Development of Anxiolytic and Depression-like Behavior in Mice Infected with Mycobacterium lepraemurium.

Murine leprosy is a systemic infectious disease of mice caused by Mycobacterium lepraemurium (MLM) in which the central nervous system (CNS) is not infected; nevertheless, diseased animals show measurable cognitive alterations. For this reason, in this study, we explored the neurobehavioral changes in mice chronically infected with MLM. BALB/c mice were infected with MLM, and 120 days later, the alterations in mice were evaluated based on immunologic, histologic, endocrine, neurochemical, and behavioral traits. We found increases in the levels of IL-4 and IL-10 associated with high bacillary loads. We also found increase in the serum levels of corticosterone, epinephrine, and norepinephrine in the adrenal gland, suggesting neuroendocrine deregulation. Mice exhibited depression-like behavior in the tail suspension and forced swimming tests and anxiolytic behavior in the open field and elevated plus maze tests. The neurobehavioral alterations of mice were correlated with the histologic damage in the prefrontal cortex, ventral hippocampus, and amygdala, as well as with a blood-brain barrier disruption in the hippocampus. These results reveal an interrelated response of the neuroimmune--endocrinological axis in unresolved chronic infections that result in neurocognitive deterioration.

Altered phagocytic capacity due to acute exposure and long-term post-exposure to pesticides used for vector-borne disease as dengue.

Spinosad and temefos are widely used pesticides for chemical control of dengue vector-borne disease (Aedes aegypti). The aim of this study was to compare the effect of acute exposure (7 days) to spinosad (0.5 mg A.I. L-1) and temefos (10 mg A.I. L-1), concentrations used by the Mexican Ministry of Health, on phagocytic capacity (PC) of mononuclear cells of guppies fish (Poecilia reticulata), as well as to assess PC in fish, at 96 days after exposure to those pesticides. Obtained results indicated that spinosad did not alter PC, while an acute exposure to temefos significantly affected phagocytosis and this parameter was maintained downed even 96 days after the acute exposure, suggesting that the immunotoxic effects of temefos may be chronic.

Death of guppy fish (Poecilia reticulata) leukocytes induced by in vivo exposure to temephos and spinosad.

Temephos and spinosad are pesticides used for control of vector-borne diseases such as dengue, chikungunya and zika. However, the inadequate use of these substances has affected the health of non-target organisms. The aim of this study was to evaluate and compare, the effects of temephos and spinosad on leukocyte viability and death, using guppy fish (Poecilia reticulate) as a model organism. Guppies were exposed to temephos (10 mg/L) and spinosad (0.5 mg/L) for 7, 14, and 21 days. Afterwards, they were placed in pesticide-free fish tanks (7, 35, and 70 days) for recovery. The results showed that exposure to temephos caused leukocyte death, even at 35 days of recovery. Contrarily, the exposure to spinosad did not cause leukocyte death. This research show, for the first time, that a single dose of temephos causes apoptosis up to 56 days post-exposition, indicating that this pesticide induces chronic effects on immune response cells.

Alterations in the non-neuronal cholinergic system induced by in-vitro exposure to diazoxon in spleen mononuclear cells of Nile tilapia (O. niloticus).

Organophosphate pesticides as diazinon disrupt the neuroimmune communication, affecting the innate and adaptive immune response of the exposed organisms. Since the target molecule of diazinon is typically the acetylcholinesterase enzyme (AChE), the existence of a non-neuronal cholinergic system in leukocytes makes them susceptible to alterations by diazinon. Therefore, the aim of this work was to evaluate the activity of AChE, acetylcholine (ACh) concentration, and the expression of nicotinic ACh receptors (nAChR) and muscarinic ACh receptors (mAChR) in spleen mononuclear cells (SMNC) of Nile tilapia (O. niloticus) exposed in vitro to diazoxon, a diazinon metabolite. SMNC were exposed in-vitro to 1 nM, 1 µM, and 10 µM diazoxon for 24 h. The enzyme activity of AChE was then evaluated by spectrophotometry, followed by ACh quantification by ultra-performance liquid chromatography. Finally, mAChR and nAChR expression was evaluated by RT-qPCR. The results indicate that AChE levels are significantly inhibited at 1 and 10 µM diazoxon, while the relative expression of (M3, M4, and M5) mAChR and (ß2) nAChR is reduced significantly as compared against SMNC not exposed to diazoxon. However, ACh levels show no significant difference with respect to the control group. The data indicate that diazoxon directly alters elements in the cholinergic system of SMNC by AChE inhibition or indirectly through the interaction with AChR, which is likely related to the immunotoxic properties of diazinon and its metabolites.

