| Summary: | 碩士 === 國立臺灣大學 === 毒理學研究所 === 104 === Paraquat (PQ) is the trade name for 1,1′-dimethyl-4,4′-bipyridinium dichloride and has a molecular formula of C12H14Cl2N2. The name paraquat is derived from the para positions of quaternary ammonium. PQ is registered and used in more than 130 countries, both developing and developed, and is considered as one of the most widely-used herbicides. PQ intoxication is caused by ingestion (swallowing) or through damaged skin, but may also be inhaled. It can cause multiple-organ failure including the lungs, heart, kidneys, adrenal glands, central nervous system, liver, muscles, as well as damaging the skin and eyes. Among which, kidney is the most sensitive via the ingestion route and has the highest concentration of PQ accumulation compared with the other organs at any detection time. Under normal homeostatic conditions, almost 100% of unlethal amount of PQ can be excreted in the urine within 24 hours of PQ ingestion. Since PQ is mainly eliminated by the kidney, damage of this organ is of particular concern to the outcome of PQ intoxication. After PQ reaches the kidney cells, they undergo a process of redox-cycling which, at the expense of NAPDH, in turn leads to the production of reactive oxygen species (ROS) such as superoxide anion, O2•̄.
A study at the emergency departments of two medical centers in southern Taiwan between January 2001 and December 2002 showed that PQ intoxication contributed 18.9% of all herbicide intoxication, which had caused a 72.1% fatality. Previous findings suggest that high dosage of PQ can cause serious kidney damage, for example, acute kidney injury (AKI). This is characterized by the symptoms of proteinuria, pyuria, azotemia and hematuria between 24 and 96 hours post-intoxication. Oliguria has also been reported after serious PQ intoxication, and this led to damages to the glomerular capillary wall and eventually glomerulonephritis (GN). Studies have shown that GN is caused by an elevated level of ROS which damages glomerular cells such as the mesangial cells. The glomerular mesangial cells constitute approximately 30 – 40% of the total glomerular cell populations, which act as contractile smooth muscle cells to modulate glomerular blood flow and filtration, as well as the uptake and clearance of macromolecules through their phagocytic / autophagic properties. Due to these reasons, the mechanisms on how PQ induces autophagy signalling in mesangial cells remains a critical research topic that needs to be unravelled.
This study used mouse mesangial cells (MMCs) as a model of examining PQ-induced autophagic signalling. Our results showed that 145μM of PQ (IC50) decreases cell survival rate. Western blot analyses demonstrated that PQ-induced LC3-II expression that peaked at 3 hours post-treatment, accompanied with the high expression of Beclin-1 and Atg5-Atg12-Atg16L1 complex, but reduction in p62 expression. Our immunofluorescence data showed the formation of autophagosomes in MMCs after PQ treatment. Further, PQ triggered autophagic signalling events mediated by GSK-3β, ERK, AKT, mTOR and p70S6K due to ROS production. Interestingly, p-eIF2α and ERK signalling was activated by endoplasmic reticulum (ER) stress. The addition of rapamycin or 3-Methyladenine (3-MA) increased cell survival by 16% or decreased by 25%, repectively. Overall, our data suggest that autophagy serves a protective role in PQ treatment of MMCs, and this is activated by an acute ROS production and ER stress. These results provide fundamental molecular mechanisms in the role of autophagy signalling in MMCs, which can be considered as an early reference in the clinical setting of PQ-induced kidney injuries.
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