Search

Currently, no reliable noninvasive methods exist for monitoring the severity of in vivo cyanide (CN) toxicity, treatment, and resulting physiological changes. We developed a broadband diffuse optical spectroscopy (DOS) system to measure bulk tissue absorption and scattering. DOS was used to optically monitor CN toxicity and treatment with sodium nitrite (NaNO2). To perform experiments, the DOS probe was placed on the hind leg of rabbits. A sodium CN solution was infused intravenously. DOS and concurrent physiologic measurements were obtained. After completion of CN infusion, NaNO2 was infused to induce methemoglobinemia (MetHb). During infusion of CN, blood gas measurements showed an increase in venous partial pressure of oxygen (pO2), and following reversal, venous pO2 values decreased. DOS measurements demonstrated corresponding changes in hemoglobin oxygenation states and redox states of cytochrome-c oxidase (CcO) during CN infusion and NaNO2 treatment. Therefore, DOS enables detection and monitoring of CN toxicity and treatment with NaNO2.

Primary cultures of rat renal proximal tubular (PT) and distal tubular (DT) cells from control and uninephrectomized (NPX) Sprague-Dawley rats were established to study whether the altered toxicological responses identified in freshly isolated cells are maintained in culture. Previous work showed that primary cultures of PT cells from hypertrophied rat kidneys maintained their differentiated properties, as evidenced by their high respiratory rate, active transport function, transport and metabolism of glutathione, and their hypertrophic phenotype. In the present study, primary cultures of PT cells from NPX rat kidneys, but to a much lesser extent DT cells, were more susceptible to cellular injury induced by either mercuric chloride, KCN, or tert-butyl hydroperoxide (tBH), than corresponding cells from normal rat kidneys. Direct comparisons of cytotoxicity and lipid peroxidation induced by tBH in freshly isolated renal cells showed that the primary cultures of cells from NPX rat kidneys retained their altered susceptibility relative to cells from control rats. These results show that primary cultures of PT cells from NPX rats are more sensitive to cellular injury induced by three mechanistically distinct toxicants, demonstrating their usefulness in the study of the molecular and biochemical basis for the altered phenotype of compensatory renal growth. This is the first report validating the use of a mammalian renal cell culture model to study the toxicological effects of compensatory renal cellular hypertrophy.

Execution of cyanide-induced apoptosis is mediated by release of cytochrome c from mitochondria. To determine how cyanide initiates cytochrome c release, Bax translocation was investigated in primary cultures of cortical neurons. Under nonapoptotic (control) conditions, Bax resided predominantly in the cytoplasm. After 300-microM cyanide treatment for 1 h, Bax translocated to the mitochondria, as shown by immunocytochemical staining and subcellular fractionation; Western blot analysis confirmed "cytosol-to-mitochondria" translocation of Bax. Temporal analysis showed that Bax translocation preceded cytochrome c release from the mitochondria, which was initiated 3 h after cyanide treatment. In double-immunofluorescence labeling for both Bax and cytochrome c, it was observed that cytochrome c was released only in cells showing Bax in mitochondria. The role of p38 mitogen-activated protein (MAP) kinase in Bax translocation was studied. The p38 MAP kinase was activated 30 min after cyanide, and its phosphorylation level of activity began to decrease 3 h later. SB203580, a p38 MAP kinase inhibitor, blocked translocation of Bax to mitochondria, whereas SB202474, a control peptide, had no effect on translocation. Inhibition of p38 MAP kinase by SB203580 blocked all downstream effects of Bax translocation, including cytochrome c release, caspase activation, and internucleosomal DNA fragmentation. These results demonstrated that Bax translocation is critical for cyanide-induced cytochrome c release and that p38 MAP kinase regulates Bax translocation from cytosol to mitochondria.

Cyanide is a well-established poison known for its rapid lethal action and toxicity. Although long-term mammalian studies examining the carcinogenic potential of cyanide have not been previously reported, cyanide was reported to be positive in Salmonella typhimurium mutagenesis assay and induced aneuploidy in Drosophila. To further evaluate the carcinogenic potential of cyanide, the ability of cyanide to induce morphological transformation in Syrian hamster embryo (SHE) cells was studied. Cyanide induced a dose-dependent increase in morphological transformation in SHE cells following a 7-day continuous treatment. A significant increase in transformation was observed at potassium cyanide doses of 200 microM and greater. Transformation induced by cyanide was inhibited in a dose-related manner by vitamin E, suggesting a role of oxidative stress in the induction of morphological transformation by cyanide. Further, it was shown that 500 microM cyanide induced oxidative DNA damage in SHE cells, evidenced by the formation of 8-hydroxy-2'-deoxyguanosine (50-66% increase over control). The induction of oxidative stress by cyanide involved an early and temporal inhibition of antioxidant enzymes (catalase and superoxide dismutase) as well as an increased production of reactive oxygen species (1.5- to 2.0-fold over control).

Isolated liver with a recirculating perfusate was used to study 3'-azido-3'-deoxythymidine (AZT) disposition in phenobarbitone-pretreated rats at 68 microM AZT concentration in the reservoir. Clearance of AZT in the livers obtained from control animals was 0.42 +/- 0.01 (mean +/- s.d.) mL min-1/10 g liver. Over the study period of 105 min, 12.7 +/- 2.6% of the dose was excreted in bile and of this 95% was recovered as 3'-azido-3'-deoxy-5'-O-beta-D-glucopyranuronosylthymidine (GAZT). The amount of GAZT found in the perfusate after 105 min of liver perfusion was < 1% of the AZT dose introduced into the reservoir. Phenobarbitone pretreatment of rats resulted in a 5.5-fold increase of AZT clearance. In addition, the area under the perfusate concentration-time curve (AUC0-105 min) for 3'-amino-3'-deoxythymidine (AMT) and for a catabolite of unknown structure was increased 3- and 10-fold, respectively, and the amount of AZT dose excreted in the bile was nearly doubled. Thus phenobarbitone was capable of stimulating both detoxification of AZT to GAZT and bioactivation of AZT to AMT, a catabolite known to be highly toxic to human bone marrow cells. This induction was the result of enhancement of AZT catabolism rather than its transport into the cells, since on incubation of AZT (0-250 microM) with rat isolated hepatocytes, a linear relationship between concentration and amount taken up by the cells was shown. In addition, the rate of AZT uptake was not influenced by KCN, dinitrophenol, or temperature, which is consistent with a simple diffusion of AZT through the hepatocellular membrane.(ABSTRACT TRUNCATED AT 250 WORDS)