Ricardo Jorge Dinis-Oliveira

Paraquat Poisonings: Mechanisms of Lung Toxicity, Clinical Features, and Treatment

Paraquat dichloride (methyl viologen; PQ) is an effective and widely used herbicide that has a proven safety record when appropriately applied to eliminate weeds. However, over the last decades, there have been numerous fatalities, mainly caused by accidental or voluntary ingestion. PQ poisoning is an extremely frustrating condition to manage clinically, due to the elevated morbidity and mortality observed so far and due to the lack of effective treatments to be used in humans. PQ mainly accumulates in the lung (pulmonary concentrations can be 6 to 10 times higher than those in the plasma), where it is retained even when blood levels start to decrease. The pulmonary effects can be 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 main molecular mechanism of PQ toxicity is based on redox cycling and intracellular oxidative stress generation. With this review we aimed to collect and describe the most pertinent and significant findings published in established scientific publications since the discovery of PQ, focusing on the most recent developments related to PQ lung toxicity and their relevance to the treatment of human poisonings. Considerable space is also dedicated to techniques for prognosis prediction, since these could allow development of rigorous clinical protocols that may produce comparable data for the evaluation of proposed therapies.

Pesticides exposure as etiological factors of Parkinson's disease and other neurodegenerative diseases—A mechanistic approach

The etiology of most neurodegenerative disorders is multifactorial and consists of an interaction between environmental factors and genetic predisposition. The role of pesticide exposure in neurodegenerative disease has long been suspected, but the specific causative agents and the mechanisms underlying are not fully understood. For the main neurodegenerative diseases such as Parkinsons disease, Alzheimers disease and amyotrophic lateral sclerosis there are evidences linking their etiology with long-term/low-dose exposure to pesticides such as paraquat, maneb, dieldrin, pyrethroids and organophosphates. Most of these pesticides share common features, namely the ability to induce oxidative stress, mitochondrial dysfunction, α-synuclein fibrillization and neuronal cell loss. This review aims to clarify the role of pesticides as environmental risk factors in genesis of idiopathic PD and other neurological syndromes. For this purpose, the most relevant epidemiological and experimental data is highlighted in order to discuss the molecular mechanisms involved in neurodegeneration.

Hair as an alternative matrix in bioanalysis

Alternative matrices are steadily gaining recognition as biological samples for toxicological analyses. Hair presents many advantages over traditional matrices, such as urine and blood, since it provides retrospective information regarding drug exposure, can distinguish between chronic and acute or recent drug use by segmental analysis, is easy to obtain, and has considerable stability for long periods of time. For this reason, it has been employed in a wide variety of contexts, namely to evaluate workplace drug exposure, drug-facilitated sexual assault, pre-natal drug exposure, anti-doping control, pharmacological monitoring and alcohol abuse. In this article, issues concerning hair structure, collection, storage and analysis are reviewed. The mechanisms of drug incorporation into hair are briefly discussed. Analytical techniques for simultaneous drug quantification in hair are addressed. Finally, representative examples of drug quantification using hair are summarized, emphasizing its potentialities and limitations as an alternative biological matrix for toxicological analyses.

Profiling of RNA Degradation for Estimation of Post Morterm Interval

An estimation of the post mortem interval (PMI) is frequently touted as the Holy Grail of forensic pathology. During the first hours after death, PMI estimation is dependent on the rate of physical observable modifications including algor, rigor and livor mortis. However, these assessment methods are still largely unreliable and inaccurate. Alternatively, RNA has been put forward as a valuable tool in forensic pathology, namely to identify body fluids, estimate the age of biological stains and to study the mechanism of death. Nevertheless, the attempts to find correlation between RNA degradation and PMI have been unsuccessful. The aim of this study was to characterize the RNA degradation in different post mortem tissues in order to develop a mathematical model that can be used as coadjuvant method for a more accurate PMI determination. For this purpose, we performed an eleven-hour kinetic analysis of total extracted RNA from murines visceral and muscle tissues. The degradation profile of total RNA and the expression levels of several reference genes were analyzed by quantitative real-time PCR. A quantitative analysis of normalized transcript levels on the former tissues allowed the identification of four quadriceps muscle genes (Actb, Gapdh, Ppia and Srp72) that were found to significantly correlate with PMI. These results allowed us to develop a mathematical model with predictive value for estimation of the PMI (confidence interval of ±51 minutes at 95%) that can become an important complementary tool for traditional methods.

