Leukocyte Viability Research Articles

In Vitro and Dietary Effects of Chitin on Cellular and Humoral Immune Parameters of Catla, Catla catla (Hamilton)

AbstractImmunostimulants increase the immunocompetency and disease resistance of fish by enhancing immune responses. This study determined the in vitro and dietary effects of chitin on immune responses of catla, Catla catla. Catla head‐kidney leukocytes were cultured in complete RPMI‐1640 medium containing 0.01, 0.1, and 1.0 mg/mL of chitin. Leucocyte viability and different cellular immune responses, namely, respiratory burst, nitric oxide production, and leucocyte peroxidase content were analyzed in vitro. In the dietary experiment, chitin‐supplemented diets (0.5, 1.0, and 2.0% of feed) were fed to catla for 2 wk, and respiratory burst, serum lysozyme, and total protein contents were measured after 1 and 2 wk of feeding. Leucocytes incubated in vitro with chitin for 72 h maintained their viability. Chitin significantly (P < 0.05) enhanced all the immune responses in vitro. The lowest dose showed the best response. Dietary chitin also significantly stimulated all the immune parameters in a dose‐dependent manner with the highest dose showing the maximum responses. All the responses were higher after 1 wk of feeding and declined thereafter. The results suggest that chitin stimulates catla cellular and humoral immune responses in vitro and in vivo.

In situ experiments to assess effects of constraints linked to caging on ecotoxicity biomarkers of the three-spined stickleback (Gasterosteus aculeatus L.).

The aim of this study was to evaluate the effects of caging constraints on multiple fish biomarkers used during ecotoxicological studies (biometric data, immune and antioxidant systems, and energetic status). Two of these constraints were linked to caging: starvation and fish density in cages, and one in relation to the post-caging handling: a short transport. Three in situ experiments were conducted with three-spined sticklebacks (Gasterosteus aculeatus). The first experiment compared the effects of three densities (low, medium, and high). The second experiment compared effects of starvation in fish fed every two days with fish that were not fed. Finally comparisons between sticklebacks which have suffered a short car transport after caging and sticklebacks killed without preliminary transport were made. The lack of food had no effect on fish energetic reserves but negatively affected their condition index and their immune system. Transport and high density induced oxidative stress, defined as an overproduction of reactive oxygen species and a stimulation of the antioxidant system. These two constraints also harmed the leucocyte viability. In order not to have any impact on ecotoxicity biomarkers during in situ experiments, it is preferable to decrease fish density in cages, prevent transport before dissections, and feed fish when the caging lasts more than two weeks.

<b><em>In vitro</em> ZnCl<sub>2</sub> cytotoxicity and genotoxicity in human leukocytes: Zero-order kinetic cellular zinc influx

Zinc (Zn) is an essential trace element for cellular viability, but concentrations above physiologic level may lead to cellular damage. The purpose of the present study was to evaluate the in vitro ZnCl 2 genotoxicity and cytotoxicity in human leukocyte cells. This was assessed in an unprecedented way that correlated the level of intracellular Zn after cell exposition with the cellular damage. The exposure to increased Zn concentrations (2.5-20 µg mL -1 ), showed significantly reduced cellular leukocyte viability. However, significant DNA damages were observed only when the Zn exposure concentrations were from 10-20 µg mL -1 . The Zn intracellular levels found in leukocytes was from 72.25-268.9 ρg cell -1 , starting to induce cytotoxicity and genotoxicity at concentrations of 95.68 and 126.2 ρg cell -1 , respectively. The relationship between the exposure concentration and intracellular levels of Zn suggests that the influx of Zn, in the form of ZnCl 2 , occurs in human leukocytes under zero-order kinetics.

