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Identification, Anaesthesia, and Euthanasia Identification of Laboratory Rodents by Ear Notching Identification of Fish by Fin Clipping Anaesthesia of Fish and Rodents Euthanasia of Rodents Euthanasia of Fish Working Sheet: Weight of Laboratory Animals Collection of Peripheral Blood Samples Reagents Materials and Equipment Procedure Removal of Organs Reagents Materials and Equipment Procedure Preparation of Cell Suspensions Extrusion of Coelomocytes in the Earthworm Hemocyte Collection in the Mollusk Cell Suspension from Peritoneal Exudate Isolation of White Blood Cells from Peripheral Blood Cell Suspensions from Lymphoid Organs Assessment of Cell Viability Determination of Cell Viability and Concentration by Trypan Blue Dye Exclusion Determination of Cell Viability and Cell Concentration with Ethidium Bromide and Acridine Orange Flow Cytometric Assessment of Cell Viability Working Sheet: Cell Viability and Concentration by Trypan Blue Dye Exclusion or Ethidium Bromide/Acridine Orange Cell Cryopreservation Reagents Materials and Equipment Procedure Special Recommendations Analysis of Results Species-Related Changes in the Protocol Technique-Related Changes in the Protocol: Phagocytosis with Yeast Statistical Tests Working Sheet: Phagocytic Function Oxidative Burst Assay Using Flow Cytometry Reagents Materials and Equipment Procedure Special Recommendations Analysis of Results Species-Related Changes in the Protocol Statistical Tests Working Sheet: Acquisition List Cell Cytotoxicity Natural Killer (NK) Cell Activity Antibody-Dependent Cellular Cytotoxicity Lymphokine-Activated Killer (LAK) Working Sheet: Acquistion List Lymphoblastic Transformation Reagents Materials and Equipment Procedure Analysis of Result Special Recommendations Species-Related Changes in the Protocol Working Sheet: Mitogenic Assay Determination of Antibody-Producing Cells to a Specific Antigen Liquid Plaque Forming Cell Assay in Mouse Model Agar Plaque Forming Cell Assay in Rat Model Working Sheet: Plaque Forming Cells (PFC) Mixed Lymphocyte Reaction (MLR) Reagents Materials and Equipment Procedure Analysis of Results Special Recommendations Species-Related Changes in the Protocol Working Sheet: Mixed Lymphocyte Reaction Intracellular Levels of Calcium Assay Using Flow Cytometry Reagents Materials and Equipment Procedure Special Recommendations Analysis of Results Species-Related Changes in the Protocol Statistical Tests Working Sheet: Acquisition List Phenotyping of Blood Mononuclear Cells Reagents Materials and Equipment Procedure Special Recommendations Analysis of Results Species-Related Changes in the Protocol Immunophenotype of Rat Peripheral Blood Lymphocytes Working Sheet Evaluation of Intracellular Level of Thiols Reagents Materials and Equipment Procedure Special Recommendations Analysis of Results Species-Related Changes in the Protocol Working Sheet: Acquisition List Apoptosis Reagents Materials and Equipment Procedure Analysis of Results

Interaction of a single dose (36 mg/kg body wt) of the organochlorine pesticide dieldrin with mouse peritoneal macrophages was examined in C57Bl/6, (C57Bl/6 X A/J)F1, and A/J strains of different genetic resistance to mouse hepatitis virus 3 (MHV3) infection. In vivo studies showed increased susceptibility to MHV3 acute disease of C57Bl/6 and (C57Bl/6 X A/J)F1 animals challenged with the pesticide. Significant decrease of mean time of death in dieldrin-exposed, MHV3-infected susceptible C57Bl/6 mice was observed similarly upon po or ip administration of a single, sublethal dose of dieldrin. In addition, decrease of humoral response to the virus was quantified by determination of anti-MHV3 IgG antibodies in spleen cell supernatant fractions and in blood sera of dieldrin-exposed C57Bl/6 mice. A single dose of dieldrin did not alter the in vivo resistance of A/J animals to acute MHV3 disease. The resistant A/J mice, however, showed increased mortality upon two subsequent exposures to dieldrin followed by infection with high lethal doses of MHV3. Phagocytic activity, cell adherence capacity, and attachment and uptake of 3H-radiolabeled MHV3 by C57Bl/6 peritoneal macrophages were determined by in vitro studies. These affector activities of peritoneal macrophages were slightly decreased or unchanged in cells originating from animals exposed to the pesticide. However, the intrinsic activity of MHV3 restriction appeared to be affected in macrophages derived from dieldrin-treated animals: (i) peritoneal C57Bl/6 macrophages collected from the early phase of acute MHV3 disease contained increased MHV3 antigen and (ii) increased cytolysis was observed after in vitro MHV3 infection of macrophages originating from dieldrin-exposed C57Bl/6 mice.

