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 (micrograms/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.

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.

Production and characterization of monoclonal antibodies against aflatoxin M1.

By using an indirect enzyme-linked immunosorbent assay, four monoclonal antibodies were selected after fusion of mouse P3-NS1-Ag4-1 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with aflatoxin M1 (AFM1) conjugated to bovine serum albumin. Two of these antibodies were found to be specific for AFM1 and were designated AMW-1 and AMW-4. The specificities of AMW-1, which had higher affinity to AFM1, were determined by a competitive direct enzyme-linked immunosorbent assay with peroxidase-AFM1 as the marker. The relative cross-reactivity of each toxin (relative to AFM1) with AMW-1, as determined by the amount of aflatoxin necessary to cause 50% inhibition of enzyme activity, was 12, greater than 40, 12, and greater than 40 for B1, B2, G1, and G2, respectively.

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.

Immunosuppression in weanling and adult Sprague-Dawley rats induced by acute exposure to 3,3',4,4'-tetrachloroazoxybenzene.

The acute immunomodulatory effects of the environmental and occupational contaminant, 3,3',4,4'-tetrachloroazoxybenzene (TCAOB), were investigated on selected immune parameters in weanling and adult Sprague-Dawley rats. Significant immunotoxic effects were found 17 days after 4 doses of 25 mg/kg TCAOB, administered i.p. The main non-immune toxic effects were decreased body, kidney, heart and testis weights, and a simultaneous increase in liver weight. The immune parameters showing significant suppression were: thymic weight, splenic plaque forming cell populations and function, pertioneal macrophage chemiluminescence, and bone marrow cellularity. Weanling animals were affected by TCAOB to a greater extent than adults on both the multiple and single dose regimens. The immunotoxic effects were found to be qualitatively similar to those of its isosteric analog, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results demonstrate that TCAOB is a very potent immunotoxic compound and may have long-term effects after a single exposure. This study is the first investigation into the effect of TCAOB on immune functions.

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.

Influence of feeding chlorocholine chloride and glyphosine on selected immune parameters in deer mice, Peromyscus 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.

The immunomodulatory effects of two plant growth regulators, cycloheximide and maleic hydrazide, in white mice.

The immunomodulatory effects of two plant regulators, cycloheximide and maleic hydrazide, were investigated by injecting the compounds twice weekly for 4 weeks in female Swiss Webster white mice. The animals were antigenically challenged with sheep red blood cells on day 24 of the study. Although humoral immunity was the primary system examined, analysis included all of the following parameters: spleen plaque forming cells (PFC's), lymphocyte viability, spleen lymphocyte counts, hemolysin titers, total plasma proteins, total white blood cells counts, hematocrit, total body weight, and liver, thymus and spleen weights. Cyclophosphamide and physiological saline were used as the respective positive and negative control substances. The dosing was as follows: cyclophosphamide at 50 mg/kg/injection, cycloheximide at 12.5, 25 and 50 mg/kg/injection and maleic hydrazide at 125 and 250 mg/kg/injection. Cycloheximide significantly (P less than .05) reduced, in a dose-dependent fashion, the thymus wt/gram of body weight, the number of PFC's/gram of spleen, total lymphocytes/gram of spleen, PFC's per 10(6) viable spleen lymphocytes and the hemolysin antibody titer levels. Maleic hydrazide significantly reduced thymus weights and moderately lowered the ratio of PFC's per 10(6) viable spleen lymphocytes. Maleic hydrazide significantly elevated total lymphocytes per gram of spleen and the hemolysin titer (up to 133% over saline control values). There was an elevation in the number of PFC's per gm of spleen by maleic hydrazide and the overall effects were dose related. Cycloheximide, a known inhibitor of protein synthesis, was distinctly the most suppressive of the two plant growth regulators. Both agents are widely utilized on agricultural products and the results suggest the need for care in their application and residue removal. Used properly, the plant growth regulators may pose little or no human health hazard, but this report documents a new biological activity for these agents.

Immunosuppression in mice induced by dioxin (TCDD) in feed.