Essential oil from Ageratum fastigiatum reduces expression of the pro-inflammatory cytokine tumor necrosis factor-alpha in peripheral blood leukocytes subjected to in vitro stimulation with phorbol myristate acetate

Ageratum fastigiatum (Gardner) R.M. King & H. Rob., a member of the Asteraceae family popularly known in Brazil as “matapasto”, is indicated in folk medicine as anti-inflammatory and analgesic. Despite its popular use, little is known about its potential effect on the parameters involved in an inflammatory response. The objective of this study was to characterize the chemical composition of the essential oil from A. fastigiatum and to evaluate the frequency of tumor necrosis factor alpha and interferon gamma producing cells in peripheral blood lymphocytes stimulated with phorbol myristate acetate in the presence of essential oil from A. fastigiatum. Non-toxic concentrations of essential oil from A. fastigiatum were evaluated in cultures of peripheral blood leucocytes using the trypan blue exclusion assay by flow cytometry. GC–MS analysis revealed that the prevalent compounds identified in the essential oil from A. fastigiatum sample were α-pinene, limonene, trans-caryophyllene, α-humulene, caryophyllene oxide, 1,2-humulene-epoxide, 1,6-humulanodien-3-ol, and α-cadinol. Results showed that exposure to essential oil from A. fastigiatum at concentrations of 0.5×10−2 and 1×10−2μl/ml caused no alterations in leukocyte viability as compared to the control group. Both concentrations lowered the percentage of tumor necrosis factor alpha (+)-lymphocytes and neutrophils. There were no changes in the percentage of lymphocytes positive for the interferon gamma cytokine. Our results suggest that part of the anti-inflammatory activity attributed to A. fastigiatum may be due to the effect of some of its components in decreasing the number of cells that produce the pro-inflammatory cytokine tumor necrosis factor alpha.

Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid

Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10−9 vs. 10−5M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10−5M, but drops to 10−9M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

Comet assay in salivary leukocytes: evaluation of early effects of air pollution exposure in pre-school children.

Comet assay in salivary leukocytes: evaluation of early effects of air pollution exposure in pre-school children.

A novel model for studies of blood-mediated long-term responses to cellular transplants

Aims. Interaction between blood and bio-surfaces is important in many medical fields. With the aim of studying blood-mediated reactions to cellular transplants, we developed a whole-blood model for incubation of small volumes for up to 48 h.Methods. Heparinized polyvinyl chloride tubing was cut in suitable lengths and sealed to create small bags. Multiple bags, with fresh venous blood, were incubated attached to a rotating wheel at 37°C. Physiological variables in blood were monitored: glucose, blood gases, mono- and divalent cations and chloride ions, osmolality, coagulation (platelet consumption, thrombin-antithrombin complexes (TAT)), and complement activation (C3a and SC5b-9), haemolysis, and leukocyte viability.Results. Basic glucose consumption was high. Glucose depletion resulted in successive elevation of extracellular potassium, while sodium and calcium ions decreased due to inhibition of energy-requiring ion pumps. Addition of glucose improved ion balance but led to metabolic acidosis. To maintain a balanced physiological environment beyond 6 h, glucose and sodium hydrogen carbonate were added regularly based on analyses of glucose, pH, ions, and osmotic pressure. With these additives haemolysis was prevented for up to 72 h and leukocyte viability better preserved. Despite using non-heparinized blood, coagulation and complement activation were lower during long-term incubations compared with addition of thromboplastin and collagen.Conclusion. A novel whole-blood model for studies of blood-mediated responses to a cellular transplant is presented allowing extended observations for up to 48 h and highlights the importance of stringent evaluations and adjustment of physiological conditions.

In vitro effects of prostaglandin E2 on leucocytes from sticklebacks (Gasterosteus aculeatus) infected and not infected with the cestode Schistocephalus solidus

