John G. Ball

Resveratrol attenuates cisplatin renal cortical cytotoxicity by modifying oxidative stress

Cisplatin, a cancer chemotherapy drug, is nephrotoxic. The aim of this study was to investigate whether resveratrol (RES) reduced cisplatin cytotoxicity and oxidative stress. Rat renal cortical slices were pre-incubated 30min with 0 (VEH, ethanol) or 30μg/ml RES followed by 60, 90 or 120min co-incubation with 0, 75, or 150μg/ml cisplatin. Lactate dehydrogenase (LDH) leakage was unchanged at 60 and 90min by cisplatin. Cisplatin increased (p<0.05) LDH leakage at 120min which was protected by RES. Cisplatin induced oxidative stress prior to LDH leakage as cisplatin depressed glutathione peroxidase and superoxide dismutase (SOD) activity, increased lipid peroxidation, protein carbonyls and 4-hydroxynonenal (4-HNE) adducted proteins within 60min. RES failed to reverse glutathione (GSH) depression by cisplatin. In order to eliminated an extracellular interaction between RES and cisplatin, additional studies (RINSE studies) allowed a 30min RES uptake into slices, transfer of slices to buffer lacking RES, followed by 120min cisplatin incubation. RES in the RINSE studies prevented LDH leakage by cisplatin indicating that RES protection was not via a physical interaction with cisplatin in the media. These findings indicate that RES diminished cisplatin in vitro renal toxicity and prevented the development of oxidative stress.

Novel Protective Mechanisms for S-Adenosyl-L-methionine against Acetaminophen Hepatotoxicity: Improvement of Key Antioxidant Enzymatic Function

Acetaminophen (APAP) overdose leads to severe hepatotoxicity, increased oxidative stress and mitochondrial dysfunction. S-adenosyl-L-methionine (SAMe) protects against APAP toxicity at a mmol/kg equivalent dose to N-acetylcysteine (NAC). SAMe acts as a principle biological methyl donor and participates in polyamine synthesis which increase cell growth and has a role in mitochondrial protection. The purpose of the current study tested the hypothesis that SAMe protects against APAP toxicity by maintaining critical antioxidant enzymes and markers of oxidative stress. Male C57Bl/6 mice were treated with vehicle (Veh; water 15 ml/kg, ip), SAMe (1.25 mmol/kg, ip), APAP (250 mg/kg, ip), and SAMe+APAP (SAMe given 1 h following APAP). Liver was collected 2 and 4 h following APAP administration; mitochondrial swelling as well as hepatic catalase, glutathione peroxidase (GPx), glutathione reductase, and both Mn- and Cu/Zn-superoxide dismutase (SOD) enzyme activity were evaluated. Mitochondrial protein carbonyl, 3-nitrotyrosine cytochrome c leakage were analyzed by Western blot. SAMe significantly increased SOD, GPx, and glutathione reductase activity at 4 h following APAP overdose. SAMe greatly reduced markers of oxidative stress and cytochrome C leakage following APAP overdose. Our studies also demonstrate that a 1.25 mmol/kg dose of SAMe does not inhibit CYP 2E1 enzyme activity. The current study identifies a plausible mechanism for the decreased oxidative stress observed when SAMe is given following APAP.

Comparison of the in vitro toxicity of dichloroaniline structural isomers

Acute exposure to certain dichloroaniline (DCA) isomers results in renal and hepatic toxicity in vivo. In the present study we examined whether dichloroaniline structural isomers were cytotoxic to liver and kidney slices in vitro and compared the toxicities of the different structural isomers. These studies were necessary in order to validate the use of an in vitro slice system for examination of the cellular mechanisms for toxicity. Renal cortical and hepatic slices were incubated for 90 min with 2,3-DCA, 2,4-DCA, 2,5-DCA, 2,6-DCA, 3,4-DCA or 3,5-DCA at a final concentration of 0-1 mm. Pyruvate-directed gluconeogenesis was measured following an additional 30-min incubation with 10 mM pyruvate. Cytotoxicity was also determined by measurement of lactate dehydrogenase (LDH) release 120 min after the addition of dichloroaniline isomers at a final concentration of 0, 0.5, 1 or 2 mM. Gluconeogenesis in renal cortical slices was inhibited by all of the isomers beginning at a concentration of 0.5 mM. Renal slice LDH leakage was elevated above control levels by 1-2 mM 3,4-DCA or 3,5-DCA. A final concentration of 2 mM was needed for 2,3-DCA, 2,4-DCA, 2,5-DCA or 2,6-DCA in order to detect a significant (P < 0.05) increase in renal slice LDH leakage. Hepatic slices incubated with 0.5-2 mM 2,3-DCA or 2 mM 2,5-DCA exhibited diminished pyruvate-directed gluconeogenesis. After exposure to 2,4-DCA, 2,6-DCA, 3,4-DCA or 3,5-DCA, pyruvate-directed gluconeogenesis was similar to that in the controls. LDH leakage was increased significantly (P < 0.05) above control values by exposure to 2 mM 3,4-DCA or 3,5-DCA. In conclusion, DCA structural isomers were toxic in vitro to liver and kidney slices. These results indicated that the kidney was more sensitive than the liver to DCA isomers, and that the most toxic isomer was 3,5-DCA. These results are similar to those previously observed in vivo.

