Resumen de Research into the mechanisms of Paraquat- induced multiple organ failure: Development and application of antidotes and antidotal pathways for the treatment of human poisonings
Paraquat (PQ) is a popular herbicide and it is probably one of the most studied pesticides. This interest is related not only to its relevance as a human poison, but also to its specific toxicological mechanisms, namely in the area of oxidative stress, making it an excellent research tool. PQ has a proven safety record when used properly for its intended purpose. Its safety in use can, at least in part, be explained by its lack of absorption either by inhalation or through the intact skin. This is because the spray droplets generated by agricultural equipment are too large in diameter (>5 μm) to be inhaled and because the skin provides an effective, impermeable barrier to the absorption of PQ. However, over the past 44 years, there have been numerous fatalities following accidental or deliberate ingestion of this weed-killer. Poisonings have received considerable attention in the medical, scientific, and popular press. The intoxication cases have often been dramatic because of the protracted and inexorable course of the illness and the absence of any effective antidote. Fatality rates have been over 50%. In addition, very little pre-clinical research has been successful in applying antidotes that shown to work in animal studies into clinical practice. PQ accumulates mainly in the lung (pulmonary concentrations can be six to ten times higher than those in the plasma), where it is retained even when blood levels start to decrease. The pulmonary effects can be readily explained by the participation of the polyamine transport system abundantly expressed in the membrane of alveolar cells type I, II and Clara cells. Further downstream at the toxicodynamic level, the molecular mechanism of PQ toxicity is based on redox cycling and intracellular oxidative stress generation.
During the last years, our research group has been a reference in the field of PQ toxicity to hospitals in the centre and north of Portugal. According, this dissertation was primarily aimed to describe a successful clinical case, regarding the intoxication of a 15- year-old girl by a presumed lethal dose of PQ. Besides the measures for decreasing PQ absorption and increasing its elimination, other protective procedures were applied aiming to reduce the production of reactive oxygen species (ROS), scavenge and repair ROS-induced lesions, and to reduce inflammation. The status-of-the-art concerning the biochemical and toxicological aspects of PQ poisoning and the pharmacological basis of the respective treatment protocol was presented. It was conducted an intensive and aggressive treatment, based on the high urinary and plasmatic PQ concentrations, and accordingly to the positive outcome, the therapeutic protocol followed could be a promising treatment of PQ human intoxications. Besides, were also good prognostic factors, young age, lesser degrees of leukocytosis and acidosis, and the absence of renal, hepatic, and pancreatic failures on admission after acute PQ poisoning.
In this thesis, the usefulness of the isolated rat lung was firstly explored. Such model was applied to characterize the toxicokinetic behaviour of PQ in this tissue after bolus injection under standard experimental conditions as well as to evaluate the influence of iso-osmotic replacement of sodium by lithium in the perfusion medium.
The obtained results showed that the isolated rat lung model is a useful technique for PQ toxicokinetic studies. It was also observed that sodium-depletion in the perfusion medium leads to a decreased uptake of PQ in the isolated rat lung although it seems that this condition does not contribute to improve the elimination of PQ once the herbicide reaches the extravascular structures of the tissue. In spite of, techniques of tissue isolation and perfusion offer an excellent alternative to characterize the kinetic profile for a tissue in a single animal and avoids the inter-individual variability in each single curve, leading as well to a corresponding reduction in curve replicates and hence a substantial reduction in the number of animals used (5-8 versus 50-80/tissue), soon we notice that PQ toxicity is a myriad of factors, together contributing to a death outcome and it would be better studied by in vivo approaches.
According to this desideratum, secondly it is described a procedure, through the induction of de novo synthesis of P-glycoprotein by the administration of a single high dose of dexamethasone (DEX) to Wistar rats, that leads to a remarkable decrease of PQ accumulation in the lung, together with an increase of its faecal excretion and a subsequent decrease of several biochemical and histopathological biomarkers of toxicity. The obtained results shown that DEX also ameliorated the biochemical and histological liver alterations induced by PQ in Wistar rats. On the other hand, these improvements were not observed in kidney and spleen of DEX treated rats. The sum of these effects was clearly positive, since it was observed an increased survival rate, which indicates that high dosage DEX treatment constitutes an important and valuable therapeutic tool to be used against PQ-induced toxicity.
Finally the role of the apoptosis, oxidative stress, platelet aggregation, nuclear factor (NF)-κB activation and fibrosis in PQ-induced lung toxicity, as well as the remarkable healing effects obtained by the administration of sodium salicylate (NaSAL, 200 mg/Kg i.p.), were assessed. The obtained results exceeded our best expectations since not only the toxicity was reverted but, most significantly, full survival of the PQintoxicated rats treated with NaSAL was observed. It may be postulated that NaSAL is the first real PQ antidote described with such degree of success.
Of note, the administrations of DEX and NaSAL were given two hours after intoxication of rats with PQ, a lag time that confers realism to be applied in humans, since this chronological time corresponds to longer biological time for humans and therefore this represent the probable time that passes between the herbicide ingestion and the begin of the medical cares.
In conclusion, the results of this dissertation suggest that high doses of DEX and/or NaSAL are therapeutic approaches with potential to be applied in humans, though, further pre-clinical studies are needed particularly those aimed to explain in more detail the mode of action of these interesting drugs in the protection against PQinduced lung damage.