Many helminth parasites have evolved strategies to evade the immune response of their hosts, which includes immunomodulation. Prostaglandin E2 (PGE2) is one of the best-described immunomodulators in mammalian helminth parasite infections. We hypothesized that also in teleost fish anti-helminthic immune responses are regulated via PGE2. We used a model system consisting of the tapeworm Schistocephalus solidus and its host, the three-spined stickleback (Gasterosteus aculeatus), to investigate in vitro effects of PGE2 on head kidney leucocytes (HKL) derived from sticklebacks that were experimentally infected with S. solidus. PGE2 was tested alone or in combination with either S. solidus antigens or bacterial lipopolysaccharides (LPS). After in vitro culture, cell viability and changes in leucocyte subpopulations (granulocytes to lymphocytes ratios) were monitored by flow cytometry and HKL were tested for their capacity to produce reactive oxygen species (ROS) with a chemiluminescence assay. In short term (2 h) HKL cultures PGE2 did not change the total numbers of live HKL, but the production of ROS decreased significantly with high (0.1 μmol L−1) PGE2 concentrations. In long-term (96 h) cultures high PGE2 concentrations induced a sharp decrease of leucocytes viability, while low (0.1 pmol L−1) and intermediate (0.1 nmol L−1) concentrations of PGE2 caused elevated leucocyte viability compared to controls. This coincided with reduced ROS production in cultures with high PGE2 and elevated ROS production in cultures with low PGE2. Granulocyte to lymphocyte ratios increased with high PGE2 concentrations alone and in combination with S. solidus antigens and LPS, most prominently with HKL from S. solidus infected sticklebacks. The present study supports the hypothesis that PGE2 might be an immunomodulator in tapeworm–fish parasite–host interactions.

Effect of Tamarindus indica L. leaves' fluid extract on human blood cells

Tamarind leaves are edible; however, their saponin content could be toxic to human blood cells. In this article, the effect of tamarind leaf fluid extract (TFE) on human blood cells was evaluated by using several tests. Results revealed that TFE did not cause significant haemolysis on human red blood cells even at the lowest evaluated concentration (20 mg/mL). Blood protein denaturalisation ratio was consistently lower than in control at TFE concentrations greater than 40 mg/mL. Erythrocyte membrane damage caused by the action of oxidative H2O2 displayed a steady reduction with increasing TFE concentrations. In the reactive oxygen species (ROS) measurement by using flow cytometry assay, leucocyte viability was over 95% at tested concentrations, and a high ROS inhibition was also recorded. Protective behaviour found in TFE should be attributed to its polyphenol content. Thus, tamarind leaves can be regarded as a potential source of interesting phytochemicals.

Окислювальний стрес у лейкоцитах крові,про/антиоксидантний статус та жирнокислотний склад ліпідів підшлункової залози за експериментального гострого панкреатиту в щурів

In an experimental model of acute pancreatitis (AP) in rats no alteration in leukocyte's viability was found by flow cytometry as compared to control. After 1 day of AP production of reactive oxygen forms in granulocytes was increased more than 5 times, but after 3 days their level was decreased. Alterations of pro/antioxidant status and specific changes in the fatty acid composition in the pancreas were established. With the development of AP, the processes of lipids peroxidation were intensified while antioxidant system was altered, that was evidenced by inflammation in the pancreas. In these conditions, the increase of phospholipase A2 activity was accompanied by significant changes of fatty acid composition of the total lipids in the pancreas. This increased relative total content of saturated fatty acids, in particular myristic, palmitic and stearic acid increased, while the total content of polyunsaturated essential fatty acids omega-3 (linolenic, eicosapentaenoic, dokozapentayenoic, docosahexaenoic) decreased. The preparation containing omega-3 polyunsaturated fatty acids partially normalized the lipid and fatty acids composition as well as prooxidant-antioxidant system.

In vitro immunotoxicity of untreated and treated urban wastewaters using various treatment processes to rainbow trout leucocytes

Municipal effluents are known to impede the immune system of aquatic organisms. The purpose of this study was to examine the immunotoxicity of urban wastewaters before and after 6 treatment processes from 12 cities toward trout leucocytes. Freshly prepared trout leucocytes were exposed to increasing concentrations of solid phase (C18) extracts of wastewaters for 24 hr at 15°C. Immunocompetence was determined by following changes in leucocyte viability and the proportion of cells able to ingest at least one (immunoactivity) and at least three (immunoefficiency) fluorescent beads. The influents were treated by six different treatment strategies consisting of facultative aerated lagoons, activated sludge, biological aerated filter, biological nutrient removal, chemically-assisted physical treatment and trickling filter/solid contact. Water quality parameters of the wastewaters revealed that the plants effectively removed total suspended solids and reduced the chemical oxygen demand. The results revealed that the effluents' immunotoxic properties were generally more influenced by the properties of the untreated wastewaters than by the treatment processes. About half of the incoming influents decreased leucocyte viability while 4 treatment plants were able to reduce toxicity. The influents readily increased phagocytosis activity for 8/12 influents while it was decreased in 4/12 influents. This increase was abolished for 4/12 of the effluents using treatments involving biological and oxidative processes. In conclusion, municipal effluents have the potential to alter the immune system in fish and more research will be needed to improve the treatments of wastewaters to better protect the quality of the aquatic environment.

