| Summary: | 博士 === 國立中興大學 === 獸醫學系 === 92 === The purpose of this work is to evaluate the pulmonary toxicity and its mechanism of purified thuringiensin, a potential pesticide, in Sprague-Dawley rats. Rats were intratracheally instillated with 0, 0.4, 0.8, 1.6, 3.2, 6.4 and 9.6 mg/kg body weight of thuringiensin. The results indicated that the acute pulmonary LD50 of thuringiensin for rats was 4.4 mg/kg. The total number of inflammatory cells and lactate dehydrogenase (LDH) activity in the bronchoalveolar lavage fluid (BALF) increased in a dose-dependent manner after thuringiensin instillation. In the time course study, thuringiensin (1.6 mg/kg)-treated animals showed a significant increase in the lung weights, lung hydroxyproline levels and fibronectin levels in BALF. The total number of cells, total protein concentrations and LDH activity in BALF showed a significant increase after 1-7 days treatment. The treated rats presented abnormal histology including distributed inflammation in the bronchioles and alveoli, bronchial cellular necrosis on day 1 and 2, and areas of septal thickening with cellular infiltration and collagen deposit in the interstitial and alveolar spaces on day 4 to 56. Rat lung weights or BALF malondialdehyde (MDA) contents were shown a significant increase in a dose-related manner at 4 hours after treated with thuringiensin (0, 0.8, 1.6 and 3.2 mg/kg). But the alkaline phosphatase (AKP), antioxidant defense superoxide dismutase (SOD) activity and GSH content in lung showed a significant decrease a dose-related manner. The proinflammatory cytokines interleukin-1 (IL-1) or tumor necrosis factor- (TNF-) in lung showed a significant increase in a dose-related manner or a significant increase in 3.2 mg/kg. Additionally, the intensity matrix metalloproteinases (MMPs) (primary MMP-9 or MMP-2 enzyme) contents or activity in BALF was significantly increased in a dose-related manner.
The effect of thuringiensin on cytotoxicity or cyclic adenosine monophosphate (cAMP) production by using Chinese hamster lung fibroblast V79 cells was performed. The median lethal concentration (LC50) of thuringiensin following 24 hours of incubation was about 9.0 mM. The activity of extracellular LDH was increased in dose-dependently by thuringiensin at concentrations of 0.25-4 mM. The addition of ATP did not reduce the cytotoxicity of thuringiensin in culture of Chinese hamster lung fibroblast V79 cells. The cAMP levels were shown dose-dependently in lung fibroblast V79 cells. Furthermore, the cAMP levels of rat cerebral cortical membrane preparation were elevated dose-dependently by thuringiensin, due to the activation of basal adenylate cyclase (AC) activity. Thuringiensin also activated basal activity of the lung AC or a commercial AC from E. coli. However, the forskolin-stimulated adenylate cyclase activity in lung or rat cerebral cortex membrane preparation was inhibited by thuringiensin or AC inhibitor (MDL-12330A), thus cAMP production decreased.
Based on those results, intratracheal instillation of above 0.8 thuringiensin mg/kg caused significant pulmonary toxicity in rats. Thuringiensin could direct injury the lung epithelial, cause epithelial or macrophage cell to release the proinflammatory cytokines (IL-1 or TNF-), and cause reactive oxygen species status to injury the lung. The thuringiensin also could induce cytotoxicity, increase LDH activity of extracellular levels, increase cAMP concentrations in V79 cells. Comparing the dose-dependent effects of thuringiensin on the basal and forskolin-stimulated AC activity, thuringiensin can be regarded as a partial agonist. Thuringiensin could activate basal AC activity and increase cAMP concentrations in rat lung. The cAMP could inhibit the GSH synthesis. In fact, changes in the lung oxidant or antioxidant status may play an import role in the thuringiensin-induced pulmonary toxicity.
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