In-vitro effect of diazoxon, a metabolite of diazinon, on proliferation, signal transduction, and death induction in mononuclear cells of Nile tilapia fish (Oreochromis niloticus).

The immune response of teleosts (bonefish) is altered by diazinon (DZN), an organophosphate pesticide. It has been suggested that such alteration is due to the extraneuronal cholinergic system in fish leukocytes that renders these cells a target of pesticides. Diazoxon (DZO), the oxon metabolite of DZN, has been attributed immunotoxic effects. Still, to date there are no reports on the effects of DZO upon parameters involved in the signaling cascade of immune response cells. Therefore, this work evaluated the effect of DZO on key parameters of cell signaling (intracellular Ca2+ flux, ERK 1/2 phosphorylation), cell proliferation, and antiproliferative processes (apoptosis, senescence, mitochondrial membrane potential) in spleen mononuclear cells of Nile tilapia fish. The results obtained show that DZO does not affect cell proliferation but causes a lack of response to stimulation with PMA and ionomycin to release intracellular calcium. In addition, it inhibits ERK 1/2 phosphorylation and causes loss of mitochondrial membrane potential, apoptosis, and senescence. These results suggest that the lack of cell response to release intracytoplasmic Ca2+ inhibits ERK which disrupts the mitochondrial membrane potential, leading to cell apoptosis and senescence. These findings prove that DZO significantly affects key parameters involved in the survival of immune response cells.

Effect of diazinon, an organophosphate pesticide, on signal transduction and death induction in mononuclear cells of Nile tilapia fish (Oreochromis niloticus).

Diazinon (DZN) is an organophosphate pesticide characterized by inhibiting the enzyme acetylcholinesterase (AChE) (E.C. 3.1.1.7), affecting the nervous system. There is currently enough evidence proving this pesticide also affects the immune response; however, the immunotoxicity mechanisms through which these substances exerts toxic effects remain unclear. For that reason, this work evaluated the effect of diazinon on the intracellular calcium flux, ERK1/2 phosphorylation (pERK1/2), apoptosis, senescence, and mitochondrial membrane potential (ΔΨm) in spleen mononuclear cells (SMNC) of Nile tilapia, a teleost fish of commercial and ecological relevance. The results obtained indicate that diazinon causes significant damage in all evaluated parameters, which play an essential role in intracytoplasmic signaling of immune cells, suggesting these signal pathways could be related with the immunotoxicity mechanism of these type of pesticides.

Muscarinic acetylcholine receptor expression in brain and immune cells of Oreochromis niloticus.

Nervous and immune systems maintain a bidirectional communication, expressing receptors for neurotransmitters and cytokines. Despite being well established in mammals, this has been poorly described in lower vertebrates as fishes. Experimental evidence shows that the neurotransmitter acetylcholine (ACh) regulates the immune response. In this research, we evaluated mRNA levels of muscarinic acetylcholine receptor (mAChR) in spleen mononuclear cells of Nile tilapia (Oreochromis niloticus) and compared the expression levels of immune cells with the brain. The mAChR subtypes (M2-M5A) were detected in both tissues, but mAChRs mRNA levels were higher in immune cells. This data have a potential use in biomedical and comparative immunology fields.

Phagocytosis and ROS production as biomarkers in Nile tilapia (Oreochromis niloticus) leukocytes by exposure to organophosphorus pesticides.

Organophosphorus pesticides (OPs) are broad-spectrum insecticides. One of the commonly used OPs is diazinon (DZN). The aim of this study was to evaluate the immunotoxic effect of DZN on phagocytic parameters of blood leukocytes using the teleost fish Oreochromis niloticus as a study model. For this purpose, fish were exposed in vivo to 0.97, 1.95 and 3.97 mg/L of DZN for 6 and 24 h. Our results indicated that phagocytic active cells decreased in fish exposed in vivo to 0.97 and 1.95 mg/L of DZN for 6 and 24 h. Regarding ROS production, H2O2 and O2- levels were higher on fish exposed to 1.95 mg/L for 6 and 24 h, while H2O2 production increased at 0.97 mg/L for 24 h. From this we can conclude that phagocytic parameters are sensitive to assess the effect of acute intoxication with organophosphorus pesticides on Nile tilapia.

