Loren D. Koller

Effects of Environmental Contaminants on the Immune System

Publisher Summary This chapter discusses the effects of environmental contaminants on the immune system. Environmental compounds, such as insecticides, pesticides, herbicides, and heavy metals are widely distributed throughout the world. The majority of these compounds are beneficial when used for specific purposes, handled properly, and applied as recommended by the manufacturer. However, many become contaminants of the environment, either by their improper application or because of their persistence in the ecosystem in soil, plants, water, and animal tissue. The degree of toxicity varies considerably among compounds and often among species. One method of initially ascertaining whether an environmental contaminant can affect the immune response of an animal is to challenge a treated animal with an LD-50 dose of an infectious agent. This procedure is simple and reliable and establishes whether the compound actually interferes with the course of disease. Infectious agents most commonly used are bacteria and viruses.

Effect of Lead, Polychlorinated Biphenyls, and Cyclophosphamide on Rat Natural Killer Cells, Interleukin 2, and Antibody Synthesis

Interleukin 2 (IL2) activity, natural killer cell (NKC) cytotoxicity, and serum antibody (Ab) levels were assessed in rats exposed to 10 or 1000 ppm lead (Pb) as lead acetate in the drinking water or 50 or 500 ppm polychlorinated biphenyls (PCB) as Aroclor 1254 in the feed for 10 weeks or injected one time with 75 mg/kg cyclophosphamide (CY). Assays for IL2 activity and NKC cytotoxicity were also performed following in vitro exposure of rat splenocytes to Pb (0.4 or 40 micrograms/ml) or PCB (0.4 or 20.0 micrograms/ml) for 24 hr in vitro. NKC cytotoxicity and IL2 activity were significantly suppressed following in vitro exposure to either Pb or PCB. Chronic exposure to PCB, but not Pb, significantly reduced NKC cytolytic activity and significantly elevated Con A-stimulated IL2 activity. Ab synthesis was significantly suppressed in groups of rats chronically exposed to Pb or PCB. CY-injected rats had significantly reduced IL2 activity, NKC cytotoxicity, and Ab levels. High background levels of IL2, presumably induced by KLH injections shortly before termination, were significantly suppressed in PCB-, Pb-, and CY-treated rats. This suppression of IL2 activity was completely reversed by in vitro stimulation with Con A in Pb- or PCB-, but not CY-, treated groups. These results indicate that PCB, Pb, and CY alter IL2 synthesis and adversely affect NKC cytotoxicity and Ab synthesis following in vivo or in vitro exposure. The effects of PCB on NKC cytotoxicity may partially explain the tumor-promoting effect of this chemical via compromising immunosurveillance.

A sensitive delayed-type hypersensitivity model in the rat for assessing in vivo cell-mediated immunity

Many drugs and other chemicals can alter cell-mediated immunity (CMI), a response that often correlates with delayed-type hypersensitivity (DTH). Several DTH assays were evaluated to determine a method best suited for assessing chemically induced modulation of CMI in rats. The effects of various antigens, adjuvants, doses, routes, and immunosuppressants were investigated. The DTH method which produced optimum results in rats uses a footpad swelling reaction elicited by specific preparations of bovine serum albumin (BSA) and Freunds complete adjuvant (FCA). The rats were sensitized with 100 micrograms BSA in FCA injected subcutaneously at the base of the tail, and were challenged 7 days later with 75 microliter of 2% heat-aggregated BSA suspension injected into the left rear footpad. Footpad swelling was measured with pressure calipers 24 h later and compared to the contralateral footpad which was sham-injected with 75 microliters of physiological saline. Antigen-injected footpads were nearly double the thickness (7-8 mm) of the control footpads (3-4 mm), and variation between animals was small (CV = 5%). Dexamethasone and cyclophosphamide significantly suppressed the DTH reaction. Histopathological examination of the DTH reaction sites revealed a mononuclear cell infiltrate which is characteristic of type IV hypersensitivity. In addition to being highly quantitative and sensitive, this method provides a simple and reproducible assessment of CMI responses in the rat.

