| Background:The toxicological change of immune system is a sensitive biomarker in safety evaluation of some exogenous chemicals. Safety evaluation will be scientific and efficient if sensitive bio markers can be detected in early phase. Due to the complexity of immune system, a set of test indicators are generally used to evaluate immunotoxicity as a whole, cellular and molecular levels. At present, many countries have established their own guidelines of immuno toxico logical safety evaluation which are mainly used in medicine, pesticides, food additives, biological products and medical devices, etc.Immune toxicology research starts late in our country. At present, the immune toxicity evaluation are involved in only Guidelines of Chemical Toxicity Identification and Management as well as some technical guiding principles such as traditional medicine, natural medicine. The latter is used for allergy and allergic reactions. There are no guidelines of immuno toxico logical evaluation on foods and pesticides. So it is necessary to establish a rapid and sensitive screening system of immunotoxicological safety evaluation.In recent years, the global pesticide industries have rapidly developed, and many countries have implemented a system of pesticide registration and management requiring specific safety data. The immune toxicity data should be provided in pesticide registration in the United States. However, immune toxicity data of new pesticide cannot be asked for in our country, because the guidelines of immuno toxicity evaluation are not established. In September 2013, JMPR expert meeting will be held to reassess some pesticides, and immunotoxicological evidences will be considered as the key part of toxicity data. In addition, the fast development of genetically modified crops have been developed rapidly all over the world, meanwhile there are some researches that have shown that genetically modified crops and proteins expressed by exogenous genes are likely to impact on immune system of experimental animals and human. Therefore, it is necessary to evaluate the immunotoxicity of pesticides and genetically modified crops.Objective:To establish a primary and secondary immunotoxicity screening system in vivo or in vitro in rodent, and provide scientific information for establishing immunotoxicity evaluation methods in our country. To study immunotoxicological effects of two kinds of pesticides such as malathion and atrazine as well as corn genetically modified with Bt CrylAh gene using the established immunotoxicity screening system, and explore the immunotoxicological target and its possible mechanism. To provide the scientific evidences on the registration and management and the formulation of maximum residue limits of malathion and atrazine, as well as the safety assessment and commercial production of corn genetically modified with Bt CrylAh gene.This study includes the following three parts:Part 1. Study on primary and secondary screening system in vivo and in vitro of immunotoxicological animal model induced by cyclophosphamide in BALB/c miceMethods:The female BALB/c mice with body weight of 18 g-22 g were used for the study. The study consisted of primary screening and secondary screening tests with nine substudies. Four substudies were used in primary screening, and the other five substudies were used in secondary screening. And each substudies was set into five groups:the control group with distilled water daily gavage; CP gavage group with 40 mg/kg bw/d by gavage; CP ip-1 group with 40 mg/kg BW/d by intraperitoneally injection (ip); CP ip-2 group with 80 mg/kg BW/d in the first three days by ip and once a week in the following period; CP ip-3 group with 200 mg/kg BW 24 h before the end of test by ip. The study lasted for 30 days. All animals were observed within 3 days of arrival, then randomly assigned to different groups with ten mice per group. Mice were housed by group with ad libitum access to water and nutritional propagation diets for 30 days. Immunotoxicological pathology parameters of primary screening included:body weight and organ indexes (liver, kidneys, spleens, thymus, and lymph nodes); hematology and clinical chemistry parameters; histopathology observation of liver, kidneys, immune organs such as spleens, thymus, lymph nodes, bone marrow. Humoral immunity included:T cell dependent antibody response (PFC) and serum immunoglobulin levels (plasma IgM and IgG levels by ELISA). Cellular immune included:ConA-induced spleen T lymphocyte proliferation and LPS-induced spleen B lymphocyte proliferation. NK cell activity was used to examine nonspecific immune. Secondary screening: immunotoxicological pathology parameters included:peripheral blood lymphocytes phenotype analysis (peripheral blood T lymphocytes, B lymphocytes and NK cells, Th cells, Ts cells percentage and CD4/CD8 using flow cytometry), bone marrow cell classification, spleen cells number, precision measurement of immune organs (periarterial lymphatic sheath and germinal center of spleen, lymph follicles and germinal center of Peryer's lymph node). Humoral