Anne Santerre

Oxidative stress in macrophages from spleen of Nile tilapia (Oreochromis niloticus) exposed to sublethal concentration of endosulfan

Endosulfan is a widely used insecticide with immunosuppressive or immunopotentiating effects which alters the immune response of fish. The effects of the acute exposure to endosulfan on a series of parameters of the innate immune response (IIR) of Nile tilapia (Oreochromis niloticus) were investigated-phagocytosis, production of oxygen reactive species, lipoperoxidation as well as spleen cell viability, relative spleen weight and splenocyte concentration-to fully document the effects of this pesticide on Nile tilapia. Juvenile Nile tilapia were exposed in vivo and for 96h to each one of nine concentrations of endosulfan in order to determine the pesticides acute toxicity level and calculate the lethal concentration of endosulfan to these organisms (LC(50)=12,795ppb). Functional assays showed that endosulfan, at a level equivalent to (1)/(2)LC(50), altered some parameters of the spleen macrophages of Nile tilapia. Phagocytosis, production of oxygen reactive species, and lipoperoxidation increased significantly in exposed fish. Spleen cell viability and relative spleen weight were lower in exposed organisms compared to non-exposed ones, without reaching statistical significance. Splenocyte concentration was not altered in the present experimental conditions. Thus, in vivo exposure (7ppb) of juvenile organisms stimulated the phagocytic activity up to significant oxidative stress levels as indicated by the increased lipid peroxidation in plasma. It can be concluded that short exposure to low concentration of endosulfan stimulated macrophage activity but that there was no significant reduction in the structural parameters of the IIR.

Endosulfan increases seric interleukin-2 like (IL-2L) factor and immunoglobulin M (IgM) of Nile tilapia (Oreochromis niloticus) challenged with Aeromona hydrophila

Endosulfan is a persistent organochlorine insecticide which is extremely toxic to fish. It is known to induce immunological alterations in juvenile Nile tilapia (Oreochromis niloticus) such as increases in phagocytic activity and reactive oxygen species production of spleen macrophages. The purpose of the present study was to demonstrate the effects of acute exposure to a sublethal concentration of endosulfan (7 ppb, 96 h) on parameters of the adaptive humoral immune response of the aforementioned aquatic organism. The effect of endosulfan on the capacity of immune cells to produce interleukin-2 like (IL-2L) factor and immunoglobulin M (IgM) in response to a challenge with (1/2) LD50 of the infectious bacteria Aeromonas hydrophila was evaluated. Experimental results indicate that short, sublethal, endosulfan exposure triggers a succession of events beginning with non-specific activation of macrophages followed by an exacerbated synthesis of the IL-2L factor by activated B cells. This leads to significantly increased secretion of IgM and could in turn facilitate autoantibody production and the development of autoimmune pathologies.

Effects of low concentration of endosulfan on proliferation, ERK1/2 pathway, apoptosis and senescence in Nile tilapia (Oreochromis niloticus) splenocytes

Endosulfan is a potent organochlorinated pesticide that is known to induce side effects in aquatic organisms, including Oreochromis niloticus (Nile tilapia). It has been previously shown that endosulfan induces oxidative stress and non-specific activation of splenic macrophages and exacerbated serum interleukin-2 synthesis in Nile tilapia. Endosulfan may promote proliferation of T cells through MAP kinase (MAPK) activated signal transductions. The ERK family of MAPKs includes ERK1 and ERK2. Phosphorylated ERK1/2 (pERK1/2) molecules are involved in many aspects of cellular survival, and are important for apoptosis or oxidative stress-induced senescence. In order to study the mechanisms by which endosulfan affects fish health, the present study was aimed at evaluating the in vitro effects of this insecticide on proliferation, the ERK1/2 pathway, apoptosis and cell senescence in splenocytes from Nile tilapia. Lymphoproliferation was evaluated by colorimetric method using the WST-1 assay. Flow cytometry was used to assess pERK1/2, apoptosis and senescence, using Annexin V-FITC and β-galactosidase respectively. Experimental data showed that exposure to 7 μg mL(-1) of endosulfan per se increased cellular proliferation, but decreased the lymphoproliferative response to mitogenic stimulus with PMA + ionomycin. Splenocytes exposed to endosulfan for 15-180 min showed significantly higher levels of pERK1/2 than the non-exposed control. Endosulfan mediated a decrease in etoposide-induced apoptosis and provoked cell senescence. In conclusion, exposure of immune cells to a low concentration of endosulfan deregulates their function and may facilitate the development of multiple diseases.

Effects of diazinon and diazoxon on the lymphoproliferation rate of splenocytes from Nile tilapia (Oreochromis niloticus): the immunosuppresive effect could involve an increase in acetylcholine levels.

The lymphoproliferation rate of spleen cells from Nile tilapia (Oreochromis niloticus) exposed to the organophosphorus pesticide diazinon, to its metabolite diazoxon and to the neurotransmitter acetylcholine, was evaluated in order to explore the immunotoxic mechanism of action of this widely used insecticide. The lymphoproliferative response of spleen cells to mitogenic stimulus was not affected by either diazinon or diazoxon, indicating that these xenobiotic substances do not have direct immunotoxic properties. Conversely, ex vivo assays showed that spleen from fish exposed to diazinon presented a lower acetylcholinesterase activity and a higher acetylcholine concentration than non-exposed controls. Lymphoproliferation assays also indicated that pre-exposure to acetylcholine depleted the proliferative function of spleen cells. Thus the combined information from in vitro and ex vivo experiments suggest that the immunotoxic properties of diazinon in Nile tilapia are indirect and could involve the cholinergic system of lymphocytes.