The immunosuppressive effects of methyl mercury (MH), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and dibenzofurans (PCDFs) are well established at high exposure levels but unclear at low exposure levels. We exposed Fischer 344 rat splenocytes, thymocytes, and peripheral blood lymphocytes in vitro for 72 h to MHg (0.1, 2 μg/ml), PCDD/PCDF mixtures (1, 15 pg/ml) of three PCDDs (2,3,7,8-tetrachlorodibenzo-p-dioxin, 1,2,3,7,8-pentachlorodibenzo-p-dioxin, and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin) and two PCDFs (2,3,7,8-tetrachlorodibenzofuran and 1,2,3,7,8-pentachlorodibenzo-furan), three Aroclor® (1242, 1254, 1260), PCB mixtures (0.01, 0.5 μg/ml), or combinations of MHg/PCB/PCDD/PCDF mixtures Mitogenic responses of lymphocytes to concanavalin A, phytohemagglutinin, or lipopolysaccharide/dextran sulfate were determined by 3H-thymidine uptake; cytotoxicity and intracellular Ca2+ were determined by flow cytometry. Methylmercury (2 μg/ml and PCB/ PCDD/PCDF mixtures with 2 μg/ml MHg decreased the viability of splenocytes to 57 and 40% at 4 and 24 h, respectively. Basal intracellular calcium ion levels were unaffected by the treatments. Methylmercury suppressed the responses of lymphocytes to T and B cell mitogens. All combinations of MHg/PCB/PCDD/PCDF mixtures decreased mitogenic responses to levels similar to those to MHg alone. In contrast, PCB and PCDD/PCDF mixtures did not suppress but augmented responses of splenocytes and peripheral blood lymphocytes to T cell mitogens. Overall, no interactive toxicity was observed with MHg/PCB/PCDD/PCDF mixtures on cytotoxicity and lymphocyte mitogenic responses. Therefore, MHg may pose a greater threat than organochlorines to the mammalian immune system.

Biological effects data with single insecticides are far more abundant than with mixtures. These data cannot be used directly to predict the effects of insecticide mixtures. Three insecticides of different chemical classes: organochlorine; dieldrin, organophosphate; malathion, and carbamate; carbofuran, previously evaluated for their immunotoxic potential, were selected for studies of combined acute exposure in C57B1/6 inbred mice. The humoral response to sheep red blood cells (SRBC) and the functional activities of peritoneal macrophages, such as phagocytosis of fluorescent beads and presentation of a single protein antigen, avidin, were examined after in vivo exposure of mice to different combinations of the selected pesticides and compared with the vehicle controls. Regarding exposure to single substances, the data confirmed the immunosuppressive potential of dieldrin and carbofuran and the immunopotentiating effect of malathion. Following the acute concomitant exposure to dieldrin/carbofuran mixture, however, values for the parameters of antigen presentation, primary IgM antibody response to SRBC antigen, and macrophage phagocytosis, returned to control or above-control values, indicating a lack of any synergistic or additive effects of the chemicals on the immune response. Thus, it was concluded the dieldrin/carbofuran mixture had an antagonistic effect on the humoral response to SRBC and the macrophage phagocytic activity, in comparison with the action of administration of each of the insecticides alone.

