| BackgroundAs one of the most important causes of foodborne diseases, food contaminants include heavy metal, natural toxins, pesticides and toxic substances produced in food processing. People used to focus on the traditional toxicity of these substances, however, little is known about the special toxicity of these substances, targetin immune system, endocrine system and neural development system. Acrylamide is one of common food contaminants produced in high temperature cooking of starchy food, the current information on acrylamide toxicology mainly focused on the neurotoxicity and developmental toxicity, but there is very little information on the immune toxicity; so this study aimed to explore the immunotoxicity of acrylamide in BALB/c mice. China national food safety risk assessment(CFSA) expert committee has listed acrylamide as a priority assessment substance in2014to reassess, our study will also provide scientific toxicological data for risk assessment of acrylamide on the health effects.With the rapid development of science and technology and the improvement of living standards, consumer’s demand for food quality has also increased. Edible biologic medicines have got the favour of the food industry because of improving immunity and special nutritional benefits of these materials. Edible biologic medicines have high value of development and utilization, however, due to the complexity of their composition, if taken in large or wrong way, these materials can cause potential harm to the body or even death.Therefore, while some safety evaluation have been carried out with edible biologic medicines, researchers also need to pay more attention to the special toxicity of medicine and food materials, for instance, neurotoxicity, immune toxicity, endocrine disruption et al. These findings on edible biologic medicines will provide scientific basis to the development of new drugs and functional food. In recent years, momordica charantia L. has attracted wide attention as one of edible biologic medicines, however, most of these research have focused on the efficacy study of momordica charantia L. and its extract. Reports on the study of the immune function are rare, therefore, this study also aims to evaluate the immunotoxicological effects of momordica charantia extract in the BALB/c mice.The immune system is the main target of toxic substances, which in turn trigger the adverse reactions to the body, including health complications such as increased susceptibility, the incidence of cancer, allergic reactions and autoimmune diseases. The adverse effect to the immune system always appeared before other harmful effects casued by exogenous chemicals (e.g. changes in lymphocyte cells, changes in lymphocyte subsets, host resistance declined, changes in specificity immune, etc). The toxicological change of immune system is a sensitive biomarker in safety evaluation of some exogenous chemicals. Investigation on immune toxicity of materials is necessary.Objective(1) In order to provide scientific data for the future risk assessment of acrylamide on health, this study used the humoral immunity, cell immunity, non-specific immunity and additional immune experiment to evaluate the immunotoxicity of acrylamide, and explore the immune toxicity targets of acrylamide and its potential toxicity mechanism based on the guidance of immune toxicity evaluation proposed by ICH;(2) According to the guidance of immune toxicity evaluation proposed by ICH, this study used the humoral immunity, cell immunity, non-specific immunity and additional immune experiment to evaluate the immunotoxicity of momordica charantia extract with intent to provide the immune toxicology data for further edible safety evaluation.Methods(1) Evaluation of the immune toxicity of acrylamide:female BALB/c mice were randomLy assigned to different groups with ten mice per group. The animals were housed with five mice per cage. Four substudies were carried out, and the experiment period for each substudy were30days; the four substudies explored general toxicity and immunophathology, humroal immunity, cellular immunity and additional immunity, respectively; each substudy had five groups:the negative control group, low, middle, and high dose groups of acrylamide, and the positive control group(cyclophosphamide). General toxicity and immunotoxicological pathology parameters were examined as follows:body weight and organ indexes (liver, kidneys, spleens, thymus, and lymph nodes); hematology and clinical chemistry parameters; histopathology observation of liver, kidneys, immune organs including spleens, thymus, lymph nodes and bone marrow. Humoral immunity parameters included:T cell dependent antibody response (PFC), serum hemolysin and serum immunoglobulin levels (plasma IgA, IgM and IgG levels by ELISA). Cellular immune indexes included:ConA-induced spleen T lymphocyte proliferation and LPS-induced spleen B lymphocyte proliferation.NK cell activity and carbon-clearance test were used to examine nonspecific immune.Additional immunity parameters included:peripheral blood lymphocytes phenotype analysis (peripheral blood T lymphocytes, B lymphocytes and NK cells, Th cells, Ts cells percentage and CD4/CD8using flow cytometry), spleen cells number, serum cytokine IL-2, IL-4, IL-6, IL-10, IL-17, gamma-IFN and TNF using flow cytometry.