immune:serum cytokine IL-2,IL-4, IL-5, IL-10, gamma-IFN and TNF using flow cytometry, and serum hemolysin. Cellular immune included:delayed-type hypersensitivity (footpad thickening test in mice) and cytotoxic T-lymphocyte assay. The phagocytic activity of macrophage was performed as carbon-clearance test in mice to examine nonspecific immune.Results:Primary screening:I mmunoto xico logical pathology parameters:Weekly body weights and the number of Peyer's patches of mice in CP gavage group, CP ip-1 group and CP ip-2 group were significantly lower than those in the negative control group (P<0.05). The numbers of Peyer's patches in CP gavage group and CP ip-1 group were significantly lower than those of CP ip-2 group and CP ip-3 group (P< 0.05). The relative weights of livers in CP gavage group and CP ip-1 group were significantly higher than those in the control group (P?0.05); the absolute weight of kidney in CP ip-1 group was significantly lower than that of the control group (P<0.05), and the relative weight of kidney in CP gavage group was significantly lower than that of the control group (P<0.05). The absolute weights of spleens and the weights of thymuses in four CP groups were significantly lower than that in the control group (P<0.05). The relative weights of spleens of CP ip-2 group and CP ip-3 group were significantly lower than that in the control group (P?0.05). The absolute and relative weights of thymuses in CP gavage group and CP ip-1 were significantly lower than those in CP ip-2 and CP ip-3 (P?0.05). The absolute weight of lymph nodes in CP ip-3 was significantly lower than the control group (P<0.05).Hematology:There were significant changes of the mean WBC counts, lymphocyte percentage, and neutrophils percentage of mice in the four CP groups were observed when compared with the control group (PO.05). There were significant changes of the basophils percentages, MCHC and RDW levels of mice in the CP gavage group, ip-1 group and ip-2 group were observed when compared with the control group (P<0.05). The PLT level in the CP ip-1 group was significantly higher than that in the control group (P<0.05).Clinical chemistry parameters:Serum TP and ALB levels in CP gavage group, CP ip-1 group and ip-2 group were significantly lower than those in the control group (P<0.05). Serum TG levels in CP gavage group and CP ip-1 group were significantly lower than those in the control group, CP ip-2 group and CP ip-3 group (P<0.05). There were significant changes of serum ALT and GLU levels of mice in CP ip-1 group were observed when compared with the control group (P?0.05). Serum ALT level of mice was significantly higher than the control group and the other CP groups (P<0.05). Histopathology:The thymus atrophy, white pulp atrophy in spleen, bone marrow hematopoietic cell decrease and lymph node atrophy were observed in mice in the four CP groups. And the liver lesions were examined in mice in CP gavage group and CP ip-1 group. The severity of thymus and spleen atrophy in mice in CP gavage group and CP ip-1 group were higher than those in CP ip-2 group and CP ip-3, however all the different administering ways of cyclophosphamide could cause obvious atrophy of Peyer's lymph nodes.Humoral immune:The number of plaque forming cells in mice in the four CP groups were significantly lower than the control group (P?0.05); especially in CP gavage group and CP ip-1 group. The levels of IgA and IgG in mice in the four CP groups were significantly lower than those in the control group (P?0.05), and the levels of IgM in CP gavage group, CP ip-1 and ip-2 groups were lower than those in the control group (P?0.05).Cellular immune:Spleen T lymphocyte and B lymphocyte proliferation abilities in the four CP groups were significantly lower than those in the control group (PO.05).Nonspecific immunity:NK cell activity in mice in CP ip-2 group was significantly lower than that in the control group (P?0.05).Secondary screening tests:Immunotoxicological pathology:Peripheral blood lymphocyte phenotype analysis:the percentages of T lymphocyte, Th cells, and the ratios ofCD4+/CDg+ in peripheral blood in CP ip-2 and ip-3 groups were significantly higher than those in the control group, CP gavage group, and CP ip-1 group (P<0.05); the percentages of Ts cells in CP gavage group, CP ip-1 and ip-2 groups were significantly higher than that in the control group (P<0.05). The percentages of NK cells in CP gavage group and CP ip-1 group were significantly higher than those in the control group, CP ip-2 group and CP ip-3 group (P<0.05). The percentages of peripheral blood B lymphocyte (CD3-CD19+) in the four CP groups were significantly higher than that in the control group (P<0.05).Bone marrow cell classification:in granulocyte, the percentages of segmented granulocyte in the four CP groups were higher than that in the control group (P?0.05); the percentages of pro granulocyte in CP gavage group and CP ip-1 group were significantly higher than those in the control group and CP ip-3 group (P<0.05).Inerythroblast, the percentages of metarubricyte in CP gavage group, CP ip-1 