum chemistry changes (ALT, BUN and CK), histopathology and integrity of genomic DNA, both quantitatively and qualitatively. Results indicate that GSPE preexposure prior to cadmium chloride and DMN provided near complete protection in terms of serum chemistry changes (ALT, BUN and CK) and inhibition of both forms of cell death. e.g., apoptosis and necrosis. DNA damage, a common denominator usually associated with both apoptosis and necrosis was significantly reduced by GSPE treatment. Histopathological examination of organs correlated strongly with the changes in serum chemistry and the DNA modification data. Surprisingly, MOCAP exposure showed symptoms of neurotoxicity coupled with serum chemistry changes in the absence of any significant genomic DNA damage or brain pathology. Although, GSPE appeared to partially protect the neural tissue, it powerfully antagonized MOCAP-induced mortality. Taken together, this study suggests that in vivo GSPE-preexposure may protect multiple target organs from a variety of toxic assaults induced by diverse chemical entities.

Protection of acetaminophen-induced hepatocellular apoptosis and necrosis by cholesteryl hemisuccinate pretreatment.

This study of acetaminophen (AAP) hepatotoxicity examined whether some aspects of the highly integrated process of drug-induced toxicity involves apoptosis, in addition to necrosis in vivo; and if so, whether cholesteryl hemisuccinate (CS) pretreatment would selectively interfere with apoptotic or necrotic liver cell death. We have previously demonstrated that CS preexposure in vivo, protects hepatocellular necrosis and necrosis-related events induced by carbon tetrachloride (CCl4) administration. Our study demonstrates that administration of hepatotoxic doses of AAP (350-500 mg/kg, i.p.) to ICR mice (CD-1) results in severe liver injury leading to cell death both by necrosis and apoptosis. AAP-induced cell death was preceded by massive elevation in serum alanine aminotransferase coupled with rapid loss of large genomic DNA (2-24 hr), fragmentation of DNA in the form of a ladder (2-24 hr), apoptotic nuclear condensation at early hours (2-6 hr) followed by massive fragmentation and margination of heterochromatin at later (6-24) hours and a near total loss of glycogen in pericentral areas. Although CS (100 mg/kg, i.p.) alone had no noticeable biochemical or morphological effects, its administration before AAP (350-500 mg/kg, i.p.) abrogated histological and biochemical diagnostics of both apoptosis and necrosis. These include near total absence of loss of large genomic DNA and glycogen, and dramatic protection from escalating levels of liver injury. CS pretreatment also arrested AAP-induced ultrastructural changes typical of both apoptosis and necrosis. Histopathological examination of periodic acid-Schiff stained liver sections mirrored the biochemical and ultrastructural findings. In conclusion, this study for the first time establishes that apoptosis, in addition to necrosis, significantly contributes to AAP hepatotoxicity in vivo, and preexposure of mice to CS prevents AAP-induced hepatic apoptosis and necrosis.

Lovastatin-acetaminophen subchronic toxicity in mice.

A subchronic, toxicity study of lovastatin-acetaminophen combination in s/w mice is reported. Oral administration of lovastatin in corn oil 30 mg/kg three times a week and acetaminophen 0.75% w/v in drinking water for 13 weeks resulted in significant increase in SGPT (ALT) in both male and female mice. Histopathology of the liver revealed centrilobular hepatocyte swelling. These results provide evidence of hepatic injury in s/w mice when concomitantly exposed to lovastatin and acetaminophen.

Methylmethacrylate: tissue distribution and pulmonary damage in rats following acute inhalation.

Distribution in blood, brain and lungs, and histopathologic changes in brain and lungs of rats following inhalation exposure to methylmethacrylate monomer (MMA) are reported. On exposing groups of four male rats to air containing 100 ppm of MMA, the concentration of MMA in the tissues determined by headspace gas chromatography, was found to be 11.14 +/- 1.05 mg % in blood, 25.24 +/- 2.04 micrograms/g in brain, 20.60 +/- 1.01 micrograms/g in lungs; these did not change significantly with different exposure periods. Interalveolar congestion/hemorrhage, pulmonary vasodilation and edema were observed with rats exposed for 2,3 and 4 hours but not for 1 hour. No histopathologic changes were seen in the brain of any of the exposed rats.

Blood n-hexane concentration following acute inhalation exposure in rats.

Blood n-hexane concentrations in rats is reported. Groups of four rats were exposed to a n-hexane vapor/air mixture, in a glass chamber. At the end of the exposure, the animals were sacrificed by decapitation, blood was collected, and the concentration of n-hexane in blood was determined by headspace gas chromatography. Results indicate that blood becomes saturated with n-hexane in 10 minutes or less and persisted in the other exposure periods, up to and including 30 minutes.

In vitro toxicity of n-hexane and 2,5-hexanedione using isolated perfused rabbit heart.

In vitro toxicity of n-hexane and 2,5-hexanedione using isolated perfused rabbit heart is reported. The hearts were perfused using Langendorf's procedure and modified Anderson's coronary perfusion apparatus. The force of cardiac contraction was significantly reduced following 1 h perfusion with 9.6 mg/l concentration of n-hexane and with 0.35% v/v concentration of 2,5-hexanedione.

In-vitro toxicity of acetone using coronary perfusion in isolated rabbit heart.

In-vitro toxicity of acetone using isolated rabbit heart is reported. The hearts were perfused using Langendorf's procedure and modified Anderson's coronary perfusion apparatus. Heart rate and force of contraction were recorded mechanically and the coronary flow was measured manually. The force of contraction was significantly reduced; heart rate and coronary flow were significantly increased with acetone concentrations of 0.5% and 1.0% V/V.

Hydrazones of isoniazid for colorimetric analysis.

Two sensitive color reactions of isoniazid are described. They are based on the formation of hydrazone Schiff bases when isoniazid is reacted with 4-nitrobenzaldehyde or pyridoxal. Linear curves were obtained with both reagents when absorbance values were plotted against concentrations.