Effect of heat-treatment on nutritional and immune factors in bovine colostrum

Studies have demonstrated that heat-treating colostrum at 140QF (60°C) for 60 minutes results in a significant reduction in colostral bacteria counts with no overall reduction in colostral IgG concentrations. Preweaned calves fed HT colostrum have enhanced efficiency of absorption of IgG and reduced morbidity. Despite these benefits, limited research exists to describe whether the heat-treatment process harms other colostrum components. The objective of Study 1, described herein, was to describe the effect of heat-treatment on concentrations of several nutrient and immune factors in bovine colostrum. The objective of Study 2 was to describe the effects of heat-treatment on leukocyte viability, alkaline phosphatase (AP) enzyme activity, and IgG concentrations in bovine colostrum.

Optimization of RNA isolation and leukocyte viability in canid RNA expression studies

For RNA transcript analysis, RNA source and quality influence the resolution and representation of true gene expression. We evaluate collection and purification methodologies for isolating gray wolf (Canis lupus) mRNA from peripheral whole blood to maximize detection and quantification of low expression genes. We specifically examine ambient-temperature stabilization of RNA in whole blood at time of collection, subsequent isolation and RNA stabilization of leukocytes to remove globin RNA, and removal of ribosomal RNA (rRNA) using commercially available kits. Ambient temperature RNA stabilization and globin RNA removal were successful with robust mRNA extraction yields and quality, verified by quantitative (q)PCR of reference housekeeping genes. Rhodopsin gene mRNA, present in low levels in peripheral blood, was only detected by qPCR when leukocyte mRNA was additionally treated for ribosomal RNA removal.

Enumeration of viable CD34+ cells by flow cytometry in blood, bone marrow and cord blood: results of a study of the novel BD™ stem cell enumeration kit

Enumeration of CD34(+) cells in leukocyte-rich cell suspensions is important for clinical decision-making in stem cell transplantation. Single-platform flow cytometry assays offer the significant advantages of speed and reproducibility, and have therefore become the gold standard in stem cell enumeration. The clinical community has recently defined the need for stem cell enumeration kits that incorporate viability dyes. The purpose of this study was to evaluate a novel assay, BD Biosciences' (BD) stem cell enumeration kit (SCE kit(‡)), in relation to Beckman Coulter's (BC) commercially available BC Stem-Kit™. Fresh/freeze-thawed samples from leukapheresis, bone marrow and cord blood, and fresh normal/mobilized blood, were analyzed with both assays (simultaneous detection of side/forward scatter and three fluorescence signals) on two flow cytometry platforms, BD FACSCanto II and BD FACSCalibur. Results. Results from both assays were highly congruent, with an overall r(2) ≥ 0.99 (all specimen types included), a linear correlation across all CD34(+) cell frequencies and concentrations, and an almost ideal steepness of the trend line. Both assays functioned reliably. Being based on single-platform International Society of Hematotherapy and Graft Engineering (ISHAGE) guidelines and similar staining methods, both assays essentially come to identical results. For most specimen types, the viability of CD34(+) cells was equal to overall leukocyte viability. In summary, in the hands of an experienced technician, the BD™ SCE kit and the BC Stem-Kit are equivalent. The infrequent user might derive benefit from the fact that counting spheres are pre-pipetted into the Trucount tube for the SCE kit, making this assay less susceptible to pipetting inaccuracy.

