Saroj Chakrabarti

Role of oxidative stress in nickel chloride-induced cell injury in rat renal cortical slices.

Nickel chloride (NiCl2) induced lactate dehydrogenase (LDH) release and lipid peroxidation (LPO) in rat renal cortical slices in vitro in a concentration- (0-2 mM) and time- (0-4 hr) dependent manner, with initial significant LDH release occurring as early as 1 hr, whereas significant increase in LPO started 3 hr after exposure, suggesting that LPO results from renal cell injury. Both NiCl2-induced LDH release and LPO were prevented significantly by glutathione and dithiothreitol, suggesting that NiCl2-induced renal cell injury is dependent on thiols. However, such injury is not dependent solely on thiols, because (a) these thiols failed to inhibit completely the uptake of Ni2+ by the renal cortex, and (b) diethylmaleate pretreatment failed to increase NiCl2-induced cell injury further. Superoxide dismutase partially reduced the NiCl2-induced LDH release without affecting LPO and glutathione, whereas catalase did not affect such LDH release and LPO. Dimethylthiourea and DMSO completely prevented NiCl2-induced LPO, but only partially reduced LDH release. Deferoxamine prevented NiCl2-induced renal cell injury without affecting LPO and without significantly reducing Ni2+ uptake by the renal cortex, suggesting that nickel chelation is not important in such prevention of injury. NiCl2-induced inhibition of para-aminohippurate uptake was prevented significantly by thiols, deferoxamine, and dimethylthiourea. NiCl2-induced loss of cellular glutathione content was prevented significantly by thiols and deferoxamine, but not by superoxide dismutase and dimethylthiourea. These results suggest that LPO was not related to NiCl2-induced lethal renal cell injury, whereas such injury may be caused by the induction of the Fenton reaction, generating hydroxyl radicals.

Modulation of Monoamine Oxidase Activity in Different Brain Regions and Platelets Following Exposure of Rats to Methylmercury

Monoamine oxidase (MAO; EC 1.4.3.4) is known to have an important role in the regulation of biogenic amines in the brain and peripheral tissues. It is also known that circulating platelets represent an excellent model for an easy assessment of the effect of MAO-B inhibitors in extracerebral tissue. The present study was carried out to determine the effects of methylmercury (MeHg) on the activity of MAO in synaptosomes of different brain regions of male Sprague-Dawley rats as well as in rat blood platelets both in vitro and in vivo. MeHg pretreatment inhibited the activity of MAO in the synaptosomes of the cortex, hypothalamus, hippocampus, striatum, cerebellum, and brain stem in a concentration-dependent (0-10 microM) manner. The threshold concentration of MeHg for such inhibition in different brain synaptosomes was found to be the same (i.e., 1 microM) except for in the rat striatum it was 2.5 microM, and the IC50 value for MeHg was found to be around 2.1 microM. Significant inhibition of the MAO activity was also observed in synaptosomes of the cortex, cerebellum, hypothalamus, and hippocampus as well as in platelets of rats 24 h after treatment by gavage with a total cumulative dose of 35 mg/kg (5 mg/kg/day for 7 days). The decrease of such activity was found to be at maximum in different brain synaptosomes and platelets 24 h following treatment with a cumulative total dose of 75 mg/kg (7.5 mg/kg/day for 10 days); the treated animals showed signs of ataxia under these conditions. The data have further shown that methylmercury is capable of inhibiting the MAO activity in different brain synaptosomes to different degrees but without showing any specificity towards any specific brain region. The present in vivo results suggest that the platelet MAO activity may be used as a potential biomarker of early neurotoxicity due to repeated exposure to MeHg in rats.

Cooperativity of warfarin binding with human serum albumin induced by free fatty acid anion.