Alteration of natural killer cell-mediated cytotoxicity in rats treated with selenium, diethylnitrosamine and ethylnitrosourea

Weanling, female Sprague--Dawley rats were divided into 14 separate groups. Three of these groups were administered 0.5, 2.0 or 5.0 ppm selenium (Se) in the drinking water for 10 weeks. Three groups received intraperitoneal injections of 1, 5 or 10 mg/kg diethylnitrosamine (DEN) twice weekly for 10 weeks. The remaining animals received 0.150% or 0.316% ethylurea (EU) in the feed and 1 or 10 ppm nitrite as sodium nitrite in the drinking water either alone or in combination. Separate groups of rats treated with cyclophosphamide (CY) were included as positive immuno-suppressed controls. Following the 10-week chemical exposure period, splenic natural killer (NK) cell-mediated cytotoxicity was assessed by a 4-h chromium release assay using YAC-1 tumor cells as targets. The NK cell cytotoxic response was enhanced in both the low and medium dose selenium-exposed groups. In contrast, rats exposed to 0.316% EU + 10 ppm NO2 had significantly depressed NK cell activity. CY treatment also resulted in a significant reduction of splenic NK cell cytotoxicity.

Effects of chlorinated phenols on immunity in rats

Female Sprague - Dawley rats were exposed to 0, 5, 50 or 500 ppm 2-chlorophenol (2-CP) or pentachlorophenol (PCP) from weaning to 3 weeks postparturition after breeding at 90 days of age. Progeny were weaned at 3 weeks of age and continued on chlorophenol treatment for 10 weeks at which time major immune functions were tested. Humoral immunity was measured by an indirect ELISA, cell-mediated immunity was monitored by delayed-type hypersensitivity (DTH) to oxazolone, and macrophage function was tested by phagocytosis of sheep red blood cells. Rats treated with cyclophosphamide were included as a positive immunosuppressed control. PCP-treated rats had significantly decreased antibody titers and DTH response and increased induced peritoneal macrophage numbers which displayed hyperphagocytic activity. Immune responses in rats treated with 2-CP were not significantly different from controls. The data indicate that (1) the immune system may be a sensitive target for PCP toxicity but not for 2-CP, (2) closely related chlorophenolic chemical isomers may exert different toxic effects on the immune system, and (3) PCP can exert depressive effects on some major immune parameters while enhancing others.

Evaluation of ELISA for detecting in vivo chemical immunomodulation

Immunotoxicology is a relatively new field of investigation and is becoming recognized and used by toxicologists involved in drug and chemical testing. The purpose of this study was to evaluate the enzyme-linked immunosorbent assay (ELISA) as a means of quantitating humoral immune responses in rats exposed to immunomodulating chemicals. The ELISA proved to be highly sensitive and quantitative, simple to perform, and reliable. The assay is also economically feasible and automated so that large numbers of samples can be analyzed at one time. The antigen used in the study was bovine serum albumin, and the immunosuppressive chemicals used to validate the system were lead, polychlorinated biphenyl, cyclophosphamide, and dexamethasone. Practical application of the procedure to immunotoxicology testing is discussed.

Effect of toxaphene exposure on immune responses in mice.

Toxaphene was fed to female weanling Swiss-Webster mice at dosages of 10, 100, and 200 ppm for 8 wk. Immunologic assays revealed depressed IgG antibody formation in those animals receiving 100 and 200 ppm toxaphene, as compared to controls. Cell-mediated immune responses were not affected in the toxaphene-exposed mice. In another experiment, mature female mice fed the same amounts of toxaphene were mated 3 wk after feeding began and were maintained on the diets until 3 wk after parturition, at which time the pups were weaned onto the control ration. Assays performed on the offspring 8 wk after their birth revealed suppressed antibody formation in the 100-ppm-toxaphene group and enhanced antibody formation in the 200-ppm group. The cell-mediated immune response was suppressed in the offspring from the 100-ppm group, while no change from the controls occurred in the other groups. Phagocytic ability of macrophages was significantly reduced in all toxaphene-treated groups, but to a greater extent in the offspring of the mice that consumed 100 ppm toxaphene.

