E. Ahmed

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.

Covalent binding of [14C]benzene to cellular organelles and bone marrow nucleic acids

Abstract Our objective was to determine whether reactive metabolites of benzene covalently bind to cellular proteins and nucleic acids of the hematopoietic organs and liver of the mouse. The distribution and binding of [ 14 C]benzene after a single dose (880 mg/kg, s.c., 0.75 mCi/kg) were investigated at 3, 6, 12, and 24 hr. Maximum levels of radioactivity were found at 3 hr in liver, spleen, bone marrow, and blood. Maximum covalent binding occurred at 6 hr in liver, spleen, and bone marrow. Chemical fractionation of label derived from [ 14 C]benzene indicated that 0.5, 7, and 17 per cent of the covalently bound label in liver, spleen, and bone marrow was recovered in the nucleic acids. Upon subcellular fractionation of liver, total radioactivity was localized chiefly in the cytosol (1723 dpm/mg protein) and mitochondria (1433 dpm/mg protein). Of the total radioactivity recovered in various organelles, mitochondria had the highest proportion covalently bound (25 per cent). These studies indicate that label derived from [ 14 C]benzene covalently binds to nucleic acids of hematopoietic cells, the major site of toxicity. In addition, the high levels of covalent binding with macromolecules of the mitochondria suggest that their functions may be impaired by benzene.

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.

Chloroacetonitrile (CAN) induces glutathione depletion and 8-hydroxylation of guanine bases in rat gastric mucosa.

Chloroacetonitrile (CAN) is detected in drinking‐water supplies as a by‐product of the chlorination process. Gastroesophageal tissues are potential target sites of acute and chronic toxicity by haloacetonitriles (HAN). To examine the mechanism of CAN toxicity, we studied its effect on glutathione (GSH) homeostasis and its impact on oxidative DNA damage in gastric mucosal cells of rats. Following a single oral dose (38 or 76 mg/Kg) of CAN, animals were sacrificed at various times (0–24 h), and mucosa from pyloric stomach were collected. The effects of CAN treatment on gastric GSH contents and the integrity of genomic gastric DNA were assessed. Oxidative damage to gastric DNA was evaluated by measuring the levels of 8‐Hydroxydeoxyguanosine (8‐OHdG) in hydrolyzed DNA by HPLC‐EC. The results indicate that CAN induced a significant, dose‐ and time‐dependent, decrease in GSH levels in pyloric stomach mucosa at 2 and 4 hours after treatment (56 and 39% of control, respectively). DNA damage was observed electrophoretically at 6 and 12 hours following CAN administration. CAN (38 mg/Kg) induced significant elevation in levels of 8‐OHdG in gastric DNA. Maximum levels of 8‐OHdG in gastric DNA were observed at 6 hours after CAN treatment [9.59 ± 0.60 (8‐OHdG/105dG) 146% of control]. When a high dose of CAN (76 mg/Kg) was used, a peak level of 8‐OHdG [11.59 ± 1.30 (8‐OHdG/105dG) 177% of control] was observed at earlier times (2 h) following treatment. When CAN was incubated with gastric mucosal cells, a concentration‐dependent cyanide liberation and significant decrease in cellular ATP levels were detected. These data indicate that a mechanism for CAN‐induced toxicity may be partially mediated by depletion of glutathione, release of cyanide, interruption of the energy metabolism, and induction of oxidative stress that leads to oxidative damage to gastric DNA.

Determination of camptothecin in biological fluids using reversed-phase high-performance liquid chromatography with fluorescence detection

Camptothecin, a plant alkaloid with antitumor activity, is a potent and rapidly acting inhibitor of DNA synthesis. The objective of this study was to develop a sensitive high-performance liquid chromatographic (HPLC) method for the detection and estimation of the camptothecin concentration in biological fluids. Using HPLC coupled with fluorescence detection, at an excitation wavelength of 370 nm and an emission wavelength of 434 nm, we found that the lower limits of detection for camptothecin in aqueous, plasma and urine samples were 0.5, 1 and 10 ng/ml, respectively. The ideal mobile phase used was methanol-10 mM potassium phosphate (75:25, v/v, pH 4.0). To determine the utilization of the method in a biological system, we studied the pharmacokinetics of camptothecin in mice. Elimination of camptothecin from mice blood was triphasic and followed first-order kinetics. The half-life of camptothecin in mouse blood was 25.7 min. Our studies indicate that HPLC with fluorescence detection for the determination of camptothecin in different media is a simple, rapid, sensitive and reproducible method.

Acrylonitrile: In vivo cytogenetic studies in mice and rats☆

Acrylonitrile (VCN), a suspect human carcinogen, does not produce significant increases in cytogenetic aberrations in the mouse-bone marrow when given orally for 4, 15 or 30 days at doses equal to 7, 14 and 21 mg/kg/day resp. or by i.p. for the same time periods at doses of 10, 15 and 20 mg/kg/day. Rats treated orally with 16 daily doses of VCN (40 mg/kg/day) or potassium cyanide (KCN) (5 mg/kg/day) showed no increase of aberrant metaphases in the bone marrow over controls.

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-.

Induction of oxidative stress and TNF-α secretion by Dichloroacetonitrile, a water disinfectant by-product, as possible mediators of apoptosis or necrosis in a murine macrophage cell line (RAW)

The water disinfectant by-product dichloroacetonitrile (DCAN) is a direct-acting mutagen and induces DNA strand breaks in cultured human lymphoblastic cells. Cellular activation by environmental agents may exert detrimental effects to the cells. Activated macrophages produce reactive oxygen intermediates such as H(2)O(2), (-)OH and O(2). Therefore, the effect of various concentrations of DCAN (100-400 microM) on the activity macrophage cells (RAW 264.7) was studied. In these cells, DCAN-induced oxidative stress was characterized by the production of reactive oxygen intermediates (ROI). Also, the ratios of intracellular GSH/GSSG was assessed and used as a biomarker for oxidative stress. The secretion of TNF-alpha was assessed since macrophages are known to secrete TNF-alpha as a result of cellular oxidative stress. Electrophoretic detection of DNA degradation and light microscopy was utilized for the characterization of DCAN-induced apoptosis. Lactate dehydrogenase (LDH) leakage and trypan blue exclusion were used as markers of cellular necrosis. Following exposure to DCAN (200 microM and 400 microM), intracellular GSSG was increased (2.5-fold of control, P<0. 05). DCAN activation of RAW cells was detected by elevated levels of intracellular ROI (1.9-2.5-fold than control, P<0.05) and increased secretion of TNF-alpha (4.5 fold-than control, P <0.05). Elecrophoresis of genomic DNA of treated cells indicated a dose-dependent increase in degradation of genomic DNA. Morphological studies also indicated that exposure of RAW cells to 100 microM or 200 microM DCAN incites apoptotic cell death. At higher concentrations (400 microM), however, significant (P<0.05) increase in LDH leakage and decrease in cell viability (55% of control) indicative of cellular necrosis, were observed. These studies indicate that DCAN induces dose-dependent apoptosis or necrosis in RAW cells that could be due to the disturbance in intracellular redox status and initiation of ROI-mediated oxidative mechanisms of cellular damage.

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.