Mechanisms of chlorpyrifos and diazinon induced neurotoxicity in cortical culture.

The main action of organophosphorous insecticides is generally believed to be the inhibition of acetylcholinesterase (AChE). However, these compounds also inhibit many other enzymes, any of which may play a role in their toxicity. We tested the neurotoxic mechanism of two organophosphorous insecticides, chlorpyrifos and diazinon in primary cortical cultures. Exposure to the insecticides caused a concentration-dependent toxicity that could not be directly attributed to the oxon forms of the compounds which caused little toxicity but strongly inhibited AChE. Addition of 1 mM acetylcholine or carbachol actually attenuated the toxicity of chlorpyrifos and diazinon, and the muscarinic receptor antagonist, atropine, and the nicotinic receptor antagonist, mecamylamine, did not attenuate the toxicity of either insecticide. These results strongly suggest that the organophosphorous toxicity observed in this culture system is not mediated by buildup of extracellular acetylcholine resulting from inhibition of AChE. The toxicity of chlorpyrifos was attenuated by antagonists of either the NMDA or AMPA/kainate-type glutamate receptors, but the cell death was potentiated by the caspase inhibitor ZVAD. Diazinon toxicity was not affected by glutamate receptor antagonists, but was attenuated by ZVAD. Chlorpyrifos induced diffuse nuclear staining characteristic of necrosis, while diazinon induced chromatin condensation characteristic of apoptosis. Also, chlorpyrifos exposure increased the levels of extracellular glutamate, while diazinon did not. The results suggest two different mechanisms of neurotoxicity of the insecticides, neither one of which involved acetylcholine. Chlorpyrifos induced a glutamate-mediated excitotoxicity, while diazinon induced apoptotic neuronal death.

Hypothermic preservation of hepatocytes. III. Effects of resuspension media on viability after up to 7 days of storage.

Hepatocyte suspensions provide a rapid method to determine how hypothermic storage affects liver cell metabolism and viability. Using these studies, improved methods of hypothermic liver preservation for transplantation may be developed. In this study, rat hepatocytes were cold-stored for up to 7 days in University of Wisconsin liver preservation solution. At the end of each day of storage hepatocytes were resuspended in Krebs-Henseleit buffer or tissue-culture medium (Liebovitz-15; Fischer's; modified Fischer's, which was similar to Fischer's but with glycine and cysteine added; or Waymouth's medium). Hepatocyte viability was assessed by rewarming and oxygenating the suspensions and measuring the percentage of leakage of lactate dehydrogenase from the cells, the cellular concentration of potassium and the stimulation of respiration by succinate, all measures of plasma membrane integrity. Additionally, concentrations of ATP and glutathione after rewarming and reoxygenation in the various resuspension media were measured. Hepatocyte permeability to lactate dehydrogenase did not increase during cold storage of 1 to 7 days (7.2% +/- 2% leakage), indicating that most of the hepatocytes remained viable during cold storage. However, when rewarmed, loss of viability (leakage of lactate dehydrogenase) was dependent on the composition of the resuspension media. In Krebs-Henseleit buffer, viability was reduced after 2 and 3 days of storage (lactate dehydrogenase leakage on rewarming = 70% to 90%). Leakage of lactate dehydrogenase was reduced significantly after resuspension in tissue-culture media. After 6 days of storage, lactate dehydrogenase leakage from hepatocytes stored in Liebovitz- 15 or modified Fischer's was only about 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycine prevention of cold ischemic injury in isolated hepatocytes.

Isolated hepatocytes suspended in a liver preservation solution (University of Wisconsin (UW) solution) and exposed to cold (5 degrees C) ischemia lose viability (LDH release) after 3 (76.5 +/- 2.6% extracellular LDH) and 4 days (90.3 +/- 5.7% extracellular LDH) storage when rewarmed (37 degrees C) in Krebs-Henseleit buffer. However, if 3 mM glycine is added to Krebs-Henseleit buffer the loss of LDH on rewarming was suppressed (% LDH = 24.4 +/- 2.2% and 33.2 +/- 3.0%, at 3 and 4 days, respectively). The protection by glycine could also be obtained by storing the hepatocytes in the UW solution containing 15 mM glycine and rewarming in the absence of glycine in Krebs-Henseleit buffer. There did not appear to be a relationship between the protection by glycine and glutathione concentration of the hepatocytes as shown by the lack of effect of a glutathione synthetase inhibitor (butathionine sulfoximine) on the protective effects of glycine. Other amino acids did not provide protection to hepatocytes exposed to cold ischemia. The mechanism of action of glycine is not known, but this compound may be important in improving cold storage of livers for transplantation.

The effect of halothane, isoflurane, and verapamil on ischemic-isolated rabbit renal tubules.

The effects of the volatile anesthetics halothane and isoflurane, and the calcium entry blocker verapamil, were studied in isolated rabbit renal tubules under nonischemic and simulated ischemic conditions. Isolated rabbit renal tubules were subjected to zero (control), 30 (I-30), or 60 (I-60) minutes of simulated ischemia following the method of Weinberg. Following the ischemic period, tubules were reoxygenated in a Gilson respirometer (simulated reperfusion) and treated with either halothane (1%) or isoflurane (1%) in the controls and at I-30, or halothane (1%, 2%, 4%) or verapamil (5 microM, 15 microM, 30 microM) at I-60. Tubules were analyzed for lactate dehydrogenase (LDH) release (measuring cell membrane integrity), intracellular potassium and adenosine triphosphate (ATP), and oxygen consumption (cellular respiratory rate). In nonischemic tubules, exposure to 1% isoflurane caused significantly reduced LDH release compared with that released by controls, indicating cell membrane protection, whereas 1% halothane had no effect on these cells. With 30 min of ischemia, 1% isoflurane was associated with significantly higher cellular LDH release and lower ATP concentration, suggesting increased cellular damage. Halothane (1%) was associated with only an increased ATP concentration in tubules exposed to 30 min of ischemia. Following 60 min of ischemia, halothane (4%) decreased LDH release by 45% (29.2 +/- 2.3% vs. 47.0 +/- 9.6% without halothane). Tubules exposed to halothane also had higher intracellular potassium and ATP concentrations, and increased respiratory rates. Halothane (2%) was less protective and only increased the ATP concentration. The release of LDH was not statistically different with or without 2% halothane.(ABSTRACT TRUNCATED AT 250 WORDS)