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Paraquat-induced membrane dysfunction in pulmonary microvascular endothelial cells
M Tsukamoto1, Y Tampo, M Sawada
1Division of Environmental Hygiene, Hokkaido College of Pharmacy, Otaru, Japan.
Abstract:
Membrane dysfunction monitored by lactate dehydrogenase release from cultured pulmonary microvascular endothelial cells of pigs, which were exposed to paraquat at different concentrations (0.1-2 mM), was examined. Paraquat caused a time-dependent increase in lactate dehydrogenase release. Lactate dehydrogenase releases after 72 hr, 32, 58, and 84% by 0.1, 0.5, and 2 mM paraquat, respectively, were well correlated with cell viability measured by cell adherence. In contrast, reductions of two tetrazolium compounds were depleted profoundly by 72 hr after exposure to 0.5 mM paraquat, suggesting depletion of intracellular reductive substances. Extracellular hydrogen peroxide began to significantly increase 56 hr or 32 hr after exposure to 0.5 mM or 1.5 mM paraquat, respectively, preceding the initial increase of lactate dehydrogenase release (64 hr by 0.5 mM or 48 hr by 1.5 mM). Lactate dehydrogenase release 72 hr after exposure to 0.5 mM paraquat was prevented strongly by catalase (1000 units/ml), but weakly by superoxide dismutase (1000 units/ml). These enzymes failed to restore the reduced acid phosphatase activity. Also, 0.1 mM desferal or alpha,alpha'-dipyridyl protected lactate dehydrogenase release. Similarly, 1 mM thiourea or dimethylthiourea, and 0.5 mM alpha-tocopherol or trolox, were effective, but diethylenetriaminepentaacetic acid (0.1 mM) and probucol (5 or 10 microM) were ineffective. Exposure of 0.5 or 1.5 mM paraquat suppressed levels of lipid peroxidation. These results indicate that membrane dysfunction by paraquat is ascribed to an iron-catalyzed reaction of extracellularly increased hydrogen peroxide. A deleterious species for the membrane dysfunction is discussed.
Insights
Paraquat exposure damages pulmonary endothelial cells, increasing lactate dehydrogenase release and decreasing cell viability. This membrane dysfunction is linked to an iron-catalyzed reaction involving extracellular hydrogen peroxide.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- Pulmonary microvascular endothelial cells (PMECs) are vital for lung function.
- Paraquat is a herbicide known for its toxicity.
- Understanding paraquat's cellular mechanisms is crucial for mitigating its effects.
Purpose of the Study:
- To investigate the mechanism of paraquat-induced membrane dysfunction in porcine PMECs.
- To identify the reactive oxygen species involved in paraquat toxicity.
- To evaluate potential protective agents against paraquat-induced cell damage.
Main Methods:
- Cultured porcine PMECs were exposed to varying concentrations of paraquat.
- Lactate dehydrogenase (LDH) release was measured to assess membrane integrity.
- Cell viability was assessed using cell adherence and tetrazolium compound reduction.
- Extracellular hydrogen peroxide levels were monitored.
- The effects of antioxidants (catalase, superoxide dismutase, desferal, alpha,alpha'-dipyridyl, thiourea, alpha-tocopherol) were evaluated.
Main Results:
- Paraquat induced a time- and dose-dependent increase in LDH release, correlating with reduced cell viability.
- Paraquat exposure led to a profound depletion of intracellular reductive substances.
- Extracellular hydrogen peroxide increased significantly before LDH release.
- Catalase and iron chelators (desferal, alpha,alpha'-dipyridyl) protected against LDH release, while superoxide dismutase had a weaker effect.
- Lipid peroxidation levels were suppressed by paraquat exposure.
Conclusions:
- Paraquat-induced membrane dysfunction in PMECs is primarily mediated by an iron-catalyzed reaction of extracellular hydrogen peroxide.
- This mechanism contributes to the observed cellular damage and loss of viability.
- Targeting iron-catalyzed reactions may offer a therapeutic strategy against paraquat toxicity.