Effects of polyamines on cellular innate immune response and the expression of immune-relevant genes in gilthead seabream leucocytes

It is well known that the polyamines spermidine and spermine, along with the diamine putrescine, are involved in many cellular processes and they are known to play an important role in the control of the innate immune response in higher vertebrates. However, to the best of our knowledge, no studies have focused on their immunological implications in other vertebrates, such as fish. For this reason, the effects of polyamines on the cellular innate immune response and immune-related gene expression were evaluated in vitro, using seabream head-kidney leucocytes (HKL). For this study, head-kidney leucocytes were incubated with the polyamines putrescine, spermine or spermidine (0.005 and 0.0025%) for 0.50, 1, 2 or 4 h. No significant effect was observed on either leucocyte viability or the innate cellular immune responses (peroxidase content and phagocytic and respiratory burst activities). The polyamines produced an increase in respiratory burst and phagocytic ability when leucocytes were incubated principally with putrescine (0.005 and 0.0025%) after 2 and 4 h of the experiment. Finally, the expression levels of immune-associated genes ( IgM, MHCIα, MHCIIα, C3, IL-1β, CD8, Hep, NCCRP-1, CSF-1 and TLR) were quantified by real-time PCR and some of them ( C3, MHCI, CD8, IgM and Hep) were up-regulated by the higher polyamine concentration. Further studies are needed to ascertain how polyamines control the immune system of seabream as well as which mechanisms are involved.

Melatonin Counteracts Alterations in Oxidative Metabolism and Cell Viability Induced by Intracellular Calcium Overload in Human Leucocytes: Changes with Age

Ageing is associated with an increased production of free radicals and alterations in the mechanisms of adaptation to oxidative stress. In fact, the free radical theory of ageing proposes that deleterious actions of free radicals are responsible for the functional deterioration associated with ageing. Moreover, a close relationship exists between calcium homeostasis and oxidative stress. The current work was aimed at proving that intracellular calcium overload induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and/or thapsigargin leads to oxidative stress. We additionally examined the effect of melatonin on the levels of reactive oxygen species (ROS) and cell viability in human leucocytes collected from young (20-30-year-old) and elderly (65-75-year-old) individuals under both basal and oxidative stress-induced conditions. Treatments with 10 nM FMLP and/or 1 microM thapsigargin induced a transient increase in cytosolic free-calcium concentration ([Ca(2 + )](c)) in human leucocytes due to calcium release from internal stores, and led in turn to oxidative stress, as assessed by intracellular ROS measurement. Non-treated leucocytes from aged individuals exhibited higher ROS levels and lower rates of cell survival when compared to leucocytes from young individuals. Similar results were obtained in FMLP and/or thapsigargin-treated leucocytes from elderly individuals when compared to those from the young individuals. Melatonin treatment significantly reduced both hydrogen peroxide (H(2)O(2)) and superoxide anion levels, likely due to its free-radical scavenging properties, and enhanced leucocyte viability in both age groups. Therefore, melatonin may be a useful tool for the treatment of disease states and processes where an excessive production of oxidative damage occurs.

Psychopharmacological Neuroprotection in Neurodegenerative Disease: Assessing the Preclinical Data

Psychopharmacological Neuroprotection in Neurodegenerative Disease: Assessing the Preclinical Data

Psychopharmacological Neuroprotection in Neurodegenerative Disease: Assessing the Preclinical Data

This manuscript reviews the preclinical in vitro, ex vivo, and nonhuman in vivo effects of psychopharmacological agents in clinical use on cell physiology with a view toward identifying agents with neuroprotective properties in neurodegenerative disease. These agents are routinely used in the symptomatic treatment of neurodegenerative disease. Each agent is reviewed in terms of its effects on pathogenic proteins, proteasomal function, mitochondrial viability, mitochondrial function and metabolism, mitochondrial permeability transition pore development, cellular viability, and apoptosis. Effects on the metabolism of the neurodegenerative disease pathogenic proteins alpha-synuclein, beta-amyloid, and tau, including tau phosphorylation, are particularly addressed, with application to Alzheimer's and Parkinson's diseases. Limitations of the current data are detailed and predictive criteria for translational clinical neuroprotection are proposed and discussed. Drugs that warrant further study for neuroprotection in neurodegenerative disease include pramipexole, thioridazine, risperidone, olanzapine, quetiapine, lithium, valproate, desipramine, maprotiline, fluoxetine, buspirone, clonazepam, diphenhydramine, and melatonin. Those with multiple neuroprotective mechanisms include pramipexole, thioridazine, olanzapine, quetiapine, lithium, valproate, desipramine, maprotiline, clonazepam, and melatonin. Those best viewed circumspectly in neurodegenerative disease until clinical disease course outcomes data become available, include several antipsychotics, lithium, oxcarbazepine, valproate, several tricyclic antidepressants, certain SSRIs, diazepam, and possibly diphenhydramine. A search for clinical studies of neuroprotection revealed only a single study demonstrating putatively positive results for ropinirole. An agenda for research on potentially neuroprotective agent is provided.

