Calpain activation and not oxidative damage mediates L-2-chloropropionic acid-induced cerebellar granule cell necrosis.

Possible biochemical events involved in L-2-chloropropionic acid (L-CPA)-induced delayed cerebellar granule cell necrosis following N-methyl-D-aspartate activation were studied in vivo. We examined whether the calcium-sensitive proteolytic enzymes, the calpains, may be activated by L-CPA or whether the generation of excess quantities of cytotoxic free radicals may play a role in the neurotoxicity produced by oral administration of L-CPA (750 mg/kg, pH 7.0). Evidence for free radical-induced cellular damage was examined using biochemical approaches such as examining brains from L-CPA-treated rats for increased lipid peroxidation, DNA damage, or protein oxidation. Second, the ability of antioxidants to provide neuroprotective activity against L-CPA-induced neurotoxicity was examined in vivo. Western blotting using antibodies against spectrin (alpha-fodrin) demonstrated evidence for calpain (EC 3.4.22.17) activation in the cerebellum, but not in the cerebral cortex of L-CPA-treated rats at 36 and 48 hr after L-CPA dosing. In contrast, there was no evidence for oxidative damage to cerebellar proteins or lipids in L-CPA-treated rat brains compared to controls. We also could not find evidence for DNA damage using the TUNEL method for the detection of single- and/ or double-strand breakage in situ in L-CPA-treated brains. We examined whether a number of reported antioxidants may be effective against L-CPA-induced neurotoxicity. The aminosteroids U74389G and U83836E, the free radical scavengers 3-methyl-1-phenylpyrazolin-5-one and N-tert-butylphenylnitrone, and the iron chelator N-ethoxy-2-ethyl-3-hydroxypyridin-4-one were all ineffective in attenuating L-CPA neurotoxicity. We suggest that L-CPA-induced cerebellar necrosis is the result of calpain activation which results in the degradation of cytoskeletal proteins and other proteins necessary for cellular biochemistry. We could find no evidence of oxidative damage to cerebellar proteins, lipids, or DNA as a result of excess amounts of free radicals, and selective antioxidants were unable to provide neuroprotection against L-CPA neurotoxicity, suggesting that oxidative stress does not play a role in the granule cell necrosis.

The effect of implantation of osmotic pumps on rat thyroid hormone and testosterone levels in the plasma, an implication for tissue 'S' phase studies.

In order to monitor the effect of the procedures required to s.c. implant osmotic pumps into rats on plasma thyroid and testosterone hormone levels, male Fischer 344 rats (8-10 weeks old) were divided into six groups of 10 rats and the groups treated in the following manner: (1) controls housed 5 per cage; (2) controls housed individually; (3) animals anaesthetised for surgery and individually housed; (4) anaesthetised, sham operated and individually housed; (5) anaesthetised, s.c. implanted with osmotic pumps containing saline and individually housed; (6) anaesthetised, s.c. implanted with osmotic pumps containing 5-bromo 2-deoxyuridine (BRDU) and individually housed. Four days after performing the surgery the study was terminated and the level of hormones in the plasma determined by radio immunoassay (RIA). Tri-iodothyronine (T3) and thyroxine (T4) plasma levels (free and total) were significantly decreased with each additional step in the procedure used for the s.c. implantation of an osmotic pump containing BRDU, when compared with the individually housed controls. Similarly, testosterone plasma levels were significantly decreased by the s.c. implantation of osmotic pumps, implying a 'stress' response might occur following implantation. These observations might need to be considered by investigators when performing toxicological research which, as part of the study, uses osmotic pumps for the delivery of the nucleotide precursor required for monitoring cells in 'S' phase.

The effect of chlorinated paraffins on hepatic enzymes and thyroid hormones.

Male rats and mice were administered chlorinated paraffins (CPs) by daily gavage in corn oil for 14 days. Chlorowax 500C (short chain CP with 58% chlorination), Cereclor 56L (short chain CP with 56% chlorination) and Chlorparaffin 40G (medium chain CP with 40% chlorination) were the CPs studied at dose levels of 0, 10, 50, 100, 250, 500 and 1000 mg/kg for both rats and mice. The no effect levels for hepatic peroxisome proliferation for the above chemicals, as determined by the CN- insensitive palmitoyl co-enzyme A beta-oxidation (PCO) assay, were calculated as 184, 600 and 473 mg/kg and 180, 120 and 252 mg/kg for rats and mice, respectively, whilst those for percent liver weight/body weight were calculated as 74, 51 and 31 mg/kg and 215, 70 and 426 mg/kg for rats and mice, respectively. The short chain CPs were more potent peroxisome proliferators than the medium chain CP, with the mouse proving to be more responsive than the rat. Rats administered the highest dose of CPs showed a depressed plasma thyroxine (T4) level, with a concomitant increase in the plasma concentrations of thyroid stimulating hormone (TSH). The decreased plasma T4 levels appeared to be the result of increased T4 glucuronidation.

Sites of deposition of aqueous aerosols: a study of efficiency of delivery systems for lung ventilation imaging in man.

A study was made of the deposition of 99Tcm-DTPA aerosol in the components of a jet nebulizer-based aerosol production system. Three impaction devices were compared: a ball-bearing separator, a virtual impactor and a step separator. In addition a comparison was made of two types of tubing which carried aerosol from nebulizer to mouthpiece: corrugated and smooth-walled tubing. The retention of aerosol following inhalation was measured in five normal volunteers using different patterns of breathing. Using an aerosol production system which included a ball-bearing separator only a mean of 11% of the radioactivity loaded into the nebulizer was emitted as an aerosol. Some 18% remained in the ball-bearing separator. The ball-bearing and step separators produced similar total outputs (7% and 6% minimum), the step separator producing marginally higher mean output/min. The virtual impactor produced a lower output than the other two impactors studied, only 1%. A larger proportion of the aerosol output was deposited on corrugated tubing (7%) compared with smooth-walled tubing (1%). The retained fraction of the aerosol inhaled by subjects ranged from 16% to 43%. A higher fraction was retained when subjects inhaled deeply and held their breath for 10 s between each breath. The efficiency of radionuclide deposition from aerosol generator to patient ranged from 1.1% to 6% and was determined more by the retention in the subject than by choice of separator or tubing.