Mohammed Y.H. Farooqui

Circadian periodicity of tissue glutathione and its relationship with lipid peroxidation in rats

Circadian fluctuations in tissue glutathione (GSH) concentrations and lipid peroxidation in male Sprague-Dawley rats were investigated. Blood and all the organs studied exhibited distinct circadian variation both in GSH concentrations and peroxidation of polyunsaturated fatty acids. There was a great variation among organs in the periodicity and amplitude of the fluctuations in GSH concentrations. Liver displayed the highest variation (approximately 50%) followed by stomach (approximately 37%), heart (approximately 25%) and kidney (approximately 19%). The changes in other organs were significant but of less magnitude. Implications of such variations and caution in interpretation of experimental results in response to the exposure of animals to xenobiotics are discussed.

Comparative toxicities of aliphatic nitriles

Aliphatic nitriles have been postulated to manifest their toxicity through cyanide liberation. We have investigated the signs of toxicity and effect of equitoxic LD50 doses of saturated and unsaturated aliphatic mono- and dinitriles on tissue and blood cyanide levels, tissue glutathione levels and cytochrome c oxidase activities. Signs of toxicity were classified into cholinomimetic effects observed with unsaturated nitriles and central nervous system effects observed with saturated nitriles and potassium cyanide (KCN). Hepatic and blood cyanide levels 1 h after treatment were highest following malononitrile (MCN) and decreased in the order of propionitrile (PCN) greater than KCN greater than butyronitrile greater than acrylonitrile (VCN) greater than allylcyanide greater than greater than fumaronitrile greater than acetonitrile. The order differed in brain where KCN preceded MCN and PCN. Hepatic and brain cytochrome c oxidase were significantly inhibited and corresponded to their cyanide levels. No significant inhibition of cytochrome c oxidase was observed in vitro. VCN was the only nitrile which significantly reduced tissue GSH levels. In conclusion, toxic expression of aliphatic nitriles depends not only upon cyanide release but also on their degree of unsaturation. With unsaturated aliphatic nitriles cyanide release plays a minimal role in their toxicity.

In vivo interactions of acrylonitrile with macromolecules in rats.

The irreversible binding of [2,3-14C]acrylonitrile (VCN) to proteins, RNA and DNA of various tissues of male Sprague-Dawley rats after a single oral dose of 46.5 mg/kg (0.5 LD50) has been studied. Proteins were isolated by chloroform-isoamyl alcohol-phenol extraction. RNA and DNA were separated by hydroxyapatite chromatography. Binding of VCN to proteins was extensive and was time dependent. Radioactivity in nucleic acids was registered in the liver and the target organs, stomach and brain. DNA alkylation, which increased by time, was significantly higher in the target organs, brain and stomach (119 and 81 pmol/mg, respectively, at 24 h) than that in the liver. The covalent binding indices for the liver, stomach and brain at 24 h after dosing were, 5.9, 51.9 and 65.3, respectively. These results suggest that VCN is able to act as a multipotent carcinogen by alkylation of DNA in the extrahepatic target tissues, stomach and brain.

Molecular interaction of acrylonitrile and potassium cyanide with rat blood

The interaction of acrylonitrile (VCN) with rat blood has been investigated at the molecular level in an attempt to understand the possible mechanism of its toxicity. The results obtained were compared to those with potassium cyanide (KCN), a compound known to liberate cyanide (CN-) in biologic conditions. The radioactivity derived from K14CN was eliminated faster than that from [1-14C]VCN. Up to a maximum of 94% of 14C from VCN in erythrocytes was detected covalently bound to cytoplasmic and membrane proteins, whereas 90% of the radioactivity from KCN in erythrocytes was found in the heme fraction of hemoglobin. Determination of specific activity showed that binding occurred more in vivo than in vitro which indicated that the VCN molecule was bioactivated inside erythrocytes. These results indicate that KCN interacts mainly through CN- liberation and binding to heme, whereas VCN, which binds to cytoplasmic and membrane proteins, may cause damage to red cells by mechanisms other than release of CN-.

Distribution and covalent interactions of [1-14C]acrylonitrile in the rat.

The tissue distribution, elimination and covalent binding of [1-14C]-acrylonitrile (VCN) have been investigated in the rat. Rats, given an oral dose of 46.5 mg/kg (0.5 LD50) VCN, excreted 40% of the 14C in urine, 2% in feces, 9% in expired air as 14CO2, 0.5% as H14CN and 4.8% as unchanged VCN in 24 h. Bile flow increased 3 times after the administration of VCN and over a period of 6 h, 27% of the 14C was recovered in bile. The red blood cells retained significant amounts of radioactivity for more than 10 days after treatment, whereas the 14C activity declined sharply in plasma. Initially, the highest levels of radioactivity were found in the stomach and stomach content followed by the intestine. In liver, kidney, brain, spleen, adrenal, lung and heart tissues the radioactivity of the acid soluble fractions declined while covalent binding to macromolecules remained unchanged. In subcellular fractions of liver, kidney, spleen, brain, lung, and heart, 20-40% of the total radioactivity was bound to nuclear, mitochondrial and microsomal fractions whereas in cytosol only 6-14% was bound over a period of 6 h.

Assessment of the acute acrylonitrile-induced neurotoxicity in rats.