Abstract The effects of oleate ion, a free fatty acid anion, on the binding characteristics of warfarin with human serum albumin have been examined using fluorescence spectroscopy. The affinity constant of the warfarin-albumin complex was found to be increased without affecting the number of warfarin binding sites in the presence of a low molar ratio (∼3) of oleate ion and serum albumin, irrespective of the concentrations of the albumin used (5–200 μM) in the study. These findings indicate the presence of a cooperative (allosteric) interaction between warfarin and the oleate ion for albumin binding and, further, suggest a conformational transition in the albumin molecule as a result of interactions. However, the results of the polarization of fluorescence of warfarin-bound albumin indicate that such cooperative effects might not result from a conformational change of the local warfarin binding sites. On the other hand, in concentrated albumin (400–600 μM) or at a molar ratio of oleate ion to albumin of greater than 5, such cooperative interactions disappear and a simple competitive inhibition of the warfarin binding results. Correlations of these results with some clinical situations have been made.

Cytogenetic effects of low-level exposure to toluene, xylene, and their mixture on human blood lymphocytes

SummaryThe present study was undertaken to investigate, both in vitro and in vivo, the genotoxic potential of short-term low-level exposure to toluene, xylene, and their mixture, for which information is limited at the present time. Five adult healthy white men were exposed for 7 consecutive hours per day over 3 consecutive days to 50 ppm toluene and 40 ppm xylene either alone or in combination in a controlled exposure chamber. Such an exposure was repeated three times at intervals of 2 weeks. Blood samples were taken before and after the termination of such exposure. Three different cytogenetic end-points were evaluated using peripheral blood lymphocytes: number of sister chromatid exchanges (SCEs), cell cycle delay, and cell mortality. No significant effects on SCEs, cell cycle delay, and cell mortality were observed following such exposure to toluene or xylene or their mixture. Similarly, exposure of human blood lymphocytes in vitro to either toluene (0–2.5 mM) or xylene (0–2 mM) or their mixture for 72 h did not result in any significant cytogenetic effects at lower concentrations, while at higher concentrations, only cell mortality was found to be significantly affected. Thus our present study indicates that simultaneous exposure to low levels (within the admissible limits) of toluene, xylene, or their mixture for a short period does not pose any potential mutagenic threat to humans.

Dose-dependent metabolism of trichloroethylene and its relevance to hepatotoxicity in rats.

Fasted male Sprague-Dawley rats pretreated with phenobarbital received an intraperitoneal injection of 0.00, 0.25, 0.50, 0.75, 1.00, 1.50, or 2.00 ml/kg of trichloroethylene (TRI) in corn oil. The metabolic fate of TRI was monitored by measuring its major urinary metabolites 24 hr following the dosing. The hepatotoxic response of TRI was evaluated by determination of the serum transaminase activities (SGOT and SGPT) 24 hr after dosing. Treatment of rats with TRI up to 0.5 ml/kg did not affect the transaminase responses, but significant response was manifested at 0.75 ml/kg dose. Further continuous increases in such responses were induced on exposure to higher doses. Dose-dependent urinary excretions of trichloroethanol and trichloroacetic acid were observed and reached an apparent saturation at 1-1.5 ml/kg dose of TRI. The percentage of dose excreted as trichloroethanol was decreased from 16% at 0.25 ml or 2.8 mmole/kg to 8% at 2 ml or 22.3 mmole/kg dose. The percentage of trichloroacetic acid was decreased from 5 to 2% for the same dose interval. A maximum of only 29% depletion of hepatic glutathione was observed at 2 hr following 1 ml/kg dose of TRI. Similarly, the excretion of urinary mercapturic acid of TRI or thioether was insignificant at any dose level. These results suggest that the conjugation of hepatic glutathione with the electrophilic intermediate of TRI does not seem to be an important determinant for TRI hepatotoxicity nor the major detoxification pathway of its reactive intermediate. TRI produced also a dose-dependent decrease in hepatic microsomal monooxygenase activities as well as cytochrome P-450 content. All these results, when combined, show that there exists an apparent saturable metabolism of TRI involving its activation/deactivation pathways which correspond to an apparent threshold or minimal toxic dose, about 1 ml/kg or 11.15 mmole/kg of TRI for its hepatotoxicity.