group and CP ip-3 group were significantly higher than that in the control group (P<0.05), and the percentages of prorubricyte in CP ip-1 group was significantly higher than those in the control group, CP ip-2 group, and CP ip-3 group (P<0.05). The percentages of lymphocyte in the four CP groups, especially CP ip-1 and CP ip-2 groups, were significantly lower than the control group (P?0.05). Splenocyte count:the number of splenocytes in the four CP groups, especially in CP ip-3 group, were significantly lower than that in the control group (P<0.05). Precision measurement of immune organs:the thicknesses of the periarterial lymphatic sheath of spleens in the four CP groups were significantly lower than that in the control group (P?0.05); the sizes of germinal center of spleens in CP gavage group and CP ip-1 were significantly lower than that in the control group (P?0.05), however those in CP ip-2 group and CP ip-3 were higher than that in the control group (P?0.05). The sizes of lymph follicles and germinal center of Peryer's patches of mice in CP gavage group and CP ip-1 group were significantly lower than those in the control group, CP ip-2 group and CP ip-3 group (P?0.05).Humoral immune:Serum cytokine:Serum IL-5 level of mice in CP gavage group was significantly lower than those in the control group and CP ip-2 (P?0.05). Serum ?-IFN levels in CP ip-2 and ip-3 groups were significantly lower than those in the control group, CP gavage group and CP ip-1 group (P?0.05). Serum TNF levels of mice in CP gavage group and CP ip-1 group were significantly higher than those in the control group, CP ip-2 and ip-3 groups (P<0.05).Serum hemolysin:There was no significant difference between the four CP groups and the control group (P>0.05).Cellular immune:The changes of 24-h footpad thickness in the four CP groups was significantly higher than the control group (P?0.05), but there was no significant difference among the four CP groups (P>0.05). No significant change of the activity of CTL of mice was found among all groups (P>0.05).Nonspecific immunity:There was no significant difference of phagocytosis activity of phagocyte among all groups (P>0.05).Conclusion:1.Some immune changes were found in four CP groups including significant decreases in immune organ weights, the number of splenocyte, the leucocyte absolute value and partial differential leucocyte percentage, serum immunoglobulin levels, the number of PFC in spleen, splenocyte proliferation activity; significant changes in the percentages of B cells and neutrophils in peripheral blood lymphocyte classification, the percentages of lymphocytes and segmented granulocyte in bone marrow cells; and significant increase in DTH. In addition, the thymus atrophy, white pulp atrophy in spleen, bone marrow hematopoietic cell decrease and lymph node atrophy were observed in mice in the four CP groups. The results of this study showed that the administration of cyclophosphamide led to significant inhibition of most of the immunopathology, cellular immune, and humoral immune. So the animal model used to evaluate the immunetoxic effect was successful.2. When comparing the four animal model methods,40 mg/kg BW CP administered by gavage or i.p. can cause the reduce of body weight and liver relative weight, the changes of clinical biochemistry parameters such as serum ALT, glucose and TG levels, and liver toxicity by pathofogical histology examination, besides the obvious immunotoxicity. There are no significant difference in immunopathology, cellular immune, humoral immune and nonspecific immune in CP ip-2 group and CP ip-3 group. But body weight loss, MCHC and RDW abnormalities can be observed in the mice in the CP ip-2 group. A single dose of CY (200 mg/kg bw) was administered via intraperitoneal injection can not only effectively reduce the immune function in mice, but has many advantages such as convenient operation, short period, small damage to animals, and good specificity. The appropriate animal model of immuno suppress ion may be established by intraperitoneal injection of 200 mg/kg CTX 24h before the end of the study.3. Primary and secondary immune toxicity screening system are carried on the comprehensive evaluation including immune pathology, cellular immunity, humoral immunity and nonspecific immune, and the results are almost consistent. Secondary screening system result is a complement to primary screening. The combination of primary and secondary screening systems of immune toxicity in vivo and in vitro can be effectively used for immunotoxicity evaluation.Part ?:Study of malathion and atrazine on immunotoxicity.Methods:The female BALB/c mice with body weight of 18 g-22 g were used for the study. All animals were observed within 3 days of arrival, then randomly assigned to different groups with ten mice per group. At first primary screening with four substudies were studiedc. And each substudies was set into five groups:the negative control group with corn oil daily gavage; low, middle, and high dose groups of malathion; low, middle, and high dose groups of atrazine; the positive control group