The potential immunotoxic effect of the carbamate pesticide aminocarb on murine bone marrow cell subpopulations was evaluated by flow cytometry, C57B1 6 mice were exposed by gavage to sublethal doses of the pesticide and lymphocyte precursors from bone marrow population were stained with PNA lectin and a panel of monoclonal antibodies against cell surface antigens. In regard to the microenvironment-dependent maturation of B lymphocytes, the pesticide effect on the lymphoproliferative potential of bone marrow was assessed by marrow transplantation from aminocarb-exposed donor mice to normal, syngeneic, X-irradiated recipient mice. The sublethal exposure of 0.08–5.0 mg/kg body wt aminocarb to donor animals did not affect regenerating bone marrow in the recipient mice. No marked effect on bone marrow cell number was noted in pesticide-exposed animals. However, a marked shift in surface IgM density on marrow B cells was noted at 0.08 and 0.31 mg/kg body wt aminocarb. This was correlated with decreased cell frequency in G 0 G 1 phase and increased frequency of cells in the S phase of the cell cycle. Thus, altered maturation of B lymphocytes, expressed as a shift in the density of surface IgM on mature B cells and not the lymphocyte count, was related to the direct effect of aminocarb and/or to the pesticide-related changes in bone marrow microenvironment. Overall, exposure to the carbamate pesticide aminocarb activated the cell maturation process in bone marrow.

Cement kiln dusts are made of a complex mixture of elements. We have evaluated the potential negative impact of those dusts on the immune system of the earthworm Lumbricus terrestris. We specifically studied cell viability and phagocytic activity of coelomocytes extruded during electrical stimulation. We used two modes of exposures: in vitro, and soil incubation using OECD artificial soil media. Extruded coelomocytes were exposed 18 h in vitro to 10, 100, and 500 mg L-1 of cement kiln dust particles. The phagocytosis and the cell viability were determined using a double-laser-flow acquisition cytometry system. Using the double laser allows us to use a dichlorofluorescein diacetate (DCFDA) marker to discriminate the biological cells from the cement kiln dusts. Dead cells are marked using propidium iodide (PI). All three exposure levels showed highly significant impacts on cell viability and phagocytic activity. The in vivo soil incubation was performed using 10, 100, and 1000 mg kg-1 of cement kiln dusts incorporated into the OECD media. Here, to discriminate the biological cells from the mineral dusts we only needed to use PI. The day-to-day variability of the in vivo assay was high and although we can observe an overall reduction in cell viability at the highest concentration tested, no statistically significant effects could be observed on either cell viability or phagocytosis. Copyright 2004 Elsevier Inc. All rights reserved.

In order to reduce their energy costs, many cement plants use fuel product substitutes (old tyres and used oil). The combustion of these products generates a metal increase (e.g. Cu, Cd, Pb and Zn) in the atmospheric emissions. After their release, these elements are deposited into the environment and could eventually accumulate up to concentrations of concern. At the Saint-Laurent cement factory (Joliette, QC, Canada), maximum deposition of these elements occurs in the direction of prevailing winds (North-East). We evaluated the potential impact of these depositions upon the immune system of three earthworm species (Lumbricus terrestris, Eisenia andrei and Aporrectodea tuberculata) exposed in a natural environment. The exposure sites were 0.5, 1.0, and 2.0 km downwind from the cement factory, along with an upwind reference site. The immune parameters studied were the cell viability and phagocytic potential of the immune cells (coelomocytes). For both L. terrestris and E. andrei, after 7 d exposure, none of the measured parameters showed significant differences among the sites. On the other hand, for the indigenous worm A. tuberculata, in the most exposed zone (at 0.5 km), we observed an increase in cell viability and phagocytic potential. This increase could possibly be attributed to physicochemical effects such as the alkaline pH of the soil, or alternatively, it could result from beneficial effects induced by an increased calcium supply.

