Ronald D. Hinsdill

Effect of polychlorinated biphenyls on the immune responses of rhesus monkeys and mice

Abstract Female rhesus monkeys were fed either normal chow or chow containing 2.5 or 5.0 ppm of Aroclor 1248 (PCB). After 6 months, the PCB-fed monkeys developed chloracne, alopecia, and facial edema. After 11 months, control and treated monkeys were immunized with sheep red blood cells (SRBCs) and tetanus toxoid (TT). Monkeys fed 5.0 ppm of PCB had significantly lower anti-SRBC antibody titers than controls at only two intervals following primary immunization. Antibody response to TT was not measurably affected by PCB exposure. Both PCB-fed groups had consistently lower γ-globulin levels than controls. These results indicate that sustained exposure to low levels of PCB could have modest to slight immunosuppressive effects, which might be important depending on the general health of the individual. Mice fed up to 1000 ppm of PCB for 3 to 5 weeks exhibited no signs of PCB intoxication other than liver hypertrophy. However, these mice, when challenged with Salmonella typhimurium , showed higher mortality and significantly greater numbers of viable organisms in the spleen, liver, and blood than did controls. Similarly, exposed mice showed an increased sensitivity to endotoxin. Thus, mice exposed to subclinical doses of PCB (i.e., doses insufficient to produce overt clinical signs of intoxication) appear to have an impaired ability to withstand challenge by pathogens and an increased sensitivity to endotoxin.

The Effect of Perinatal Exposure to Tetrachlorodibenzo-p-Dioxin on the Immune Response of Young Mice

The immunocompetence of 5 week old offspring from mice fed control chow or chow containing 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was evaluated. The 5 ppb maternal feeding level was the only level that produced symptoms of intoxication in the offspring (i.e., facial alopecia and periorbital edema). Mice from mothers fed either 2.5 or 5 ppb of TCDD demonstrated thymus cortex atrophy and a significantly reduced spleen anti-SRBC plaque forming cell (PFC) response, but had normal serum anti-SRBC antibody levels following primary and secondary immunization. Contact sensitivity response to DNFB was significantly reduced only in offspring from mothers fed 5 ppb of TCDD. The blastogenic response of splenic T- and B-lymphocytes to concanavalin-A and E. coli lipopolysaccharide was unaffected by perinatal TCDD exposure. This correlated with the normal appearance of the T- and B-cell dependent areas of the spleens from these animals. There was no significant difference in the differential white blood cell counts between control and TCDD-exposed offspring. Offspring from mothers fed up to 5 ppb of TCDD withstood a live Listeria challenge as well as controls. However, maternal feeding levels as low as 1 ppb of TCDD rendered offspring more sensitive to an endotoxin challenge.

Aldicarb immunomodulation in mice: An inverse dose-response to parts per billion levels in drinking water

The effects of aldicarb (a carbamate insecticide and nematocide) on selected immune and non-immune parameters of mice were examined. The primary objective was to determine whether exposure to low levels of aldicarb through drinking water could affect selected immune functions. Aldicarb was administered via drinking water either at 10, 100, and 1,000 parts per billion (ppb)2 for 14 days or at 1, 10, 100, and 1,000 ppb for 34 days. The 34-day experiment was repeated twice with minor variations. Aldicarb significantly and repeatedly suppressed the splenic plaque forming cell response to sheep red blood cells at the lowest concentrations tested: the 1 ppb level elicited greater suppression of plaque forming cells and other immune factors than did 1,000 ppb of aldicarb. This inverse dose-response represents a dramatic departure from the classic toxicologic dose-response curve, where toxicity increases with increasing dose levels.

Oil and related toxicant effects on mallard immune defenses

A crude oil, a petroleum distillate, and chemically dispersed oil were tested for their effects on resistance to bacterial infection and the immune response in waterfowl. Sublethal oral doses for mallards were determined for South Louisiana crude oil, Bunker C fuel oil, a dispersant--Corexit 9527, and oil/Corexit combinations by gizzard intubation. Resistance to bacterial challenge (Pasteurella multocida) was significantly lowered in mallards receiving 2.5 or 4.0 ml/kg of Bunker C fuel oil, 4.0 ml/kg of South Louisiana crude oil, and 4.0 ml/kg of a 50:1 Bunker C fuel oil/Corexit mixture daily for 28 days. Ingestion of oil or oil/Corexit mixtures had no effect on mallard antibody-producing capability as measured by the direct spleen plaque-forming assay.