(2) Preliminary study on the mechanism of immune toxicity of acrylamide:female BALB/c mice were randomLy assigned to different groups with ten mice per group.The animals were housed with five mice per cage. The experiment period for the study was30days; there was five groups in this experiment, which was the negative control group, low, middle, and high dose groups of acrylamide, and positive control group (cyclophosphamide). Mice were sacrificed after the study, the spleen and thymus was cut into two part immediately, with one-half fixed in10%formalin for pathological analysis, The other half of the the spleen and thymus were quickly frozen in liquid nitrogen and then stored at-80℃for RT-PCR and Western blotting. The level of cell apoptosis was detected by TUNEL in spleen and thymus; Bax, Bcl-2, caspase-3, PARP mRNA levels were determined by RT-PCR. Bax, Bcl-2, caspase-3, PARP proteins expression were determined by Western blotting.(3) Evaluation of the immune toxicity of Momordica charantia extract (MCE):the female BALB/c mice were randomLy assigned to different groups with ten mice per group. The animals were housed with five mice per cage. Four substudies were carried out, and the experiment period for each substudy were30days; the four substudies explored general toxicity and immunophathology, humroal immunity, cellular immunity and additional immunity respectively; each substudy had five groups:the negative control group, low, middle, and high dose groups of Momordica charantia extract, and the positive control group (cyclophosphamide). General toxicity and immunotoxicological pathology were examined as follows:body weight and organ indexes (liver, kidneys, spleens, thymus and lymph nodes); hematology and clinical chemistry parameters; histopathology observation of liver, kidneys, immune organs including spleens, thymus, lymph nodes and bone marrow. Humoral immunity parameters included:T cell dependent antibody response (PFC), serum hemolysin and serum immunoglobulin levels (plasma IgA, IgM and IgG levels by ELISA). Cellular immune indexes included:ConA-induced spleen T lymphocyte proliferation and LPS-induced spleen B lymphocyte proliferation.NK cell activity and carbon-clearance test were used to examine nonspecific immune. Additional immunity parameters included: peripheral blood lymphocytes phenotype analysis (peripheral blood T lymphocytes, B lymphocytes and NK cells, Th cells, Ts cells percentage and CD4/CD8using flow cytometry), spleen cells number, serum cytokine IL-2, IL-4, IL-6, IL-10, IL-17, gamma-IFN and TNF using flow cytometry.Results(1) General toxicity and immunotoxicological pathology examination showed that Peyer’s lymph nodes, absolute weight and relative weight of spleen and thymus were significantly lower in cyclophosphamide (CP) groups than those in the negative control group, there were moderate atrophy and extramedullary hemopoiesis cells decrease in the spleen. The germinal centers in lymphoid nodule were shrinking or disappeared. There were mild or moderate atrophy in the thymus; the Peyer’s lymph was atrophy, and follicular structure was not clear, the germinal centers were shrink or disappeared. Hematopoietic cells in bone marrow decreased, and ratio of lobulated nuclear cells in bone marrow increased, other type of cells also decreased. Hematology results showed that WBC, MCV, LYM, LYM(%) significantly decreased compared with those in the negative control group (P<0.05); MCHC, MPV, PDW, MON(%),NEUT(%) significantly increased compared with those in the negative control group.Clinical biochemical parameters showed that ALB, A/G significantly decreased compared with those in the negative control group; CHO significantly increased compared with those in the negative control group. Cellular immunity toxicity tests showed that ConA and LPS-induced spleen T lymphocyte proliferation in CP group significantly decreased compared with negative control group. Humoral immunity toxicity tests showed that PFC, HC50, serum IgG and IgM level in CP group significantly decreased compared with negative control group. According to the results of peripheral blood lymphocyte classification, the percentages of T lymphocyte (CD3+CD19-), Th(CD4+)cells, and the ratios of Th/Ts in peripheral blood in CP group significantly higher than those in the negative control group. Serum levels of cytokines detection showed that IL-6level in CP group significantly lower than those in the negative control group.