3,4-Dicholoroaniline acute toxicity in male Fischer 344 rats

The aromatic amine, 3,4-dichloroaniline (DCA) is an important intermediate in the chemical production of agricultural chemicals. A previous study had shown that nephrotoxicity was apparent 48 h after injection of 3,4-DCA. The purpose of this study was to examine the potential for 3,4-DCA to be toxic to the kidney, liver and urinary bladder 24 h after acute administration. Male Fischer 344 (F344) rats were injected (intraperitoneal (i.p.)) with 0.4, 0.8 or 1.0 mmol/kg 3,4-DCA hydrochloride (HCl) salt (2.5 ml/kg, 25% ethanol). Nephrotoxicity was apparent within 24 h in the 0.8 and 1.0 mmol/kg 3,4-DCA treated group and was characterized by elevated (P < 0.05) blood urea nitrogen (BUN) and kidney weight. Renal cortical slice accumulation ofp-aminohippurate (PAH) was also decreased in the 0.8 and 1.0 mmol/kg 3,4-DCA treated group relative to pair fed controls (PFC). Cellular changes were noted in the liver and bladder 24 h after 3,4-DCA administration. Plasma alanine transaminase (ALT) activity was elevated (P < 0.05) above PFC values 24 h after treatment with 0.8 or 1.0 mmol/kg indicating liver damage was apparent within 24 h. Morphological damage was apparent along the centrilobular region. Hematuria was observed in the 0.8 and 1.0 mmol/kg 3,4-DCA treated groups. Infiltration of erythrocytes and polymorphonuclear leukocytes was apparent within the urinary bladder upon examination by light microscopy. These results indicated that 3,4-DCA was toxic within 24 h and that the target tissues were the kidney, liver and urinary bladder. In vitro studies were conducted to compare the toxicity of two forms of 3,4-DCA, the free base and hydrochloride salt to determine whether chemical form contributes to renal cortical slice toxicity. Lactate dehydrogenase (LDH) release was elevated above control by 120 min exposure to 2 mM 3,4-DCA free base or hydrochloride salt. Pyruvate directed gluconeogenesis in renal slices was decreased relative to control by 0.5 mM 3,4-DCA free base and hydrochloride salt. The results from the in vitro studies indicates that the chemical form did not modify in vitro renal cortical slice toxicity.

Acute renal and hepatic toxicity of 2-haloanilines in Fischer 344 rats

Aniline and its halogenated derivatives are widely used as chemical intermediates. The purpose of this study was to determine the hepatotoxic and nephrotoxic potential of the 2-haloanilines. Male Fischer 344 rats (n > or = 4) were injected (i.p.) with 1.0 or 1.25 mmol/kg of: aniline (A), 2-fluoroaniline (2-FA), 2-chloroaniline (2-ClA), 2-bromoaniline (2-BrA), 2-iodoaniline (2-IA) or vehicle (0.9% saline, 2.5 ml/kg). All compounds were injected as hydrochloride salts. Renal and hepatic function was monitored 24 h after treatment. All of the 2-haloanilines induced oliguria, diminished kidney weight, tubular casts and decreased renal cortical slice accumulation of organic anions. Blood urea nitrogen (BUN) levels were increased (P < 0.05) by treatment with 1.0 or 1.25 mmol/kg of 2-FA, 2-ClA or 2-BrA. Hepatic alterations were also observed and characterized by elevated plasma ALT/GPT activity and altered morphology in the centrilobular region. The nephrotoxic and hepatotoxic potentials were similar among the 2-haloanilines but aniline was less toxic than its 2-halo derivatives. These results demonstrated that halogen substitution at the 2-position of aniline increased hepatic and renal toxicity. However, the severity of toxicity was not influenced by the nature of the halogen substituent.

