Cyclophilins modify their profile depending on the organ or tissue in a murine inflammatory model.

Inflammation is the leading subjacent cause of many chronic diseases. Despite several studies in the last decades, the molecular mechanism involving its pathophysiology is not fully known. Recently, the implication of cyclophilins in inflammatory-based diseases has been demonstrated. However, the main role of cyclophilins in these processes remains elusive. Hence, a mouse model of systemic inflammation was used to better understand the relationship between cyclophilins and their tissue distribution. To induce inflammation, mice were fed with high-fat diet for 10 weeks. In these conditions, serum levels of interleukins 2 and 6, tumour necrosis factor-α, interferon-ϒ, and the monocyte chemoattractant protein 1 were elevated, evidencing a systemic inflammatory state. Then, in this inflammatory model, cyclophilins and CD147 profiles in the aorta, liver, and kidney were studied. The results demonstrate that, upon inflammatory conditions, cyclophilins A and C expression levels were increased in the aorta. Cyclophilins A and D were augmented in the liver, meanwhile, cyclophilins B and C were diminished. In the kidney, cyclophilins B and C levels were elevated. Furthermore, CD147 receptor was also increased in the aorta, liver, and kidney. In addition, when cyclophilin A was modulated, serum levels of inflammatory mediators were decreased, indicating a reduction in systemic inflammation. Besides, the expression levels of cyclophilin A and CD147 were also reduced in the aorta and liver, when cyclophilin A was modulated. Therefore, these results suggest that each cyclophilin has a different profile depending on the tissue, under inflammatory conditions.

Reevaluation of the acute toxicity of palytoxin in mice: Determination of lethal dose 50 (LD50) and No-observed-adverse-effect level (NOAEL).

Palytoxin is an emergent toxin in Europe and one of the most toxic substances know to date. The toxin disrupts the physiological functioning of the Na+/K+-ATPase converting the enzyme in a permeant cation channel. Human intoxications by PLTX after consumption of contaminated fishery products are a serious health issue and can be fatal. Several reports have previously investigated the oral and intraperitoneal toxicity of PLTX in mice. However, in all cases short observation periods (24 and 48 h) after toxin administration were evaluated. In this work, single oral or intraperitoneal doses of PLTX were administered to healthy mice and surviving animals were followed up for 96 h. The data obtained here allowed us to calculate the oral and intraperitoneal lethal doses 50 (LD50) which were in the range of the values previously described. Surprisingly, the oral NOAEL for PLTX was more than 10 times lower than that previously described, a fact that indicates the need for the reevaluation of the levels of the toxin in edible fishery products.

Subacute immunotoxicity of the marine phycotoxin yessotoxin in rats.

Yessotoxin (YTX) is a marine phycotoxin produced by dinoflagellates and accumulated in filter feeding shellfish. YTX content in shellfish is regulated by many food safety authorities to protect human health, although currently no human intoxication episodes have been unequivocally related to YTX presence in food. The immune system has been proposed as one of the target organs of YTX due to alterations of lymphoid tissues and cellular and humoral components. The aim of the present study was to explore subacute immunotoxicity of YTX in rats by evaluating the haematological response, inflammatory cytokine biomarkers and the presence of YTX-induced structural alterations in the spleen and thymus. The results showed that repeated administrations of YTX caused a decrease of lymphocyte percentage and an increase of neutrophil counts, a reduction in interleukine-6 (IL-6) plasmatic levels and histopathological splenic alterations in rats after four intraperitoneal injections of YTX at doses of 50 or 70 µg/kg that were administered every 4 days along a period of 15 days. Therefore, for the first time, subacute YTX-immunotoxicity is reported in rats, suggesting that repeated exposures to low amounts of YTX might also suppose a threat to human health, especially in immuno-compromised populations.

In vivo cardiomyocyte response to YTX- and AZA-1-induced damage: autophagy versus apoptosis.

Yessotoxins (YTX) and azaspiracids (AZAs) are marine toxins produced by phytoplanktonic dinoflagellates that get accumulated in filter feeding shellfish and finally reach human consumers through the food web. Both toxin classes are worldwide distributed, and food safety authorities have regulated their content in shellfish in many countries. Recently, YTXs and AZAs have been described as compounds with subacute cardiotoxic potential in rats owed to alterations of the cardiovascular function and ultrastructural heart damage. These molecules are also well known in vitro inducers of cell death. The aim of this study was to explore the presence of cardiomyocyte death after repeated subacute exposure of rats to AZA-1 and YTX for 15 days. Because autophagy and apoptosis are often found in dying cardiomyocytes, several autophagic and apoptotic markers were determined by western blot in heart tissues of these rats. The results showed that hearts from YTX-treated rats presented increased levels of the autophagic markers microtubule-associated protein light chain 3-II (LC3-II) and beclin-1, nevertheless AZA-1-treated hearts evidenced increased levels of the apoptosis markers cleaved caspase-3 and -8, cleaved PARP and Fas ligand. Therefore, while YTX-induced damage to the heart triggers autophagic processes, apoptosis activation occurs in the case of AZA-1. For the first time, activation of cell death signals in cardiomyocytes is demonstrated for these toxins with in vivo experiments, which may be related to alterations of the cardiovascular function.

