The immunosuppressive effects of 10 mg/kg cyclophosphamide in Wistar rats

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Abstract

The purpose of this study is to identify the immunosuppressive effects of cyclophosphamide (a known immunosuppressant) in male Wistar rats which were administered orally for 30 days repeatedly, and to find a proper dose of cyclophosphamide at which the immunosuppressive effects is identified. The dose of cyclophosphamide is 2, 5, 10 mg/kg bw, respectively. The results showed that 10 mg/kg cyclophosphamide treatment induced decreases in body weight, body weight gain, relative weight of spleen and thymus, antibody plaque-forming cells, delayed-type hypersensitivity reaction, natural killer cell activity, lipopolysaccharide-induced B-cell proliferation, and Concanavalin A-induced T-cell proliferation. The histopathology observation on spleen and thymus revealed as decreased cellularity. But, no significant clinical symptoms of toxicities and stress were observed and no rats died. These results indicated that the immunosuppressive effects of 10 mg/kg cyclophosphamide dosed for 30 days orally were identified in male Wistar rats. Based on the results, it is considered that the dose of 10 mg/kg of cyclophosphamide orally for 30 days in male Wistar rats could be used as the dose of positive control group for chemicals immunotoxicity assessment tests. This study also proved that the testing procedures and the experimental techniques established in our laboratory for immunotoxicity tests in male Wistar rats are reliable and feasible.

Introduction

Immunotoxicology is the study of the potentially harmful effects that chemicals may have on the immune system, by causing immunosuppression, hypersensitivity (allergic) reactions, or autoimmunity. Immunosuppression are detected as marked decreases in immune function measured as an effect on humoral, cellular, or nonspecific parameters of the immune system. Many environmental chemicals suppress the immune response. Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), biphenyls (PCBs), some pesticides, some solvents, and so on, are shown to be environmental pollutants of potential immunosuppression. Immunosuppressive chemicals exposed under certain doses and durations, may cause increased incidences of infectious diseases and cancers. For example, it was reported that virus-associated mass mortalities among seals inhabiting northwestern Europe was associated with mixtures of environmental contaminants including PCBs, PCDFs, and PCDDs (Van Loveren et al., 2000, Barron et al., 2003). Thus, for the protection of human health and the environment, assessment of immunotoxicities of chemicals is needed.

OPPTS 880.3550 Immunotoxicity (OPPTS, 1996) is a guideline that has been developed by the Office of Prevention, Pesticides and Toxic Substances (OPPTS), United States Environmental Protection Agency. It is used in the testing of pesticides and toxic substances. The guideline is a harmonized one. It blended the testing guidance and requirements that existed in the OPPTS and appeared in Title 40, Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) which appeared in publications of the National Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development (OECD).

In our laboratory, the immunotoxicity assessment tests for a test substance are usually performed according the guideline of OPPTS 880.3550. A commonly used experimental animal strain in most toxicology assessment tests, the Wistar rats, is usually used and the animals are usually divided into five animal groups which including a vehicle-treated control group, a positive control group dosed with a known immunosuppressant and three dose level groups of the test substance. The animals of each group are treated orally by gavage for 30 days repeatedly. At the termination of the treatment, both non-functional and functional tests which demanded by the guideline of OPPTS 880.3550 are conducted to identify the immunosuppressive effects of the test substance. The non-functional parameters mainly are clinical signs of toxicities, body weight, haematology, relative weights of lymphoid organs, histopathology of lymphoid organs. The functional parameters usually are antibody plaque-forming cells (PFCs) assay for assessment of the effect of test substance on humoral immunity, the delayed-type hypersensitivity (DTH) reaction assay for assessment of the effect of test substance on specific cell-mediated immunity and natural killer (NK) cell activity assay for assessment of the effect of test substance on non-specific cell-mediated immunity. Finally, a no-observed-adverse effect level (NOAEL) are obtained and used for the immunotoxicological assessment.

The concurrent positive control group is necessary to demonstrate the sensitivity and reliability of the testing procedures. In addition, the dose of the known immunosuppressant used for positive control group should be appropriate. In one hand, it should induce significant changes of the endpoints described above. In the other hand, it should not induce obvious stress, malnutrition, or fatalities, but should ideally produce some measurable sign of general toxicity (loss of body weight). Cyclophosphamide, a known immunosuppressant, was recommended by the guideline of OPPTS 880.3550 for use in the positive control group. The proper dose of cyclophosphamide which could induce Wistar rat immunosuppression by orally treatment for 30 days repeatedly was not found in the reported researches. Thus, the purpose of this study is to find a proper dose of cyclophosphamide at which the immunosuppressive effects in male Wistar rats are identified. Then, the dose of cyclophosphamide could be used as the dose of positive control group in chemicals immunotoxicity tests. This study will also prove if the testing procedures and the experimental techniques established in our laboratory for the immunotoxicity assessment tests in male Wistar rats are reliable and feasible.

Section snippets

Animals and maintenance

Male Wistar rats (specific pathogen-free, SPF) with body weight of 160–180 g were purchased from Beijing Weitong Lihua Laboratory Animal Technology Ltd. Corporation (certificate no.: SCXK (Jing) 2002-0003).

Animals were kept in a barrier-sustained animal room and the certificate number of environmental condition was SYXK (Jing) 2005-0023. Stainless steel wire-mesh cages were used. Each cage had 5 rats of the same sex. The feed were supplied ad libitum and all rats were free to access to feed and

Results

The rats of each dose group did not show any obvious sign of general toxicities except that the body weight and body weight gain were decreased in the high dose group (Table 1). No rats died during the treatment. Thus, 10 mg/kg cyclophosphamide is a dose that did not produced significant stress, malnutrition, or fatalities, but ideally produced some measurable sign of general toxicity (loss of body weight), which accord with the demand of the guideline of OPPTS 880.3550.

The relative weights of

Discussion

Assays of the PFCs, DTH reaction, NK cell activity, LPS-induced B-cell proliferation and ConA-induced T-cell proliferation, were used in this study to identify the immunosuppressive effects of cyclophosphamide. The five assays in combination were reported to have a high degree of concordance with the predicted immunotoxic properties of chemicals (Krzystyniak et al., 1995).

The results showed that 10 mg/kg cyclophosphamide had induced decreases in body weight, body weight gain, relative weights of

Acknowledgements

This work was supported by National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention. The authors thank Prof. Tao Li, Prof. Yuxin Zheng and Prof. Bin Li for all-around support to this study, Dr. Yunchang Guo and Mr. Chunming Du for some technological instruction, Prof. Qing Gao for histopathology preparation and examination.

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