Influence of long-chain free fatty acids on the binding of warfarin to bovine serum albumin☆

Abstract The effects of the concentration of long-chain free fatty acids and bovine serum albumin on the binding characteristics of warfarin with albumin have been studied by adsorption and fluorescence spectroscopy using the method of ultrafiltration. The binding affinity of warfarin is higher in a concentrated than in a dilute albumin solution. In a dilute solution of serum albumin (55 μM or less), the binding constant of the warfarin-albumin complex is increased in the presence of 100 μM (or less) of either lauric or oleic acid or a mixture of these two acids, but higher concentrations of the fatty acids decrease such binding. A mixture of saturated and unsaturated fatty acids shows the same effects as those by either fatty acid. Simultaneous measurements of the fluorescence peak intensity and polarization of fluorescence of warfarin bound to albumin in a dilute solution indicate that low and high concentrations of free fatty acids induce different conformational states of the same warfarin-binding sites on the albumin molecule. Such a dualistic behavior of free fatty acids could be best interpreted in terms of allosteric interactions involving heterotropic effects. On the other hand, in a concentrated solution of serum albumin, the binding constant of warfarin is decreased in the presence of both low and high concentrations of free fatty acids. Such a decrease in warfarin binding with concentrated albumin in the presence of free fatty acids is not due simply to a displacement of warfarin by free fatty acids from the warfarin-binding sites, but also could result simultaneously from a further conformational change of warfarin-binding sites caused by the interaction of warfarin and free fatty acids (a negative heterotropic effect).

In vivo nephrotoxic action of an isomeric mixture of S-(1-phenyl-2-hydroxyethyl)glutathione and S(2-phenyl-2-hydroxyethyl)glutathione in Ficher-344 rats

An isomeric mixture of S-[(1 and 2)-phenyl-2-hydroxyethyl]glutathione (PHEG), a glutathione conjugate of styrene, is moderately nephrotoxic. Its in vivo nephrotoxicity was characterized by significant elevations in the urinary excretion of glucose, gamma-glutamyl transpeptidase, glutamate dehydrogenase, N-acetyl-beta-D-glucosaminidase and lactic dehydrogenase 24 h after an i.v. administration of PHEG (0.5 mmol/kg) in male Fischer-344 rats. The histologic alterations consisted of moderate tubular damage with proximal tubule vacuolization and accumulation of tubular cast material, indicating an early sign of tubular necrosis. The data suggest that nephrotoxic injury induced by PHEG lies preferentially at the tubular region of the rat kidney involving several subcellular targets. The nephrotoxicity of PHEG was blocked by acivicin, a specific inhibitor of gamma-glutamyl transpeptidase, by phenylalanylglycine, an inhibitor of cysteinylglycine dipeptidase, as well as by probenecid, a competitive inhibitor of renal organic anion transport system. On the other hand, pretreatment with aminooxyacetic acid, a specific inhibitor of renal cysteine conjugate beta-lyase, failed to inhibit the nephrotoxicity of this glutathione conjugate. Similarly, prior administration of alpha-ketobutyrate, an inducer of renal cysteine conjugate beta-lyase, failed to potentiate its nephrotoxicity, suggesting an insignificant role of beta-lyase in such toxicity. A modest decline in renal cellular GSH due to PHEG but without any concomitant oxidation of GSH to GSSG and without any increase in lipid peroxidation indicates that oxidative stress may not be an important mechanism of its nephrotoxicity. Therefore, the following steps at least, are involved in the development of its nephrotoxicity: (1) renal tubular accumulation of PHEG via a probenecid-sensitive transport process; and (2) its renal metabolism via gamma-glutamyl transpeptidase and cysteinylglycine dipeptidase to the corresponding cysteine-S-conjugate.