Evaluation of immunomodulatory chemicals: Alteration of macrophage function in vitro

In an effort to develop a useful screening procedure for detecting potential modulators of macrophage function, 25 chemicals were tested for their ability to alter the nonspecific phagocytosis and killing of Saccharomyces cerevisiae by elicited mouse peritoneal macrophages (PM). Parallel studies monitored PM viability so as to distinguish between effective modulation of phagocytic function and changes due solely to cytotoxicity. Dextran sulfate, iota-carrageenan, lambda-carrageenan, dipropyltin dichloride, and di-n-octyltin dichloride, at concentrations which did not produce overt toxicity, significantly reduced the percentage of PM capable of ingesting yeast. The carrageenans also decreased the average number of yeast ingested per PM, while increasing the ability of PM to kill yeast. Microbicidal activity was suppressed in PM treated with indomethacin and dextran sulfate. Di-n-octyltin dichloride and dextran sulfate diminished the adherence of PM, whereas levamisole enhanced PM adherence. Vanillin, propyl gallate, lead acetate, hydrocortisone 21-phosphate, and hydrocortisone 21-hemisuccinate decreased the percentage of PM capable of ingesting yeast, but not below 50% of the control. Gallic acid, methyl paraben, mercuric chloride, cadmium chloride, nickel chloride, chromic chloride, dimethyltin dichloride, diethyltin dichloride, dibutyltin dichloride, tetra-n-octyltin, gibberellic acid, cyclophosphamide, and azathioprine did not alter PM phagocytic function at noncytotoxic doses. The results indicate that chemicals can be grouped into three broad categories as either ineffective, weak modulators, or effective modulators of PM function.

Perinatal PCB Exposure and its Effect on the Immune System of Young Rabbits

Antibody mediated and cell mediated immune functions of offspring from New Zealand white rabbits fed control chow or chow containing 10, 100 or 250 ppm of Aroclor 1248 (PCB) were assessed. Only those offspring from mothers fed 250 ppm of PCB had a significantly lower contact sensitivity response to 2,4-dinitro, 1-fluorobenzene (DNFB). There was no significant decrease in either the plaque forming cell (PFC) response to sheep red blood cells (SRBC) or serum anti-SRBC antibody titer at any exposure level. Histopathologic examination of the spleen and thymus did not reveal any changes in cellularity that might be associated with immuno-suppression; nor did differential white blood cell counts or mitogen response of peripheral blood lymphocytes to PHA or Con-A differ significantly from controls.

Influence of feeding chlorocholine chloride and glyphosine on selected immune parameters in deer mice, peromycscus maniculatus

Exposure to the plant growth regulators, chlorocholine chloride (CCC) and glyphosine (GPS), resulted in significant immunomodulatory effects after feeding to deer mice (Peromyscus maniculatus) for 28 days. Cyclophosphamide (CP) and saline controls were included. Both CCC and GPS feeding levels were equivalent to 1, 10, 20, 40 and 80 mg/kg mouse/day. The parameters assessed were: spleen plaque forming cell (PFC) assays, hemolysin titers, white blood cell counts, bone marrow cellularity, hematocrits, plasma proteins, and spleen, liver, kidney, thymus and body weights. GPS significantly raised the ratio of liver/body wt, lymphocytes/g of spleen (at high doses only) and hemolysin titers (at low doses only). Lymphocyte viability was significantly reduced, while WBC counts and plasma protein levels were moderately lowered. CCC significantly reduced plasma proteins, PFCs/g of spleen, lymphocyte viability and hemolysin titers. It also affected thymus weights, WBC counts, lymphocytes/g of spleen and PFCs/10(6) lymphocytes. Most of the effects for both compounds followed a typical dose-response curve, but GPS gave a bimodal response in certain tests. The results demonstrate that CCC and GPS can act as immunomodulatory agents and show the feasibility of using deer mice for immunotoxicity studies of environmental contaminants and agricultural chemicals.