The effect of lead and polychlorinated biphenyl exposure on rat natural killer cell cytotoxicity.

Splenic natural killer (NK) cell cytotoxicity was assessed in rats chronically exposed to lead (Pb) as lead acetate in the drinking water or polychlorinated biphenyl (PCB) as Aroclor 1254 in the feed. Rats treated with cyclophosphamide were included as positive immunosuppressed controls. Weanling, male Sprague-Dawley rats exposed to 50 and 500 ppm PCB in the feed for ten weeks exhibited significantly suppressed (P less than 0.01) splenic NK activity. Cyclophosphamide injected i.p. six days prior to termination at a dose of 75 mg/kg also significantly inhibited splenic NK activity. NK cell activity was reduced, though not significantly, in spleen cells isolated from animals exposed to 10 and 1000 ppm Pb as Pb acetate in the drinking water for ten weeks. In vitro exposure of rat spleen cells to PCB at concentrations of 0.4 and 20.0 micrograms/ml similarly resulted in a significant depression of splenic NK cell activity. In addition, in vitro exposure to lead at the same concentrations resulted in suppressed NK cell cytotoxicity of rat splenocytes. These results indicate that two environmental contaminants have the ability to adversely affect NK cell cytotoxicity. The effects seen here with Pb and PCB on NK cells may in part explain the tumor inducing effect these chemicals are suspected of possessing via compromising the immune surveillance system.

Multiple Immunoassay in a Single Animal: A Practical Approach to Immunotoxicologic Testing

A battery of immunoassays were developed for the rat which measured the major arms of the immune system, namely, humoral immunity, cell-mediated immunity (CMI), and macrophage function. Humoral immunity was assessed by the enzyme-linked immunosorbent assay (using keyhole limpet hemocyanin as antigen). CMI was measured by delayed-type hypersensitivity to heat-aggregated bovine serum albumin using a footpad swelling technique. Macrophage function was assessed by measuring the ability of macrophages to phagocytize sheep red blood cells in vitro. The feasibility of performing all three immunoassays in the same animal was investigated by treating groups of rats with all combinations of antigenic exposure used to induce each type of response. Multiple antigenic treatment regimens did not significantly alter responses to individual antigens used in this study. Animals exposed to the concurrent antigen treatment regimens remained sensitive to immunosuppressants. These data suggest that multiple immunoassays can be successfully performed utilizing a single animal which has been concurrently treated with different antigens. The inclusion of additional assays into this protocol and the efficacy to immunotoxicologic testing is discussed.

Neoplasia Induced in Male Rats Fed Lead Acetate, Ethyl Urea, and Sodium Nitrite

Sprague-Dawley rats were exposed to 0, 26, or 2600 ppm lead as lead acetate in drinking water for 76 weeks. At 28 weeks of lead exposure, a portion of each group was exposed simultaneously to 6.36 g/kg ethyl urea (EU) and 2.0 g/kg sodium nitrite (NaNO2) for a duration of 20 weeks, and then continued an additional 28 weeks on standard diet free of EU and NaNO2. The animals were observed for incidence, latency, and distribution of tumors. Rats exposed to 2600 ppm lead alone had 81% renal tumors, while rats given 2600 ppm lead in combination with EU/NaNO2 had a 50% incidence. Renal tumors did not occur in the EU/NaNO2 only or EU/NaNO2-26 ppm lead groups. The major tumor type found in EU/NaNO2-exposed rats was lymphosarcoma. ***Lead did not appear to be syncarcinogenic to the activity of ethylnitrosourea, the carcinogen formed by oral exposure to EU and NaNO2. The lead-induced renal neoplasms were histologically similar to those which occur spontaneously in man and, therefore, may serve as an animal model to study human disease.