Postmortem Analyses Unveil the Poor Efficacy of Decontamination, Anti-Inflammatory and Immunosuppressive Therapies in Paraquat Human Intoxications

Background Fatalities resulting from paraquat (PQ) self-poisonings represent a major burden of this herbicide. Specific therapeutic approaches have been followed to interrupt its toxic pathway, namely decontamination measures to prevent PQ absorption and to increase its excretion from organism, as well as the administration of anti-inflammatory and immunosuppressive drugs. Until now, none of the postmortem studies resulting from human PQ poisonings have assessed the relationship of these therapeutic measures with PQ toxicokinetics and related histopathological lesions, these being the aims of the present study. Methodology/Principal Findings For that purpose, during 2008, we collected human fluids and tissues from five forensic autopsies following fatal PQ poisonings. PQ levels were measured by gas chromatography-ion trap mass spectrometry. Structural inflammatory lesions were evaluated by histological and immunohistochemistry analysis. The samples of cardiac blood, urine, gastric and duodenal wall, liver, lung, kidney, heart and diaphragm, showed quantifiable levels of PQ even at 6 days post-intoxication. Structural analysis showed diffused necrotic areas, intense macrophage activation and leukocyte infiltration in all analyzed tissues. By immunohistochemistry it was possible to observe a strong nuclear factor kappa-B (NF-κB) activation and excessive collagen deposition. Conclusions/Significance Considering the observed PQ levels in all analyzed tissues and the expressive inflammatory reaction that ultimately leads to fibrosis, we conclude that the therapeutic protocol usually performed needs to be reviewed, in order to increase the efficacy of PQ elimination from the body as well as to diminish the inflammatory process.

An effective antidote for paraquat poisonings: the treatment with lysine acetylsalicylate.

Sodium salicylate (NaSAL) has been shown to have a multifactorial protection mechanism against paraquat (PQ)-induced toxicity, due to its ability to modulate inflammatory signalling systems, to prevent oxidative stress and to its capacity to chelate PQ. Considering that currently there is no pharmaceutical formulation available for parenteral administration of NaSAL, the aim of the present study was to evaluate the antidotal feasibility of a salicylate prodrug, lysine acetylsalicylate (LAS), accessible for parenteral administrations. PQ was administered to Wistar rats by gavage (125mg/kg of PQ ion) and the treatment was performed intraperitoneally with different doses (100, 200 and 400mg/kg of body weight) of LAS. Survival rate was followed during 30 days and living animals at this endpoint were sacrificed for lung, kidney, liver, jejune and heart histological analysis. It was shown, that the salicylate prodrug, LAS, available in a large number of hospitals, is also effective in the treatment of PQ intoxications. From all tested LAS doses, 200mg/kg assured animals full survival. Comparatively to 60% of mortality observed in PQ only exposed animals, the lethality was higher (80%) in the group that received 400mg/kg of LAS 2h after PQ administration. The dose of 100mg/kg of LAS showed only a modest protection (60% of survival). Collagen deposition was observed by histological analysis in survived animals of all experimental groups, being less pronounced in animals receiving 200mg/kg of LAS, reinforcing the importance of this dose against tissue damage induced by PQ. The results allow us to suggest that LAS should be considered in the hospital treatment of PQ poisonings.

Chronic exposure to ethanol exacerbates MDMA-induced hyperthermia and exposes liver to severe MDMA-induced toxicity in CD1 mice

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) is an amphetamine derivative drug with entactogenic, empathogenic and hallucinogenic properties, commonly consumed at rave parties in a polydrug abuse pattern, especially with cannabis, tobacco and ethanol. Since both MDMA and ethanol may cause deleterious effects to the liver, the evaluation of their putative hepatotoxic interaction is of great interest, especially considering that most of the MDMA users are regular ethanol consumers. Thus, the aim of the present study was to evaluate, in vivo, the acute hepatotoxic effects of MDMA (10mg/kg i.p.) in CD-1 mice previously exposed to 12% ethanol as drinking fluid (for 8 weeks). Body temperature was continuously measured for 12h after MDMA administration and, after 24h, hepatic damage was evaluated. The administration of MDMA to non pre-treated mice resulted in sustained hyperthermia, which was significantly increased in ethanol pre-exposed mice. A correspondent higher increase of hepatic heat shock transcription factor (HSF-1) activation was also observed in the latter group. Furthermore, MDMA administration resulted in liver damage as confirmed by histological analysis, slight decrease in liver weight and increased plasma transaminases levels. These hepatotoxic effects were also exacerbated when mice were pre-treated with ethanol. The activities of some antioxidant enzymes (such as SOD, GPx and Catalase) were modified by ethanol, MDMA and their joint action. The hepatotoxicity resulting from the simultaneous exposure to MDMA and ethanol was associated with a higher activation of NF-kappaB, indicating a pro-inflammatory effect in this organ. In conclusion, the obtained results strongly suggest that the consumption of ethanol increases the hyperthermic and hepatotoxic effects associated with MDMA abuse.