with 200 mg/kg BW 24 h before the end of test by ip. The high dosage of malathion and atrazien was mainly based on the results of the oral acute toxicity in mice,1/4 LD50 and 1/12 LD50, respectively. The dosages of low, middle and high dose groups of malathine were 16 mg/kg BW,65 mg/kg BW,258 mg/kg BW, respectively. The dosages of low, middle and high dose groups of atrazine were 23 mg/kg BW,90 mg/kg BW,360 mg/kg BW, respectively. Mice were housed by group with ad libitum access to water and nutritional propagation diets. If one of primary screening results is positive, it is needed to continue to carry on secondary screening in which there are five substudies. The parameters and methods of immunopathology, humoral immunity, cellular immunity and nonspecific immunity of primary and secondary screening were accordance with those of Part ?Results:Malathion:primary screening system:relative and absolute weights of liver as well as serum CHO and ALT of mice in high dose group were higher than those in the control group, low and middle dose groups (P<0.05); however whole blood leukocyte count, basophils and its percentage in whole blood decreased significantly. Histopathological examination showed that malathion could cause hepatocyte swelling and thymus atrophy. Serum total protein and albumin level of mice in the high dose group of malathion were significantly lower than those in the negative control group (P<0.05). Relative and absolute weights of thymus in low and middle dose group were higher than those in the control group, but neutrophils count in whole blood decreased significantly (P<0.05). Humoral immunity toxicity tests showed PFC of spleen in the high dose group was significantly lower than that in the negative control group(P<0.05), IgG level in plasma in the high dose group was higher than those in the negative control group, low and middle dose groups(P<0.05). There were no significant difference of T lymphocyte and B lymphocyte proliferation ability as well as NK cell activity between the three dose groups of malathion and the negative control group (P>0.05). Secondary screening system:polychromatic erythroblast and mononuclear cells of bone marrow smear in the three dose groups were lower than those in the negative control group (P?0.05). The B lymphocytes proportion in peripheral blood can markedly increase in the high dose group (P?0.05). Humoral immunity toxicity tests showed that serum IL-10 levels in low and middle dose groups as well as serum IL-5 level in middle dose group markedly changed. There were no significant difference of CTL, DHT, and macrophage phagocytosis between the three dose groups of malathion and the negative control group (P>0.05).Atrazine:Primary screening system:weekly body weights at week 1-week 3 in high group were lower than those in the negative control group, and there was an obvious dose-response relationship among the three dose groups of atrazine (P?0.05). The thymus weight of mice markedly increased in low dose group, and thymus atrophy was found in BALB/c mice in middle and high dose groups by histopathofogic examination. The leukocyte counts in whole blood in the three dose groups were significantly lower than that in the negative control group (P?0.05). The numbers and percentages of lymphocytes and monocytes in the high group were significantly lower than the negative control group, and there was a dose-response relationship (P<0.05); when compared with the negative control group, the numbers of neutrophils significantly decreased in low and middle dose groups, but it increased in high dose group(P<0.05). Serum total protein and albumin levels significantly increased in low dose group compared with negative control group, but those indexes decreased in high dose group, and there was a dose-response relationship (P?0.05). The relative weights of liver and kidney in high dose group were significantly higher than those in negative control group and other atrazine groups, serum BUN and CHO in high dose group was significantly higher than those in the negative control group (P<0.05). Histopathological results showed that hepatocyte swelling could be observed in BALB/c mice in middle and high dose groups. Humoral immunity toxicity tests showed that PFC of spleen in the high dose group was significantly lower than that in the negative control group(P<0.05), IgG level in plasma in the high dose group was higher than those in the negative control group, low and middle dose groups(P<0.05). Cellular immunity toxicity tests showed that T lymphocyte and B lymphocyte proliferation ability in high dose group was lower than those in the negative control group, low and middle dose groups (P<0.05). There was no significant difference of NK cell activity between the three dose groups of malathion and the negative control group (P>0.05). Secondary screening system:polychromatic erythroblast and mononuclear cells of bone marrow smear in the three dose groups were lower than those in the negative control group (P<0.05). The number of splenocytes in high dose group significantly reduced (P<0.05), and there