(2) Result of immunotoxicity of acrylamide in BALB/c mice:for the acrylamide immunotoxicological tests, the general toxicity and immunotoxicological pathology examination showed that weekly body weights, terminal body weight, the number of Peyer’s patches, absolute weight of kidney, absolute weight and relative weight of spleen and thymus of mice in high dose group of acrylamide significantly decreased compared with those in the negative control group. White pulp of spleen was atrophy in high dose group of acrylamide, organization structure of spleen in other dose groups was clear, red pulp, white pulp and the fringe area was clear, no obvious abnormalities were observed. Thymus was mild atrophy, no obvious abnormalities were observed in Peyer’s patches and bone marrow. No pathologycial changes were observed in low and middle dose groups. Hematology results showed that RBC, HGB, HCT, RDW in middle dose group of acrylamide were significantly lower than those in the negative control group, RBC, HGB, MCHC, RDW, LYM, LYM(%), MON(%) in high dose group of acrylamide were significantly lower than those in the negative control group(P<0.05), MCV in high dose group of acrylamide were significantly higher than those in the negative control group. Other indicators were no statistical singnificance between each dose group of acrylamide and negative control group. Clinical biochemical parameters showed that TP, ALB, CHO in high dose group of acrylamide were significantly higher than those in the negative control group, TG were significantly lower than those in the negative control group. Cellular immunity toxicity tests showed that ConA-induced spleen T lymphocyte proliferation in high dose group of acrylamide significantly decreased compared with negative control group. Humoral immunity toxicity tests showed that HC50in high dose group of acrylamide significantly decreased compared with negative control group (P<0.05). No obvious effects on NK cell activity were observed in each dose group of acrylamide. According to the results of peripheral blood lymphocyte classification, the percentages of T lymphocyte (CD3+CD19-) in peripheral blood in each dose group of acrylamide were significantly higher than those in the negative control group, the percentages of NK cell were significantly lower than those in the negative control group. Serum levels of cytokines detection showed that IL-6level in high dose group of acrylamide was significantly lower than those in the negative control group, IL-17level in high dose group of acrylamide was significantly higher than those in the negative control group, IFN-y level in low and middle dose group of acrylamide was significantly higher than those in the negative control group.(3) Result of the mechanism of immune toxicity of acrylamide in BALB/c mice:Tunel test results showed tha the cell apoptosis rate (AI) of spleen and thymus in middle and high dose groups of acrylamide was significantly higher than those in the negative control group. The AI of spleen in the positive control group animals significantly increased compared with the negative control group.According to the RT-PCR test, the expression level of Caspase-3in spleen in low and high dose groups of acrylamide significantly decreased compared with those in the negative control group, however, the expression level of Caspase-3in spleen in the positive control group significantly increased compared with those in the negative control group. The expression level of PARP in thymus in low dose group of acrylamide and positive control group significantly increased compared with those in the negative control group.There were no significant differences in expression levels of Bcl-2, Bax, Caspase3, PARP gene between other groups of acrylamide and the negative control group. Western blotting tests showed that the expression level of Bcl-2in spleen in middle dose group of acrylamide was significantly higher than that in the negative control group, the expression level of Bax and PARP in the positive control group were lower than those in the negative control group. The expression level of Caspase-3in each dose group of acrylamide and positive control group singnificantly decreased than those in the negative control group. The expression level of Bcl-2and Caspase-3in thymus in each dose group of acrylamide and positive control group was significantly lower than those in the negative control group, the expression level of PARP in the middle and high dose group of acrylamide and positive control group singnificantly decreased than those in the negative control group,the expression level of Bax in the middle dose group of acrylamide and positive control group singnificantly decreased than those in the negative control group.