Paraquat research: do recent advances in limiting its toxicity make its use safer?

The use of the herbicide paraquat (1,1′‐dimethyl‐4,4′‐bipyridylium dichloride; PQ) has been fiercely challenged due to its severe acute toxicity, putative neurotoxicity after long‐term exposure and lack of antidotes. Breakthrough research on PQ is therefore required for an effective risk control and to allow a safer use of PQ in the future. The silencing or inhibition of quinone oxidoreductase 2, a NAD(P)H‐independent flavoenzyme, was shown to significantly attenuate PQ toxicity in vitro, in primary pneumocytes and astroglial U373 cells, and to strongly antagonize PQ‐induced systemic toxicity and animal mortality. The novel results reported in this issue of BJP, added to recent findings using sodium salicylate and lysine acetylsalicylate, in which full survival of PQ‐intoxicated rats was also achieved, open the door for new preventative and therapeutic strategies that may lead to safer use of this effective pesticide.

Metabolomics of drugs of abuse: a more realistic view of the toxicological complexity

Metabolomics (also known as metabolic profiling or metabonomics) is a relatively new field of ‘omics’ technology, representing genomics, transcriptomics and proteomics other approaches. Metabolomics is primarily concerned with the ‘global’ qualitative and quantitative characterization of small molecule metabolites of 100 samples/day is possible). In this regard, LC-MS has short history in metabolomics comparatively to GC-MS, but has becoming increasingly popular since it is able to analyze nonvolatile, temperaturesensitive and polar metabolites without the need of a derivatization step [4]. As a result, virtually all compounds can be analyzed by LC-MS. Besides the analytical technique, metabolomics also use multivariate statistical methods (e.g., principal component analysis, partial least-squares discriminate analysis, orthogonal partial least-squares discriminate analysis, clustering) to examine patterns in the data (without bias) that exhibit the maximum variance. Metabolomics entered in the area of toxicology for about 10–12 years ago, later showing a rapid growth aiming the comprehensive analysis of the urine, plasma and tissue extracts metabolome [5]. Recently, it has been attracted the interest of many clinical and forensic toxicology researchers for studying the all ‘picture’ of drugs of abuse, since it is an effective approach to examine xenobiotic metabolism as well as the associated metabolic effects. Overall, this editorial aims to highlight that mapping the biochemical changes after drugs of abuse exposure, can complement traditional approaches by revealing potential biomarkers of organ toxicity, discovering new metabolites in timeand dose-dependent manner and different pharmacodynamic targets, as well as by giving insights about the pathways implicated in the mechanism of action, adverse effects and variability of Metabolomics of drugs of abuse: a more realistic view of the toxicological complexity

Antioxidant Properties and Associated Mechanisms of Salicylates

The pharmacological action of salicylates has been historically related to their ability to inhibit cyclooxygenases, thereby blocking the synthesis of prostaglandins and thromboxane A2. On the other hand, several studies have suggested that salicylates have a multitude of cyclooxygenase-independent actions specially related with their antioxidant properties, which might contribute to the overall salutary effects of these compounds. Although salicylates are well-known antioxidants through their ability to scavenge hydroxyl radical, their antioxidant mechanisms of action have not been fully compiled and characterized. In this context, several mechanisms of action have been suggested, namely i) scavenging of hydroxyl radical and chelation of transition metals; ii) upregulation of nitric oxide; iii) increased synthesis of lipoxins; iv) inhibition of neutrophil oxidative burst; v) inhibition of NF-κB and AP-1 protein kinases; and vii) inhibiton of lectin-like oxidized LDL receptor-1. The newly discovered acetyl salicylic acid-triggered lipoxins probably play a key role in the maintenance of the oxidative stress balance. Furthermore, salicylates have shown to protect low-density lipoprotein from oxidation and elicit an inhibitory effect on the expression of lectin-like receptors on endothelial cells. This review aims to provide an overview of the various proposed antioxidant mechanisms of salicylates.