Subacute Cardiotoxicity of Yessotoxin: In Vitro and in Vivo Studies.

Yessotoxin (YTX) is a marine phycotoxin produced by dinoflagellates and accumulated in filter feeding shellfish. Although no human intoxication episodes have been reported, YTX content in shellfish is regulated by many food safety authorities due to their worldwide distribution. YTXs have been related to ultrastructural heart damage in vivo, but the functional consequences in the long term have not been evaluated. In this study, we explored the accumulative cardiotoxic potential of YTX in vitro and in vivo. Preliminary in vitro evaluation of cardiotoxicity was based on the effect on hERG (human ether-a-go-go related gene) channel trafficking. In vivo experiments were performed in rats that received repeated administrations of YTX followed by recordings of electrocardiograms, arterial blood pressure, plasmatic cardiac biomarkers, and analysis of myocardium structure and ultrastructure. Our results showed that an exposure to 100 nM YTX for 12 or 24 h caused an increase of extracellular surface hERG channels. Furthermore, remarkable bradycardia and hypotension, structural heart alterations, and increased plasma levels of tissue inhibitor of metalloproteinases-1 were observed in rats after four intraperitoneal injections of YTX at doses of 50 or 70 µg/kg that were administered every 4 days along a period of 15 days. Therefore, and for the first time, YTX-induced subacute cardiotoxicity is supported by evidence of cardiovascular function alterations related to its repeated administration. Considering international criteria for marine toxin risk estimation and that the regulatory limit for YTX has been recently raised in many countries, YTX cardiotoxicity might pose a health risk to humans and especially to people with previous cardiovascular risk.

Subacute Cardiovascular Toxicity of the Marine Phycotoxin Azaspiracid-1 in Rats.

Azaspiracids (AZAs) are marine toxins produced by Azadinium spinosum that get accumulated in filter feeding shellfish through the food-web. The first intoxication was described in The Netherlands in 1990, and since then several episodes have been reported worldwide. Azaspiracid-1, AZA-2, and AZA-3 presence in shellfish is regulated by food safety authorities of several countries to protect human health. Azaspiracids have been related to widespread organ damage, tumorogenic properties and acute heart rhythm alterations in vivo but the mechanism of action remains unknown. Azaspiracid toxicity kinetics in vivo and in vitro suggests accumulative effects. We studied subacute cardiotoxicity in vivo after repeated exposure to AZA-1 by evaluation of the ECG, arterial blood pressure, plasmatic heart damage biomarkers, and myocardium structure and ultrastructure. Our results showed that four administrations of AZA-1 along 15 days caused functional signs of heart failure and structural heart alterations in rats at doses ranging from 1 to 55 µg/kg. Azaspiracid-1 altered arterial blood pressure, tissue inhibitors of metalloproteinase-1 plasma levels, heart collagen deposition, and ultrastructure of the myocardium. Overall, these data indicate that repeated exposure to low amounts of AZA-1 causes cardiotoxicity, at doses that do not induce signs of other organic system toxicity. Remarkably, human exposure to AZAs considering current regulatory limits of these toxins may be dangerously close to clearly cardiotoxic doses in rats. These findings should be considered when human risk is estimated particularly in high cardiovascular risk subpopulations.

In vivo arrhythmogenicity of the marine biotoxin azaspiracid-2 in rats.

Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellate Azadinium spinosum that accumulate in several shellfish species. Azaspiracid poisoning episodes have been described in humans due to ingestion of AZA-contaminated seafood. Therefore, the contents of AZA-1, AZA-2 and AZA-3, the best-known analogs of the group, in shellfish destined to human consumption have been regulated by food safety authorities of many countries to protect human health. In vivo and in vitro toxicological studies have described effects of AZAs at different cellular levels and on several organs, however, AZA target remains unknown. Very recently, AZAs have been demonstrated to block the hERG cardiac potassium channel. In this study, we explored the potential cardiotoxicity of AZA-2 in vivo. The effects of AZA-2 on rat electrocardiogram (ECG) and cardiac biomarkers were evaluated for cardiotoxicity signs besides corroborating the hERG-blocking activity of AZA-2. Our results demonstrated that AZA-2 does not induce QT interval prolongation on rat ECGs in vivo, in spite of being an in vitro blocker of the hERG cardiac potassium channel. However, AZA-2 alters the heart electrical activity causing prolongation of PR intervals and the appearance of arrhythmias. More studies will be needed to clarify the mechanism by which AZA-2 causes these ECG alterations; however, the potential cardiotoxicity of AZAs demonstrated in this in vivo study should be taken into consideration when evaluating the possible threat that these toxins pose to human health, mainly for individuals with pre-existing cardiovascular disease when regulated toxin limits are exceeded.