Rat splenocyte mixed leukocyte reaction (MLR), splenic natural killer (NK) cell activity, and phagocytic activities of splenic, peritoneal, and peripheral blood leukocytes (PBLs) were evaluated in vitro to determine the immunotoxicity of mixtures containing low levels of methylmercury (MeHg), polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and Aroclor polychlorinated biphenyls (PCBs). The mixtures were based on the concentrations of the chemicals in fish flesh. Leukocytes from male Fischer rats were exposed to MeHg (0.1-2 microg/ml), PCDD/PCDF mixtures (1-15 pg/ml) of three PCDDs (2,3,7,8-tetrachlorodibenzo-p-dioxin, 1,2,3,7,8-pentachlorodibenzo-p-dioxin, and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin) and two PCDFs (2,3,7,8-tetrachlorodibenzofuran and 1,2,3,7,8-pentachlorodibenzofuran), three Aroclor PCB (Aroclor 1242, 1254, and 1260) mixtures (0.01-0.5 microg/ml), or combinations of MeHg/PCB/PCDD/PCDF mixtures for 24 or 72 h before immunological assays. Phagocytosis and NK cell cytotoxicity were evaluated with a flow cytometer, and MLR of Fischer rat responder splenocytes cultured with mitomycin C-treated Long-Evans splenocytes by [3H]thymidine uptake. Exposure to MeHg (2 microg/ml) alone or with PCB/ PCDD/PCDF resulted in significant cytolethality in rat splenocytes, peritoneal leukocytes, and PBLs at 24 h exposure. Treatment with Aroclor PCB mixtures, PCDD/PCDF mixtures, 0.1 microg MeHg/ml (noncytolethal), or PCB/PCDD/PCDF mixtures with 0.1 microg MeHg/ml caused no suppression of splenocyte MLR response, splenic NK cell-mediated lysis of Yac-l cells, or phagocytosis of fluorescent beads by splenic, peritoneal, and peripheral blood phagocytic cells. The results indicate that in vitro exposure of rat leukocytes to low levels of MeHg, Aroclor PCB mixtures, PCDD/PCDF mixtures, or MeHg/PCB/PCDD/PCDF mixtures had no suppressive effects on the immune functions assayed, and thus produced no additive immunotoxicity. However, in order to predict the potential risk of these chemical mixtures to the human immune system, in vivo animal studies with blood (tissue) levels compatible with the levels of MeHg, PCBs, and PCDDs/PCDFs in exposed human populations should be evaluated.

The antiviral drug acyclovir [9-(2-hydroxyethoxymethyl)guanine (ACV)], its 7-isomer (7-ACV) and its two derivatives: N2-acetyl ACV (ac-ACV) and N2,O-diacetyl ACV (diac-ACV) were examined for their potential in vitro lymphotoxicity and in vivo immunotoxicity in mice. In vitro lymphotoxicity of ACV and its acetylated derivatives was low, whereas the 7-ACV isomer enhanced the in vitro cell proliferation in PHA-stimulated cultures. Addition of 2'-deoxyguanosine (dGuo) did not exhibit any inhibitory potential of ACV. However, reduction in the absolute number of CD3+, CD8+, and CD25+ cells, but not Ig+ cells, was noted at high concentrations of ACV and its derivatives, suggesting a selective T cell cytotoxicity. Similarly, the in vivo exposure revealed selective T cell immunotoxicity of ACV and its derivatives since the reduced number of Thy 1.2+ and CD8+ cells was not accompanied with any marked changes in the Ig+ population. The CD4+/CD8+ ratio was affected both in vitro and in vivo by high concentrations of ACV.

Induction of contact hypersensitivity (CH) by molybdenum chloride (MoCl5) was determined by auricular lymph node (ALN) test in C57B1/6 mice. The ALN test was further improved by immunophenotyping and cytometric analysis of subset-specific cell in the draining node. Skin sensitization was induced by topical ear exposure to 1.0-50% oxazolone and resulted in a strong dose-related ALN reaction. Analogous exposure to MoCl5 resulted in a weaker but marked dose-related reaction, also manifested as an increase in cell number/ALN. Other differences between the oxazolone-induced strong sensitization and the MoCl5-related ALN reaction were: (1) an increase in the total number of Ig+ cells, which was, however, unchanged in the MoCl5-exposed mice; (2) a significant increase in the total number of large/activated T-cell subsets; and (3) a marked shift in the relative percentage of gated large/activated subsets of ALN cells, which was not observed in the MoCl5-exposed animals. Thus, it appeared that the molybdenum exposure induced a nonspecific increase in the cell number/ALN and was not accompanied by any marked activation of the T-cell subsets. Immunotoxicity of a 14 day subchronic exposure to MoCl5 at 1-100 ppm in food was studied by quantification of splenic humoral IgM response to sheep erythrocytes (SRBC). Plaque-forming cells (PFC) and enzyme-linked immunosorbent assay (ELISA) revealed unchanged humoral exposure in MoCl5-exposed mice. Cytometric assay of fluorescent beads uptake showed unchanged phagocytic activity of peritoneal macrophages from the MoCl5-exposed mice. Immunophenotyping of CD4+, CD8+, Thy 1.2+ and Ig+ cells revealed no effect of MoCl5 exposure on the total count of cell subsets in the ungated populations of spleen, lymph nodes and peripheral blood cells. Molybdenum chloride should thus be considered as a non-immunotoxic and a weak, nonspecific contact irritant.

