Further evaluation of the incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats in a standard toxicology study.

ABSTRACT

A previous study (Ladics et al., 1995) conducted in our laboratory using the known immunosuppressant agent, cyclophosphamide, indicated that a functional assay for assessment of humoral immunity may be conducted in rats in a standard toxicology study. The objective of this study was to further examine the feasibility of conducting an immunotoxicological assay for assessing humoral immunity in rats in a standard toxicology study using a chemical, carbon tetrachloride (CCl4), whose principal target organ of toxicity is not the immune system. Specifically, the previous study and this study were done to determine whether the conduct of an assay for assessing humoral immune function would affect standard toxicological endpoints. Male CD rats were untreated or dosed orally for 30 or 90 days, excluding weekends, with vehicle or 12.5 or 25 mg/kg CCl4. Six days prior to sacrifice, selected rats were injected intravenously with sheep red blood cells (SRBC) for assessment of humoral immune function. One day prior to necropsy, blood for hematological and clinical chemical measurements was collected from each rat. On the day of necropsy standard protocol tissues were collected, weighed, processed to slides, and later examined microscopically. One-half of each spleen was used to assess spleen cell numbers and quantitate lymphocyte subsets (Thelper; Tcyt/sup; total T- and B-cells) by flow cytometry. Serum was analyzed for anti-SRBC IgM antibody by using an enzyme-linked immunosorbent assay. Administration of 12.5 and 25 mg/kg CCl4 for 30 days decreased SRBC-specific serum IgM levels 42 and 45%, respectively, while 25 mg/kg CCl4 for 90 days increased SRBC-specific IgM levels by 50%. CCl4 did not alter splenic lymphocyte subset numbers nor the weight nor morphology of lymphoid organs. Exposure to 25 mg/kg CCl4 did increase liver weight and serum sorbitol dehydrogenase levels, as well as produce centrilobular fatty change. SRBC administration did not alter any hematological or clinical chemistry parameters, nor lymphocyte subset numbers. With the expected exception of the spleen (slight increase in number and size of germinal centers), administration of SRBC did not significantly alter the weights or morphology of routine protocol tissues. Furthermore, administration of SRBC did not mask the rather mild hepatotoxic effects of CCL4 exposure observed in this study. Based on these and previous findings, it appears that a functional assay for assessing humoral immunity may be conducted in animals on standard toxicology study without altering standard toxicological endpoints.