was a dose-response relationship. The thickness of the periarterial lymphatic sheath of spleen in high dose group was significantly lower than that in the negative control group (P?0.05), and there was a dose-response relationship; the size of germinal center of spleen in high dose group was significantly lower than that in the negative control group (P?0.05). Humoral immunity toxicity tests showed that serum IL-5 level in the three dose groups markedly decreased (P?0.05), and there was a dose-response relationship. The activity of DTH in high dose group was significantly lower than that in the negative control group (P?0.05), and there was a dose-response relationship. There were no significant difference of CTL and macrophage phagocytosis between the three dose groups of atrazine and the negative control group (P>0.05).Conclusion:Malathion has an impact on some immunopathology and humoral immunity parameters in BALB/c mice; low-dose malathion significantly impacts on thymus weight, bone marrow cell classification and serum cytokines. It can preliminarily conclude that LOAEL of malathion is 16 mg/kg BW. Atrazine has an impact on some immunopathology, humoral immunity, and cellular immunity parameters in BALB/c mice; low-dose atrazien led to significant decreases of weekly body weight at week l-week 3, the numbers of leukocyte and neutrophil in whole blood, the percentages of polychromatic erythroblast and mononuclear cells, and serum cytokine IL-5 level. It can preliminarily conclude that LOAEL of atrazine is 23 mg/kg BW.Part III:Study of corn genetically modified with Bt CrylAh gene on immunotoxicity.Methods:The female BALB/c mice with body weight of 18 g-22 g were used for the study. All animals were observed within 3 days of arrival, then randomly assigned to different groups with ten mice per group. The study consisted of primary screening and secondary screening tests with nine substudies. And each substudies was set into four groups:the negative control group, the parental corn group, the GM corn group and the positive control group (200 mg/kg of cyclophosphamide via intraperitoneal injection 24 h before the termination of the study). Mice in the GM corn group and the parental corn group were fed with diets containing 70% corresponding corn. Mice in the negative control group and the positive control group were fed with AIN93G diet. Mice were housed by group with ad libitum access to water and nutritional propagation diets. The parameters and methods of immunopathology, humoral immunity, cellular immunity and nonspecific immunity of primary and secondary screening were accordance with those of Part I.Results:Primary screening system:when compared with the negative control group, immunopathology examination showed that weekly body weight at week 4 of mice fed with corn genetically modified with Bt CrylAh gene increased significantly, the number of mononuclear cells in peripheral blood decreased significantly, but there was no significant difference between the GM group and the parental group. Corn genetically modified with Bt CrylAh gene had no effects on the weights and histopathology of spleen, thymus, lymph nodes, liver and kidney as well as clinical biochemical parameters in BALB/c mice. And it also had no effects on the PFC and IgA, IgM, and IgG levels reflecting humoral immunotoxicity, proliferation activity of T lymphocyte and B lymphocyte reflecting cellular immunotoxicity, and NK cell activity reflecting nonspecific immunotoxicity. Secondary screening system:Immunopathology examination showed that the percentages of lymphocytes and lobulated granulocytes in bone marrow cells in the GM group decreased significantly, the percentage of rod granulocytes of mice in the GM group increased when compared with the negative control group (P<0.05). The thickness of the periarterial lymphatic sheath of spleen in the GM group was significantly lower than that in the negative control group (P?0.05), the size of germinal center of spleen in the GM group was significantly higher than that in the negative control group (P<0.05). Humoral immunity toxicity tests showed that serum IL-5 level in the GM group was lower than that in the negative control group (P<0.05). However, there was no significant difference of above mentioned parameters between the GM group and the parental group (P>0.05). There were no significant difference of CTL, DTH, and macrophage phagocytosis among the GM group, the parental group, and the negative control group (P>0.05).Conclusion:The corn genetically modified with Bt CrylAh gene has no effects on the immunopathology, cell immunity, humoral immunity and nonspecific immunity in BALB/c mice by primary and secondary screening systems. The corn genetically modified with Bt CrylAh gene is considered consistent with the parental corn in terms of the effects on immune system in BALB/c mice, and there is a "substantial equality". It is concluded that the corn genetically modified with Bt CrylAh gene is as safe as its parental corn in immunotoxicological evaluation. |
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