Immunocompetence and alterations in hepatic gene expression in rainbow trout exposed to CdS/CdTe quantum dots

Cadmium (Cd) tellurium quantum dots (QDs) are long-lived fluorescent nanocrystals that have the ability to produce potentially toxic reactive oxygen species at the surface of the nanoparticle and release toxic cadmium ions. The purpose of this study was to examine the sublethal effects of CdS/CdTe QDs and dissolved Cd on the immune system of rainbow trout. Changes in hepatic gene expression were also monitored to provide insight on the mode of action of both forms of cadmium. Oncorhynchus mykiss fish were exposed to increasing concentrations of CdS/CdTe or dissolved Cd (CdSO(4)) for 48 h at 15 degrees C. The anterior head kidney was analyzed for leukocyte count, viability and phagocytic activity. The livers were harvested and prepared for gene expression analysis using a DNA microarray comprised of 207 stress-related genes. An analysis of total Cd in the aquarium water revealed that the nominal concentrations corresponded well to the actual concentrations and that a small proportion (0.4%) of Cd in the QDs was found in the dissolved fraction. Exposure to QDs led to significant reductions in leukocyte counts, viability and both resting and active phagocytic activity. On a mass concentration basis, QDs were more potent than dissolved Cd in suppressing immunocompetence in rainbow trout. The analysis revealed that both forms of Cd were strong inducers of metallothionein and CP2K1 gene expressions, which are respectively involved in metal detoxification processes and xenobiotic transformation/inflammation conditions. The analysis revealed different modes of action for each form of Cd. For QDs, 25 genes specific to QDs and related to the immune endpoints were found. The genes were involved in inflammation, xenobiotic biotransformation and endocrine system (including the induction of vitellogenin and its receptor). The effect for dissolved Cd was narrower than for QDs, with 9 genes specific to dissolved Cd being strongly correlated with the observed effects on immunocompetence. The involved genes were binding and transport of various solutes (urea, sodium, potassium) and the complement system. The present study revealed that each form of Cd produced a different pattern of gene expression and lowered fish immunocompetence.

Allium sativum L. extract prevents methyl mercury-induced cytotoxicity in peripheral blood leukocytes (LS)

Adenosine deaminase (ADA) is involved in purine metabolism and plays a significant role in the immune system. The focus of this investigation was to examine the effects of low concentrations of organic mercury on ADA activity in human leukocytes and to investigate the relationship between these effects and cell death. We have examined the protective potential effects of Allium sativum extract (GaE) against Methylmercury (MeHg)-induced cytotoxic effects on human leucocytes under in vitro conditions. MeHg (0.05–10 μM) significantly decreased leukocyte viability (58.97% for MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and 51.67% for Alamar Blue (AB) and this decrease was positively correlated to the MeHg-induced inhibition of ADA activity. N-acetylcysteine (NAC) and GaE prevented both the MeHg-induced cytotoxic effects on leukocytes according to MTT and AB assays and the effects on the ADA activity. The present results suggest that the protective effects of GaE against MeHg-induced leukocyte damage is related to the removal of oxidant species generated in the presence of MeHg due to the antioxidant efficacy of garlic constituents. It is important to point out that the intense presence of ADA in Leukocyte suspension (LS) highlights the relevant effects in the immune system and in vitro cytotoxicity of MeHg exposure.