Traditional infrageneric classification of Gymnopilus is not supported by ribosomal DNA sequence data

The traditional classification of Gymnopilus (Agaricales) recognizes two primary groups, Annulati and Gymnopilus, based on the presence or absence of a membranous partial veil. While our analyses of DNA sequence data from the nuclear ribosomal ITS1–5.8S-ITS2 (ITS) gene supports the monophyly of the genus, these traditional subgroups were not recovered. Five well-supported clades within the genus were identified through these analyses: 1) the spectabilis-imperialis group; 2) nevadensis-penetrans group; 3) a clade formed by G. underwoodii, G. validipes and G. cf. flavidellus; 4) aeruginosus-luteofolius group; and 5) lepidotus-subearlei group. Relationships among these subgroups were not resolved.

A chronic combinatory stress model that activates the HPA axis and avoids habituation in BALB/C mice

A detailed protocol is described to induce chronic stress in BALB/c mice, which affects the hypothalamic-pituitary-adrenal (HPA) axis. The protocol is based on a combination of two mild physical stressors: restraint stress and forced swimming. Physical stressors were applied on an alternative schedule: one day restraint, next day swimming, during a 28-day period. Mice were sacrificed at days 7, 14, 21 and 28 and plasma was obtained. Optimized chromatographic system with electrochemical detection and a commercially available enzyme immunoassay kit were used to measure catecholamines [representative of the activation of the autonomic nervous system (ANS)] and corticosterone (representative of the activation of the HPA axis). Corticosterone levels increased in mice under stress and remained significantly higher in stressed mice compared to control animals throughout the experimental procedure, indicating that mice did not show habituation to the combined stress. In our experimental conditions, catecholamine levels were not useful as an index of stress. The stress model applied here provoked a steady activation of the HPA axis resulting in the constant secretion of corticosterone from the adrenal gland, which may prevent the activation of the ANS axis. The simple and economic stress model presented here affected the HPA axis but not the ANS of BALB/c mice and is useful for the study of metabolic stress-related gastric pathology and stress hormone secretion in mice.

Genetic diversity within a declining natural population of Ferocactus histrix (DC) Lindsay

Ferocactus histrix is a barrel cactus that is widespread in Mexico. A population located in Llanos de Ojuelos, a semiarid zone representative of many disturbed regions in north-central Mexico, was studied. Over a period of 10 years (1997 to 2007), the average number of individuals decreased from 21.95 to 3.53 plants per 300 m2. A change in population size structure was also registered over this period of time. In 2008, a plot selected on the basis of plant abundance was established within the population and a genetic analysis was conducted with ISTR and ISSR markers. This analysis revealed low levels of genetic diversity (expected heterozygosity (HE) = 0.073, Shannon index (I) = 0.113 and HE = 0.178, I = 0.271, respectively) compared with those of most studied cacti species. The genetic diversity between the different life stages was also evaluated, and a gradual decrease in levels of genetic variation was observed from adults to juveniles and seedlings (HE = 0.130, I = 0.192 to HE = 0.103, I = 0.157). These differences, however, were not significant. Loci fixation and a decrease in the frequency of rare alleles were observed in seedling and juvenile classes. The decline in genetic variation may be associated with recent bottlenecks experienced by the population of F. histrix. If the sizes of local populations of F. histrix continue to decrease, genetic variation will be gradually lost, and the risk of extinction will increase.

Endosulfan decreases cytotoxic activity of nonspecific cytotoxic cells and expression of granzyme gene in Oreochromis niloticus

The effect of the organochlorinated insecticide endosulfan, on the cytotoxic activity of Nile tilapia nonspecific cytotoxic cells (NCC) was assessed. Juvenile Nile tilapia were exposed to endosulfan (7 ppb) for 96 h and splenic NCC were isolated. Flow cytometric phenotyping of NCC was based on the detection of the NCC specific membrane signaling protein NCCRP-1 by using the monoclonal antibody Mab 5C6; granzyme expression was evaluated by quantitative RT-PCR. The cytotoxic activity of sorted NCC on HL-60 tumoral cells was assessed using propidium iodide (PI) staining of DNA in HL-60 nuclei, indicating dead cells. Nile tilapia splenic NCC had the ability to kill HL-60 tumoral cells, however, the exposure to endosulfan significantly reduced, by a 65%, their cytotoxic activity when using the effector:target ratio of 40:1. Additionally, the exposure to endosulfan tended to increase the expression of NCCRP-1, which is involved in NCC antigen recognition and signaling. Moreover, it decreased the expression of the granzyme gene in exposed group as compared with non-exposed group; however significant differences between groups were not detected. In summary, the acute exposure of Nile tilapia to sublethal concentration of endosulfan induces alteration in function of NCC: significant decrease of cytotoxic activity and a tendency to lower granzyme expression, severe enough to compromise the immunity of this species.