The role of nitric oxide in paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line.
Paraquat (PQ) is a well-known pneumotoxicant that exerts its toxic effect by elevating intracellular levels of superoxide. In addition, production of pro-inflammatory cytokines has possibly been linked to PQ-induced inflammatory processes through reactive oxygen species (ROSs) and nitric oxide (NO). However, the role of NO in PQ-induced cell injury has been controversial. To explore this problem, we examined the effect of NO on A549 cells by exposing them to the exogenous NO donor NOC18 or to cytokines; tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma, as well as PQ. Although the exogenous NO donor on its own had no effect on the release of lactate dehydrogenase (LDH), remarkable release was observed when the cells were exposed to high concentrations of NOC18 and PQ. This cellular damage caused by 1 mM NOC18 plus 0.2 mM PQ was ascertained by phase contrast microscopy. On the other hand, NO derived from 25-50 microM NOC18 added into the medium improved the MTT reduction activity of mitochondria, suggesting a beneficial effect of NO on the cells. Incubation of A549 cells with cytokines increased in inducible NO synthase (iNOS) expression and nitrite accumulation, resulting in LDH release. PQ further potentiated this release. The increase in nitrite levels could be completely prevented by NOS inhibitors, while the leakage of LDH was not attenuated by the inhibition of NO production with them. On the other hand, ROS scavenging enzymes, superoxide dismutase and catalase, inhibited the leakage of LDH, whereas they had no effect on the increase in the nitrite level. These results indicate that superoxide, not NO, played a key role in the cellular damage caused by PQ/cytokines. Our in vitro models demonstrate that NO has both beneficial and deleterious actions, depending on the concentrations produced and model system used.
Tomita
M
M
Departments of Legal Medicine, Kawasaki Medical School, Kurashiki 701-0192, Japan. tomita@bcc.kawasaki-m.ac.jp
Okuyama
T
T
Ishikawa
T
T
Hidaka
K
K
Nohno
T
T
D000230:Adenocarcinoma / Q000473:pathology*; D015415:Biomarkers; D002374:Catalase / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006540:Herbicides / Q000494:pharmacology* / Q000506:poisoning; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008168:Lung / Q000187:drug effects* / Q000378:metabolism; D008175:Lung Neoplasms / Q000473:pathology*; D008928:Mitochondria / Q000187:drug effects; D009363:Neoplasm Proteins / Q000096:biosynthesis; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D009603:Nitroso Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D010269:Paraquat / Q000494:pharmacology* / Q000506:poisoning; D013482:Superoxide Dismutase / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects*
D015415:Biomarkers; D004791:Enzyme Inhibitors; D006540:Herbicides; C093285:NOC 18; D009363:Neoplasm Proteins; D020030:Nitric Oxide Donors; D009573:Nitrites; D009603:Nitroso Compounds; D013481:Superoxides; D009569:Nitric Oxide; D007770:L-Lactate Dehydrogenase; D002374:Catalase; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D013482:Superoxide Dismutase; D010269:Paraquat