Cholinergic alterations by exposure to pesticides used in control vector: Guppies fish (Poecilia reticulta) as biological model.

Spinosad and temephos are two of the most used pesticides in Mexico for the control of vector causing disease such as dengue, chikungunya and Zika. The aim of this study was to compare the neurotoxic effects of these two pesticides using guppy fish (Poecilia reticulata) as a model organism. Guppies were exposed for 7 and 21 days to technical grade temephos and spinosad at 1.0 and 0.07 g/L, respectively, (10 and 0.5 mg/L of active substance; concentrations recommended by the Ministery of Health of the State (Secretaría de Salud de Nayarit (SSN) Mexico)). Subsequently, acetylcholinesterase activity (AChE) and acetylcholine concentrations (ACh) in muscle tissue were determined. Temephos exposure decreased AChE activity and increased ACh concentration, whereas exposure to spinosad only increased ACh concentration. Though cholinergic alterations were more severe in fish exposed to temephos, both pesticides were equally lethal during the first seven days after exposure. Nonetheless, temephos was more lethal after 21 days.

Effects of diazinon on the lymphocytic cholinergic system of Nile tilapia fish (Oreochromis niloticus).

Fish rearing under intensive farming conditions can be easily disturbed by pesticides, substances that have immunotoxic properties and may predispose to infections. Organophosphorus pesticides (OPs) are widely used in agricultural activities; however, the mechanism of immunotoxicity of these substances is unclear. The aim of this study was to evaluate the effect of diazinon pesticides (OPs) on the cholinergic system of immune cells as a possible target of OP immunotoxicity. We evaluated ACh levels and cholinergic (nicotinic and muscarinic) receptor concentration. Additionally, AChE activity was evaluated in mononuclear cells of Nile tilapia (Oreochromis niloticus), a freshwater fish mostly cultivated in tropical regions around the world. The obtained results indicate that acute exposure to diazinon induces an increase in ACh concentration and a decrease in nAChR and mAChR concentrations and AChE activity in fish immune cells, This suggests that the non-neuronal lymphocytic cholinergic system may be the main target in the mechanism of OP immunotoxicity. This study contributes to the understanding of the mechanisms of immunotoxicity of pollutants and may help to take actions for animal health improvement.

Oxidative damage in gills and liver in Nile tilapia (Oreochromis niloticus) exposed to diazinon.

Agricultural activity demands the use of pesticides for plague control and extermination. In that matter, diazinon is one of the most widely used organophosphorus pesticides (OPs). Despite its benefits, the use of OPs in agricultural activities can also have negative effects since the excessive use of these substances can represent a major contamination problem for water bodies and organisms that inhabit them. The aim of this paper was to evaluate oxidative damage in lipids and proteins of Nile tilapia (Oreochromis niloticus) exposed acutely to diazinon (0.97, 1.95 and 3.95ppm) for 12 or 24h. The evaluation of oxidative damage was determined by quantifying lipid hydroperoxides (Fox method) and oxidized proteins (DNPH method). The data from this study suggest that diazinon induces a concentration-dependent oxidative damage in proteins, but not lipids, of the liver and gills of Nile tilapia. Furthermore, the treatment leads to a decrease in the concentration of total proteins, which can have serious consequences in cell physiology and fish development.

Assessment of pollution of the Boca de Camichin Estuary in Nayarit (Mexico) and its influence on oxidative stress in Crassostrea corteziensis oysters.