(4) Result of immunotoxicity of Momordica charantia extract(MCE) in BALB/c mice: The general toxicity and immunotoxicological pathology examination showed that no significant difference in terminal body weight, number of Peyer’s lymph nodes, organ weight, liver/body ratio, kidney/body ratio, Spleen/body ratio, thymus/body ratio in each dose group of MCE was observed when compared with negative control group; No pathologycial changes were observed in liver, kidneys, spleen, thymus, lymph nodes and bone marrow in each dose group of MCE animals; MCE had no effects on hematology and clinical biochemical parameters. Humoral immunity toxicity tests showed that serum IgG level in the middle dosage group of MCE was significantly higher than the negative control group, the serum IgA level in the middle dose group of MCE was significantly higher than the negative control group. Cellular immunity toxicity tests showed that ConA-induced spleen T lymphocyte proliferation in low dosage group of MCE significantly increased compared with negative control group. According to the results of peripheral blood lymphocyte classification, the percentage of NK cells (CD3-CD49+) in each group of MCE were significantly lower than those in the negative control group. Serum levels of cytokines detection showed that IL-6, IL-4, TNF in low dosage group of MCE level was significantly higher than those in the negative control group.Conclusion(1) The immunotoxicity of acrylamide was systematic evaluated for the first time at home and aborad in this study. Acrylamide has obvious toxic effects on spleen, thymus and lymph nodes, and has obvious inhibitory effect on some immunopathology, humoral immunity, and cellular immunity parameters in BALB/c mice; no obvious toxic effect of non-specific immune functions was observed; high-dose acrylamide led to significant decreases of weekly body weight at week1～week4, the number of Peyer’s patches, absolute weight and relative weight of spleen and thymus, the effect of acrylamide on Th cell was more sensitive than on other T cells, acrylamide can inhibit the secretion of Th2type cytokines IL-6, and improve the secretion of Th17type IL-17cytokines level, which can increase the risk of autoimmune diseases. Low-dose acrylamide led to significant decrease of the percentages of T lymphocyte and NK cell in whole blood, the percentages of polychromatic erythroblast and mononuclear cells, and significant increase of serum cytokine IFN-y level. It can preliminarily conclude that LOAEL of acrylamide is4mg/kg BW. CFSA expert committee has listed acrylamide as a priority assessment substance in2014to reassess, results of this study can provide data support for acrylamide reevaluation and supplement immunotocixity data of acrylamide.(2)Apoptosis pathway is one of the mechanism of the immune toxicity action which cause immune organ injury. The mechanism of acrylamide toxicity on immune system may be through excessive activation of Caspase-3protein in thymus and spleen and suppression of normal expression of Bcl-2in thymus induced by in thymus by itself or its metabolites, which induced Bcl-2and Bax coordination role imbalance and resulted in the excessive apoptosis of spleen and thymus, finally led to the atrophy of spleen and thymus and triggered the immune function disorder; However, other molecular mechanisms might be also involved in the process, further investigation on the influence of acrylamide exposure of the body for a long time and comprehensive molecular mechanism are wanted.(3) The immunotoxicity of Momordica charantia extract was systematic evaluated for the first time at home and aborad in this study.Under the dosage in this study, there were no obvious adverse effects of Momordica charantia extrac on immune function in normal BALB/c mice, Momordica charantia extract promote the secretion of serum immunoglobulin IgA, IgM, and the cytokines IL-4, IL-6, and low dose group of Momordica charantia extract elevated the ConA-induced spleen T lymphocyte proliferation, which indicates that Momordica charantia extract can modulate the immune function of BALB/c mice, especially for humoral immunity and cellular immunity. |
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