Aquat. Toxicol. ISI Document Delivery No.: NX445 Times Cited: 30 Cited Reference Count: 25 Voccia, i krzystyniak, k dunier, m flipo, d fournier, m Elsevier science bv Amsterdam; Cytotoxic and immunotoxic effects of mercury chloride and methylmercury on blood and head kidney leucocytes of rainbow trout (Oncorhynchus mykiss) were evaluated in vitro, for a broad dose range (10(-4)-10(-9) M Hg) of the chemicals. Mercury-related impairment of functional cellular parameters were measured using mixed leucocyte reaction (MLR) and mitogen stimulation of the cells by phytohaemagglutinin (PHA), concanavalin A (Con A) and lipopolysaccharide (LPS). Non-specific defense mechanisms of blood neutrophils and pronephros macrophages were analysed using flow cytometric assay of phagocytosis and respiratory burst. Impairment of the functional cellular activities appeared to be limited almost exclusively to cytotoxic concentrations of the mercurials. Similarly, phagocytic and respiratory burst activities of blood and head kidney phagocytes were markedly impaired by high, cytotoxic concentrations of the mercurials. The in vitro cytotoxic potential of methylmercury; greater-than-or-equal-to 10(-5) M Hg, appeared to be at least ten times higher over the cytotoxicity of mercury chloride greater-than-or-equal-to 10(-4) M Hg. At lower mercury concentrations, the data showed mostly normal- or above-normal values of the assayed functional activities of the cells. Overall, a nonspecific, poisoning-related impairment of leucocyte and phagocyte functions was concluded for the in vitro cytotoxic concentrations of mercurials.

Macrophages from mice peritoneal exudate were isolated on basis of specific adherence on epichlorohydrin cross-linked amylose (CLA), a chromatographic gel presenting a high susceptibility to advanced amylolysis with alpha-amylase. The cell suspension, containing predominantly macrophages and lymphocytes, was applied onto the column and incubated for 30 min at 37 degrees C for the adherence of macrophages. After this interval the non-adherent cells were eluted with buffered medium and the CLA support was incubated in the column with an alpha-amylase-buffered solution liquefying the matrix and releasing, in situ, the adherent cell population containing 90% macrophages with a viability higher than 90%.

Effect of selected organochlorine, organophosphorus, and carbamate pesticides on the humoral immune IgM response was examined upon immunization of inbred C57B1/6 mice with neutral, polyvalent, T-dependent sheep erythrocytes (SRBC) and T-independent lipopolysaccharide (LPS). In addition, a pathogenic antigen, mouse hepatitis virus 3 (MHV3) was used for determination for the interaction of selected pesticides on the primary IgG immune response in a model of viral infection, of the genetically resistant A/J mouse strain. Single, sublethal doses of dieldrin, carbofuran, and matacil induced a marked immunosuppression of the humoral responses to both neutral and pathogenic antigens. The data showed that single, sublethal doses (0.4 ≤ LD 50 ≤ 0.6) of dieldrin, carbofuran, and matacil inhibited the number of SRBC-primed cells without any direct cytotoxic effect on the activated plasmocyte, as the titer of specific antibody measured by an enzyme-linked immunosorbent assay (ELISA) per activated cell, was constant. In contrast, exposure to malathion at 10–14 days prior to the assay increased the number of plaque-forming cells (PFC) and the anti-SRBC IgM and anti-MHV3 IgG antibody titer, suggesting therefore an increase in the humoral response to neutral and pathogenic antigens in C57B1/6 and A/J mice. Immunomodulation of the humoral IgM response by selected pesticides was shown to take place at a stage prior to antibody secretion from the activated cell, as the ELISA/PFC index was similar to the control value. The data obtained for dieldrin-induced inhibition of the humoral response to SRBC and LPS antigens suggest a mechanism of immunosuppression common for both T-dependent and T-independent antigens. Good correlations were obtained for the immunomodulatory effects of selected pesticides, as measured by PFC and ELISA, which encourages support for the latter technique in the immunotoxicological screening of pesticides.