Altered Regulation of Dopaminergic Activity and Impairment in Motor Function in Rats After Subchronic Exposure to Styrene

Animal and human studies suggest a dopamine-mediated effect of styrene neurotoxicity. However, the results reported to date are incomplete and not consistent. As such, the mechanism of its neurotoxicity is still unclear. The present study has, therefore, reexamined the central dopaminergic system in relation to some neurobehavioral effects in rats following subchronic exposure to styrene. Groups of adult male Sprague-Dawley rats received 0, 0.25, or 0.5 g styrene per kg b.wt. by gavage for 13 consecutive weeks. Twenty-four hours after cessation of such treatment with the higher dose (0.5 g/kg), the contents of dopamine (DA) and its metabolites were significantly reduced in the corpus striatum, hypothalamus, and lateral olfactory tract regions. In vitro styrene showed a significant increase in DA release from rat striatal synaptosomes similar to that of tyramine. Significant loss of motor function was observed on days 56, 70, and 84 during the styrene treatment with the higher dose, and lasted over a month after such treatment. However, the treated animals recovered their motor function within 45-60 days after cessation of such treatment, along with the recovery of normal levels of dopamine and its metabolites. Furthermore, styrene-induced initial impairments in measures of dopaminergic activity cannot be attributed to altered regulation of tyrosine hydroxylase activity. Specific [3H]-spiroperidol binding was also unaltered 7 or 15 days after subchronic treatment with styrene. These data imply that despite the dopaminergic neuronal loss due to styrene, dopaminergic transmission was not reduced to a level that would result in an overall development of dopamine receptor supersensitivity in the striatum. Collectively, these studies indicate that the subchronic neurotoxic action of styrene may be primarily presynaptic in nature and may involve impaired regulation of DA content and stimulation of DA release.

Rapid high-performance liquid chromatographic assay of acetaminophen in serum and tissue homogenates

The quantitation of a hepatorenal toxic drug, acetaminophen, in blood and target organ tissues is needed for toxicokinetic and distribution studies. A rapid, sensitive and simple method is described to assay acetaminophen in rat serum and liver or kidney homogenates by reversed-phase high-performance liquid chromatography, using an octadecyl (3 micron particle size) Apex column, a mobile phase consisting of a mixture of distilled water-acetonitrile (86:14) and ultraviolet detection at 245 nm. Short retention times of ca. 3.75 and 6.25 min are observed for acetaminophen and the internal standard (sulfamerazine), respectively. A sensitivity of 50 ng/ml is easily achieved for 100-microliter serum and liver or kidney homogenate samples. The proposed method proved to have satisfactory recovery, precision and accuracy. The preliminary results obtained with human plasma of volunteers and of patients treated with various drugs show that the assay, with a sensitivity of 25 ng/ml, would be of considerable interest in clinical monitoring of acetaminophen.

Effects of different styrene metabolites on cytotoxicity, sister-chromatid exchanges and cell-cycle kinetics in human whole blood lymphocytes in vitro

5 metabolites of styrene were tested in vitro for their cytotoxic effects, induction of SCEs and changes in cell-cycle progression in cultured human blood lymphocytes. Fresh heparinized peripheral blood (0.3 ml) from normal volunteers was cultured for a total of 72 h in 5 ml of RPMI 1640 medium containing 10% fetal calf serum, 0.1% garamycine, 1% glutamine and 1% phytohaemagglutinin. Styrene-7,8-oxide (SO), styrene glycol (SG), phenylglyoxylic acid (PGA), S-[1,2-phenyl-2-hydroxyethyl]-glutathione (PEG) (a glutathione conjugate of styrene oxide), N-acetyl-S-[1,2-phenyl-2-hydroxyethyl]-cysteine (NAPEC) in dimethyl sulfoxide (DMSO) were injected into the cultures 36 h after initial culture, so that the exposure time for these metabolites was 36 h. The final concentration of SO was 100 microM and those of the other metabolites were 500 microM. 24 h before harvest, BrdU (10 micrograms/ml) was added into the cultures for assessing cytogenetic endpoints. SO showed significant induction of SCEs and cell-cycle delay as well as a significant decline of cell survival. The same phenomena, but of less magnitude, were also observed with NAPEC, a cysteine derivative of SO. On the other hand, SG, PGA and PEG failed to produce any significant changes of these endpoints compared to the control. Thus, the present results have demonstrated that, in addition to SO, NAPEC possesses some cytogenotoxic potential and hence, these two metabolites together could contribute to the genotoxicity of styrene in human blood lymphocytes.