In vitro nephrotoxicity induced by chloronitrobenzenes in renal cortical slices from Fischer 344 rats.

Chloronitrobenzenes are important chemical intermediates in the manufacture of industrial, agricultural and pharmaceutical agents. Toxicity induced by the various chloronitrobenzene isomers in vivo includes hematotoxicity, immunotoxicity, hepatotoxicity and nephrotoxicity. The purpose of the study was to determine the direct nephrotoxic effects of nitrobenzene and ten chlorinated nitrobenzene derivatives using renal cortical slices as the in vitro model. Renal cortical slices were prepared from kidneys of untreated, male Fischer 344 rats and incubated with nitrobenzene (1.0-5.0 mM), a chloronitrobenzene (0.5-5.0 mM) or vehicle for 2 h. At the end of the 2 h incubation, tissue gluconeogenesis capacity (pyruvate-stimulated gluconeogenesis) and lactate dehydrogenase (LDH) release were determined as measures of cellular function and cytotoxicity. Based on decreased pyruvate-stimulated gluconeogenesis and increased LDH release, the order of decreasing nephrotoxic potential was trichloronitrobenzenes>dichloronitrobenzenes>monochloronitrobenzenes>nitrobenzene. Among the mono- and dichloronitrobenzenes, 1-chloro-3-nitrobenzene and 3,4-dichloronitrobenzene were the most potent nephrotoxicants, while the two trichloronitrobenzenes tested exhibited similar nephrotoxic potentials. These results demonstrate that chloronitrobenzenes are directly nephrotoxic in vitro and that increasing the number of chloro groups increases the nephrotoxic potential of the resulting chloronitrobenzene derivative.

Characterization of 2-amino-4,5-dichlorophenol (2A45CP) in vitro toxicity in renal cortical slices from male Fischer 344 rats

2-Amino-4,5-dichlorophenol (2A45CP) is a major, aromatic ring hydroxylated metabolite of the renal toxicant, 3,4-dichloroaniline. 3,4-Dichloroaniline is nephrotoxic with primary damage located to the proximal tubules. The purpose of this study was to first characterize the in vitro toxicity of 2A45CP in renal cortical slices. Second, the effect of antioxidants and sulfhydryl containing agents on the severity of 2A45CP toxicity was explored since part of the mechanism of toxicity for aminophenols may involve redox cycling. Renal tissue was isolated from male Fischer 344 rats (190--220 g). Renal slices were rinsed three times for 3 min each in 5-ml Krebs buffer. Tissues were then incubated for 90--120 min with varying concentrations of 2A45CP between 0 and 0.5 mM. In a separate series of experiments, the slices (50--100 mg) were preincubated for 30 min with 1 mM dithiothreitol (DTT), 1 mM glutathione (GSH) or 2 mM ascorbic acid prior to exposure to 0, 0.05, 0.1 or 0.25 mM 2A45CP. 2A45CP produced a concentration and time dependent increase in LDH leakage from renal cortical slices. Total glutathione levels were diminished by 0.5 mM 2A45CP within 30 min. Renal slices incubated for 60 and 120 min with 0.05 and 0.1 mM 2A45CP had lower malondialdehyde levels than control. Pretreatment with DTT did not alter 2A45CP toxicity. Pretreatment of renal cortical slices with GSH or ascorbic acid reduced 2A45CP toxicity. These findings indicate that 2A45CP is directly toxic to renal cortical slices and that cytotoxicity is at least partially mediated by a reactive intermediate.

In vitro toxicity of 2- and 4-chloroaniline : Comparisons with 4-amino-3-chlorophenol, 2-amino-5-chlorophenol and aminophenols