Boca de Camichin Estuary is one of the main producers of Crassostrea corteziensis oysters in Mexico, but the presence of pollutants can affect oyster production. Molluscs produce reactive oxygen species (ROS) in response to changes in the environment and pollution. These ROS induce oxidative damage in biomolecules. The main objective of this study was to evaluate pollution in the estuary and the subsequent oxidative stress in C. corteziensis oysters during the 2010 production cycle. For this aim, we performed monthly samplings in the oyster farms from January to May. We took water samples to quantify polycyclic aromatic hydrocarbon (PAH) and metal content; also, we evaluated oxidative damage (lipoperoxidation, lipidic hydroperoxides, protein oxidation) and enzyme activity (CAT, SOD, GPx, GST and AChE) in oyster gills. The results show the presence of Cu, Fe, Mn, naphthalene, benz[a]anthracene, pyrene, benz[a]pyrene and benzo[k]fluoranthene. On the other hand, AChE activity was not inhibited, which suggests that organophosphorus pollutants or carbamates were absent. Regarding oxidative stress, oysters from the estuary had oxidative damage in lipids, not proteins, and altered antioxidant enzyme activity, when compared to control organisms. Interestingly, we did not observe any correlation between the pollutants and the oxidative stress parameters evaluated in this study. Thus, we cannot rule out that a synergistic effect between the environmental variables and the pollutants is causing the oxidative stress in these oysters.

Modulation of immune response by organophosphorus pesticides: fishes as a potential model in immunotoxicology.

Immune response is modulated by different substances that are present in the environment. Nevertheless, some of these may cause an immunotoxic effect. In this paper, the effect of organophosphorus pesticides (frequent substances spilled in aquatic ecosystems) on the immune system of fishes and in immunotoxicology is reviewed. Furthermore, some cellular and molecular mechanisms that might be involved in immunoregulation mechanisms of organophosphorus pesticides are discussed.

Influence of the cholinergic system on the immune response of teleost fishes: potential model in biomedical research.

Fishes are the phylogenetically oldest vertebrate group, which includes more than one-half of the vertebrates on the planet; additionally, many species have ecological and economic importance. Fish are the first evolved group of organisms with adaptive immune mechanisms; consequently, they are an important link in the evolution of the immune system, thus a potential model for understanding the mechanisms of immunoregulation. Currently, the influence of the neurotransmitter acetylcholine (ACh) on the cells of the immune system is widely studied in mammalian models, which have provided evidence on ACh production by immune cells (the noncholinergic neuronal system); however, these neuroimmunomodulation mechanisms in fish and lower vertebrates are poorly studied. Therefore, the objective of this review paper was to analyze the influence of the cholinergic system on the immune response of teleost fish, which could provide information concerning the possibility of bidirectional communication between the nervous and immune systems in these organisms and provide data for a better understanding of basic issues in neuroimmunology in lower vertebrates, such as bony fishes. Thus, the use of fish as a model in biomedical research may contribute to a better understanding of human diseases and diseases in other animals.

Evaluation of pollution in Camichin estuary (Mexico): pro-oxidant and antioxidant response in oyster (Crassostrea corteziensis).

The physiological system of molluscs, particularly pro-oxidant and antioxidant mechanisms, could be altered by pollutants and induce disturbance on health status and productive parameters of aquatic organisms, such as oyster. Therefore, the aim of this study was to evaluate the chemical contamination in water (total metals and polycyclic aromatic hydrocarbons) and oxidative stress parameters in oysters (Crassostrea corteziensis) in Camichin estuary, located in Mexican Tropical Pacific. The results obtained showed the presence of arsenic, lead and zinc, as well as naphthalene, pyrene and benzo[a]pyrene in concentrations relatively higher than criteria established by local and international guidelines. Regarding the biomarkers of oxidative stress response (H2O2 and O2 concentration, catalase activity, lipid peroxidation, and hydroperoxide concentration), differences between oyster from estuary and control group were significant. These results indicate that these pollutants could be related with oxidative stress detected in oyster.

Organophosphate pesticides increase the expression of alpha glutathione S-transferase in HepG2 cells.

Chlorpyrifos and methyl parathion are among the most widely used insecticides in the world. Human populations are constantly exposed to low doses of both due to their extensive use and presence in food and drinking water. Glutathione S-transferase (GST) catalyzes the conjugation of glutathione on electrophilic substrates and is an important line of defense in the protection of cellular components from reactive species. GST alpha1 (GSTA1) is the predominant isoform of GST expressed in the human liver; thus, determining the effect of insecticides on GSTA1 transcription is very important. In the present study, we analyzed the effects of methyl parathion and chlorpyrifos on GSTA1 gene expression in HepG2 cells using real time PCR, and activity and immunoreactive protein assays. The results demonstrated that exposure to methyl parathion and chlorpyrifos increased the level of GSTA1 mRNA, GSTA1 immunoreactive protein and GST activity relative to a control. These results demonstrated that these insecticides can increase the expression of GSTA1. In conclusion, HepG2 cell cultures treated with methyl parathion and chlorpyrifos could be a useful model for studying the function of GSTA1 and its role in the metabolism of xenobiotics in the liver.