Acrylonitrile (VCN) is an aliphatic nitrile which is used extensively in manufacturing of synthetic fibers, plastics, and rubber. Although the neurotoxicity of VCN is recognized, no thorough characterization of this effect has been reported. Current studies were designed to quantitatively characterize the acute phase of VCN-induced cholinomimetic neurotoxicity, and to determine the effects of dose, route of administration, and atropine on such toxicity. Administration of a single gavage or subcutaneous doses of 20, 40, or 80 mg VCN/kg to male Sprague-Dawley rats causes two distinctive phases of acute neurotoxic effects. Signs observed in the early phase had a rapid onset, and were cholinomimetic in nature. They included salivation, lacrimation, chromodacryorrhea, polyuria, miosis, vasodilatation in face, ears and extremities, increased gastric secretion, and diarrhea. A late phase developed hours after VCN dosing, and the toxic signs included depression, convulsions, and respiratory failure followed by death at high doses. These results revealed that the cholinomimetic toxicity induced by VCN was dose related regardless of the route of administration. In another study, rats were pretreated with atropine (1 mg/kg, IP) prior to VCN (40 mg/kg) in order to investigate the role of the cholinergic system. Atropine protected rats against VCN-induced cholinomimetic neurotoxicity, suggesting possible involvement of the cholinergic system. Finally, this work provides essential basic information for studying the biochemical, pharmacological, and neurological basis of VCN-induced neurotoxicity in the rat.

Toxicokinetics and molecular interaction of [14C]-formaldehyde in rats.

The excretion, tissue distribution, and binding of [14C]-formaldehyde were studied at different time intervals in male rats following a single intraperitoneal injection of 72 mg CH2O (14.7 μCi)/ kg body weight. Within 30 min, 10% of the total dose was recovered in expired air as14CO2 and by the end of 72 hr, 41% of the administered dose was eliminated through expired air. The total elimination of14CH2O activity in urine and feces in 72 hr was 15%. Erythrocytes retained significant amounts of radioactivity, even at the end of 72 hr. Substantial levels of radioactivity were detected in most tissues one hr after administration, indicating a fast absorption and rapid distribution. Subcellular fractionation of the tissues showed that the highest levels of relative percent binding was in the microsomal fraction, whereas cytosol fractions contained lowest levels of bound radioactivity. DNA, RNA, protein and lipid fractions of liver and spleen tissues showed significantly elevated levels of14C-incorporation as compared to other tissues. Thein vivo incorporation of14C-activity showed an increased association of14CH2O with RNA in all the tissues. The maximum registration of radioactivity in RNA was at 48 hr after administration. Significantly higher amounts of14C-activity were registered in DNA of all tissues. The maximum registration of radiolabel in DNA of most tissues was at 12 hr after the14CH2O administration. The liver DNA showed maximal levels at 3 hr with a second peak at 48 hr.Substantial amounts of bound radioactivity in nucleic acids of all the tissues were observed even 72 hr after dosing. The relationship between macromolecular association and formaldehyde toxicity has been discussed.

Lung injury and repair: DNA synthesis following 1,1-dichloroethylene

Injury and cellular proliferation in the lung were examined following administration of 1,1-dichloroethylene (1,1-DCE) or vinylidene chloride. C57BL/6 male mice were treated orally with 200 mg/kg of 1,1-DCE prior to a single pulse of tritiated thymidine [( 3H]TdR). Necrosis and exfoliation of Clara cells of bronchiolar epithelium were evident by 1 day after chemical administration, and increased in severity by 2 days. A regenerative response was observed at 3 days after 1,1-DCE administration, and by 7 days the epithelium was substantially restored. At 30 days after 1,1-DCE, re-epithelization was achieved and areas devoid of epithelium were not observed. Changes in cellular proliferation were calculated from measurements of [3H]TdR incorporation into total pulmonary DNA. Activity of [3H]TdR was significantly inhibited at 1 day after chemical administration and thereafter increased: a peak of synthesis occurred between 3 and 5 days. At 7 days after 1,1-DCE administration, incorporation of [3H]TdR decreased to levels that were not significantly different from those of control animals. Autoradiographic examination of 0.5 micron thick plastic-embedded lung sections showed that [3H]TdR was incorporated into the DNA of bronchiolar epithelial cells, macrophages, interstitial, endothelial and Type II alveolar cells. However, the majority of the label was taken up by the nonciliated bronchiolar epithelial cells. The increased [3H]TdR incorporation into whole lung correlated with repopulation of bronchioles which was observed following injury. The results demonstrated that 1,1-DCE-induced damage to Clara cells of the bronchiolar epithelium was severe and rapid; re-epithelization was achieved in a relatively short time whereas differentiation was a prolonged process.

The effects of acrylonitrile on hemoglobin and red cell metabolism

The effects of acrylonitrile (VCN) on hemoglobin and red cell metabolism were studied in vitro and in vivo using male Sprague-Dawley rats. Reduced glutathione (GSH) was rapidly depleted by VCN. The reaction between VCN and GSH to form S-cyanoethyl glutathione is both enzymic and nonenzymic. GSH depletion induced oxidation of considerable amount of hemoglobin to methemoglobin. Incubation of nitrite-treated erythrocytes with VCN (2-10 mM) resulted in a significant decrease in methemoglobin reduction. VCN initiated hemolysis in vitro at a concentration of 0.05 M, and at concentrations lower than 0.05 M rendered erythrocytes susceptible to osmotic fragility even at higher concentration of NaCl. Following oral administration of VCN (80 mg/kg), significant perturbations of levels of red-cell GSH, 2,3-diphosphoglycerate, adenosine triphosphate, pyruvate, lactate, and oxidized glutathione occurred within 1 h. These changes returned to normal levels between 6 and 24 h. A strong correlation between the depletion of GSH in vivo and covalent binding [2,3-14C]VCN to hemoglobin was observed. These in vivo and in vitro results suggest that chronic exposure to VCN may lead to methemoglobinemia and consequently may cause impaired delivery of oxygen to various tissues.