Screening for immunomodulators: effects of xenobiotics on macrophage chemiluminescence in vitro.

Macrophage chemiluminescence (CL) was evaluated as a primary screening assay by assessing the modulatory activity of 17 different chemicals. The chemicals were either known immunomodulatory drugs or environmental toxicants with reported immunomodulatory activity. Elicited mouse peritoneal macrophages were exposed to the chemicals in vitro, and CL was measured in response to an opsonized yeast stimulus. Ten chemicals (hydrocortisone, dextran sulfate, di-n-octyltin dichloride, dimethyltin dichloride, azathioprine, lambda carrageenan (l-carrageenan), lead, N-propyl gallate, gallic acid, and indomethacin) were identified as effective modulators of CL. The polyanions dextran sulfate and l-carrageenan either suppressed or enhanced CL, depending on the experimental conditions, while the remaining modulators were inhibitory. A series of secondary assays was used to verify this modulatory activity and to explore different mechanisms of action. Each effective modulator altered only a few specific components of the more complex CL response, and the following general mechanisms were apparent. At least 2 chemicals showed distinct antioxidant activity and thus probably did not alter functional aspects of macrophage CL. Chemicals which blocked Fc receptor function delayed the peak CL of macrophages stimulated by opsonized yeast. Nine of the 10 modulators inhibited hydrogen peroxide release, but only 3 inhibited the release of superoxide. Finally, some effective modulators were chemicals known to interact with cell membranes or specific membrane receptors, and these were able to directly induce a CL response without the addition of opsonized yeast as a stimulus. Thus, macrophage CL was a simple, quantitative, yet sensitive immunotoxicologic screening assay capable of identifying many known immunomodulatory drugs. Supplementary assays were useful both in confirming the functional effects of these chemicals on CL and in delineating potential mechanisms by which modulation of CL can occur.

Evaluation of chemicals for immunomodulatory effects using an in vitro antibody-producing assay

Abstract Several chemicals were examined for immunomodulatory effects using the Mishell — Dutton in vitro antibody-producing assay. The chemicals studied were either of environmental concern and/or of special interest to the food industry. A number of these compounds have been shown by others to be immunosuppressive in whole animal exposure studies, at least in certain species and in relatively high doses. Azathioprine (Imuran) (0.5 μg/culture), gallic acid (7 μg/culture), dextran sulfate (100 μg/culture), methyl paraben (100 μg/culture), and vanillin (200 μg/culture) were all found to directly suppress the in vitro anti-sheep red blood cell (SRBC) antibody response at noncytotoxic doses. All of these chemicals appeared to interrupt an early phase of the immune response, and had no effect on the actual release of specific anti-SRBC antibody. The Mishell — Dutton in vitro antibody-producing assay was found to be a sensitive detection system for direct-acting immunosuppressive chemicals. Chemicals that require activation to immunosuppressive intermediates are not detectable in this assay.

An analysis of risk from exposure to aldicarb using immune response of nonuniform populations of mice

The immunomodulation response of mice to low levels of aldicarb in drinking water was investigated in four series of studies. The splenic plaque forming cell (PFC) response to red sheep cells were measured for treatment levels of 0.01 to 1,000 ppb (μg/kg). Based on their beginning and end body weights, the animal populations were uniform in all series of tests, but based on their net body weights and PFC counts they were highly nonuniform in the 30 and 60 day tests and uniform in the 90 and 180 day tests.The mean PFC counts for animals in each treatment were calculated and compared with the mean PFC counts for animals in the controls in all four series of tests. This approach ignores the variability and nonuniformity in the animal population. The outcomes using this approach were stimulatory for the 30 and 60 day tests and inhibitory for the 90 and 180 day tests.An alternative approach was developed based on the analysis of the distributions of the relative PFC counts of each animal in a treatment with each animal in a control, and specifically addresses the variability and nonuniformity in animal population as integral parts of the analysis. The distribution peaks were estimated by maximum likelihood and kernel/bootstrap procedures and were used to summarize the tests. The outcomes were consistently inhibitory, indicating immune suppression. The outcome of this approach converged to the outcome of the mean PFC approach for the 90 and 180 day tests where the animal populations were uniform.