Phytoremediatory effect and growth of two species of Ocimum in endosulfan polluted soil.

Endosulfan is a hazardous organochlorine pesticide banned or restricted in several countries. However, it has been found in the environment and in animal samples. To study a potential way to bioremediate soils contaminated with this pesticide, two plant species of the genus Ocimum were studied: Ocimum basilicum L. and Ocimum minimum L., since they are economically feasible and well adapted to the climatic conditions of the Nayarit zone (Mexican pacific coast). Young plants were transplanted into soil experimentally polluted with endosulfan. Growth of both species was not affected by endosulfan, the plants grew, flourished, and produced seeds; 30 days later, endosulfan concentration was lower in the soil with O. basilicum than in the soil without plants. On day 90, no differences in endosulfan concentrations were found between soil with or without O. minimum. At day 1, plants in the polluted soil showed lipoperoxidation, as measured by thiobarbituric acid-reactive species (TBARS). Interestingly, a higher TBARS value was observed at day 3 in transplanted plants as compared to non-transplanted plants. In conclusion, both species can endure endosulfan pollution (as high as 1 g kg(-1)) in soils. O. basilicum seems to be an adequate candidate for bioremediation of soils polluted with endosulfan.

Acetylcholinesterase and metallothionein in oysters (Crassostrea corteziensis) from a subtropical Mexican Pacific estuary.

Substantial efforts have been devoted to developing and applying biomarkers for ecological risk assessment. Bivalve mollusks, such as mussels and oysters, are commonly used in environmental monitoring programs because of their wide geographical distribution, great sensitivity to environmental pollutants, and ability to accumulate anthropogenically derived chemicals at a high rate. Acetylcholinesterase (AChE) activity and metallothionein (MT's) content are representative specific biomarkers that indicate the presence of anticholinesterasic compounds (like organophosphorus and carbamate pesticides) and metals, respectively. The aim of this study was to evaluate AChE activity and MT's content in Crassostrea corteziensis from Boca de Camichín estuary. The results obtained here showed that AChE activity was 65% lower in oysters from Boca de Camichín than in control organisms. In contrast, MT's content in collected organisms was not statistically different from that in control organisms. AChE activity and MT's content in oysters could be used as early biomarkers of effects and exposure to pesticides and heavy metals, respectively, in aquatic environments.

Immunologic parameters evaluations in Nile tilapia (Oreochromis niloticus) exposed to sublethal concentrations of diazinon.

Fish resistance to microorganisms depends basically on the immune response. Although there are several studies on the diazinon mammalian immunotoxicity, in the case of fish there are only few. The aim of present study was to evaluate the effect of diazinon on immunological parameters (relative spleen weight, splenocytes count, lysozyme activity, respiratory burst and IgM concentration) in Nile tilapia. Diazinon at sublethal concentrations (0.39 and 0.78 mg/L) did not alter RSW, splenocytes count or lysozyme activity. However, at the highest concentration tested (1.96 mg/L) diazinon significantly increased respiratory burst and IgM concentration. In summary, diazinon (and perhaps other pesticides) could alter immunological response and induce oxidative stress.

Effect of sub-lethal concentrations of endosulfan on phagocytic and hematological parameters in Nile tilapia (Oreochromis niloticus).

The effect of endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepin-3-oxide), an organochlorine pesticide, was evaluated on phagocytic (phagocytic index and percentage of active cells) and hematological parameters in Nile tilapia. Experimental data showed that an acute exposure to endosulfan (4.0 and 7.0 microg/L) induces a significant decrease in the phagocytic index and the percentage of active cells in peripherical blood of Nile tilapia. However, hemoglobin concentration (Hb), hematocrit (Hto), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were not significantly altered in fish exposed to endosulfan compared with control group.