Chloroanilines have been associated with renal and hepatic toxicity. This study (a) examined the in vitro hepatic and renal toxicity of 2-chloroaniline and 4-chloroaniline, (b) further examined whether aromatic ring hydroxylation would increase toxicity of the parent compound and (c) compared toxicity between respective aminochlorophenol and aminophenol. Renal and hepatic slices were exposed to varying concentrations of 2-chloroaniline, 4-chloroaniline. 4-amino-3-chlorophenol, 2-amino-5-chlorophenol, 2-aminophenol or 4-aminophenol. Toxicity was monitored by measurement of pyruvate-directed gluconeogenesis and leakage of lactate dehydrogenase (LDH). Hepatic tissue was less susceptible to toxicity than kidney tissue for all compounds since LDH leakage was elevated only in renal tissue. Gluconeogenesis was reduced in renal cortical slices exposed to 0.1 mum aminochlorophenols or 4-aminophenol, whereas a concentration of 0.5 mum was necessary for the chloroanilines and 2-aminophenol. LDH release was increased in renal slices by aminochlorophenols and aminophenols but not by the chloroanilines. The nephrotoxic potential in renal cortical slices was 4-aminophenol > 2-amino-5-chlorophenol > 4-amino-3-chlorophenol > 2-aminophenol > 2-chloroaniline = 4-chloroaniline. These results suggest that aromatic ring hydroxylation increased in vitro toxicity of the chloroanilines. Comparison of aminophenols with aminochlorophenols indicated that addition of a halogen can have variable effects on toxicity.

Cephaloridine nephrotoxicity in streptozotocin induced diabetic fischer 344 (F344) rats

The purpose of this study was to determine if cephaloridine nephrotoxicity is attenuated in streptozotocin (STZ)-induced diabetic rats. Fischer 344 (F344) rats (205-250 g) were given a single injection (i.p.) of STZ (27-35 mg/kg) or citrate buffer. The nephrotoxicity of (750 mg/kg) cephaloridine (i.p.) was then compared with normoglycemic and 14-day diabetic rats. Increased blood urea nitrogen (BUN) levels as well as diminished renal cortical slice accumulation of tetraethylammonium (TEA) and lactate-stimulated p-aminohippurate (PAH) were measured (P less than 0.05) in normoglycemic rats 48 h after cephaloridine administration. Cephaloridine failed to alter BUN levels and organic ion accumulation in diabetic rats. Diabetes did not totally protect against cephaloridine toxicity since kidney weights were elevated in normoglycemic and diabetic rats 48 h after administration of 750 mg/kg cephaloridine. A series of experiments also measured BUN levels, kidney weight and renal cortical slice uptake of PAH and TEA 24, 48 and 72 h after (1500 mg/kg) cephaloridine administration. Cephaloridine increased (P less than 0.05) kidney wt and decreased PAH and TEA uptake (P less than 0.05) in the normoglycemic group at 24-72 h. No change in kidney wt, PAH or TEA uptake was observed in the diabetic rats. These data indicate diabetes reduces cephaloridine nephrotoxicity.

Nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol in Fischer 344 rats: comparisons with 2- and 4-chloroaniline and 2- and 4-aminophenol☆

Nephrotoxicity occurs following intraperitoneal (i.p.) administration of 2-chloroaniline or 4-chloroaniline hydrochloride to Fischer 344 rats, but the nephrotoxicant chemical species and mechanism of nephrotoxicity are unknown. The purpose of this study was to evaluate the in vivo and in vitro nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol, metabolites of 4-chloroaniline and 2-chloroaniline. A comparison was also made between the nephrotoxic potential of the aminochlorophenols and the corresponding aminophenols to examine the effect of adding a chloride group on the nephrotoxic potential of the animophenols. Male Fischer 344 rats (4/group) were given an i.p. injection of a chloroaniline or aminochlorophenol hydrochloride (1.5 mmol/kg), and aminophenol (1.0 or 1.5 mmol/kg), or vehicle, and renal function monitored at 24 and 48 h. Both aminochlorophenols induced smaller and fewer renal effects that the parent chloroanilenes in vivo. Also, 4-aminophenol was markedly more potent as a nephrotoxicant that 4-amino-3-chlorophenol, while 2-aminophenol and 2-amino-5-chlorophenol induced only mild change in renal function. In vitro, the phenolic compounds reduce p-aminohippurate accumulation by renal cortical slices at bath concentrations of 0.01 mM, while a bath concentration of 0.50 mM or greater was required for the chloroanilines. However, all compounds reduced tetraethylammonium accumulation at bath concentrations of 0.1-0.5 mM or greater. These results indicate that extrarenally-produced aminochlorophenol metabolites do not contribute to the mechanism of chloroaniline nephrotoxicity. Also, the reduced nephrotoxic potential of 4-amino-3-chlorophenol compared to 4-aminophenol could result from an altered ability of the aminochlorophenol to redox cycle or form conjugates.