Juvenile and adult mice (4 and 7 weeks old, respectively) were fed various levels of 2, 3, 7, 8-tetracholorodibenzo-p-dioxin (TCDD) incorporated in mouse feed for five weeks or more. Animal parameters monitored included body weight, organ weights, white blood counts, hematocrits, certain serum protein levels (see below), symptoms of overt toxicity and mortality. High exposure levels (100 ppb) produced marked suppression in total serum protein, gamma globulin and albumin, but an increase in the beta-globulins. Feeding levels of 10 ppb TCDD or more reduced the primary and secondary antibody response to both tetanus toxoid and sheep erythrocytes. The amount of suppression appeared to be dose related, with juvenile animals showing greater suppression than adults. Antibody suppression from the 10 ppb feed level was roughly equivalent to that observed from a single high dose (200 mg/kg) of cyclophosphamide (CY). No evidence of enhanced IgE synthesis was obtained from TCDD exposed animals. TCDD feeding also lowered contact sensitization to dinitrofluorobenzene and resistance to challenge with either Salmonella typhimurium or Listeria monocytogenes.

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.

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.

Murine macrophage-lymphocyte interactions: scanning electron microscopic study.

Light and scanning electron microscopic observations revealed murine macrophage-lymphocyte interactions involving the initial contact of peritoneal, spleen, or thymus lymphocytes with peritoneal macrophage processes or microprocesses followed by clustering of lymphocytes over the central nuclear area of the macrophages. Lymphocyte-lymphocyte clustering was not observed in the absence of macrophages. Attachment and subsequent clustering appeared not to require the presence of serum or antigen; the attachment of allogeneic or xenogeneic lymphocytes was comparable to that seen in the syngeneic system, but central clustering of these lymphocytes failed to occur. No attachment or clustering was observed when thymic lymphocytes were cultured with thymus derived fibroblasts rather than with peritoneal macrophages. Lymphocyte attachment to immune, antigen-activated, syngeneic macrophages occurred more rapidly than that to normal unstimulated syngeneic macrophages; however, lymphocytes attached to the "activated" macrophages appeared to be killed by a nonphagocytic mechanism. A similar increase in the rate of lymphocyte attachment to macrophages occurred in the presence of migration inhibitory factor. Subsequent lymphocyte clustering on macrophages was observed in the migration inhibitory factor-stimulated cultures. In addition, lymphocyte-macrophage interactions similar to those in vitro were observed to occur in vivo on intraperitoneally implanted cover slips.

Preparation of cultured cells for SEM: air drying from organic solvents.

Air evaporation from organic solvents of differing polarities and surface free energies was used in the preparation of cultured murine peritoneal macrophages for scanning electron microscopy (SEM). The surface structural features of these cells were compared to the surfaces of similar cells prepared by the critical-point procedure. In general, all organic solvents produced a marked collapse of cell structure resulting in an increase in surface irregularity. Fracture surfaces and sharply defined contours including numerous flaps and ridges were characteristic of the non-polar solvent dehydrated samples. Polar solvent dehydrated samples, including those dried from solvents of low surface free energy, exhibited a secondary flowing and settling of the cell membrane. Primary collapse was apparent but cell contours were smoothed and rounded. Overall cell shape and cell-to-cell relationships were retained regardless of solvent type. It is suggested that solvent evaporation may prove useful in certain cases, though investigators are advised to use caution when interpreting the results obtained by such procedures.

Production of antibody against ochratoxin A.

Antibody against ochratoxin A was obtained after repeated injection of different protein-ochratoxin A conjugates to rabbits. Among many protein-ochratoxin conjugates tested, bovine serum albumin-ochratoxin A was found to be the best antigen. The antibody is specific for ochratoxins A, C, and T, but is not specific for ochratoxins B, alpha, and other coumarin derivatives. The sensitivity for ochratoxin A detection using a binding assay is in the range of 0.5 to 10 ng/0.5-ml sample. Detailed methods for the preparation of protein-ochratoxin conjugates, preparation of immune serum, and methods for antibody titers are described.

Alterations in mouse macrophage migration: a function of assay systems, lymphocyte activation product preparation, and fractionation.

Supernatants from mouse spleen cell and peritoneal cell cultures were tested for the presence of lymphocyte activation products. Supernatants from mouse spleen cell and peritoneal cell cultures incubated with brucella antigens contained a macrophage migration inhibition factor(s) and a macrophage spreading factor(s) only if the cells were harvested from Brucella-infected mice. After dialysis and freeze-drying, the supernatants were fractionated by preparative acrylamide-gel electrophoresis. Three fractions with lymphocyte activation product activity were obtained from the fractionated supernatants of mouse spleen cells and peritoneal cells harvested from Brucella-infected mice and cultured with brucella antigen. One fraction inhibited mouse macrophage migration from capillary tubes but not from agarose wells. A second fraction not only inhibited macrophage migration from both agarose wells and capillary tubes, but also contained an activity(s) that stimulated macrophage migration through Nuclepore filters and induced macrophage spreading. A third fraction timulated macrophage migration from agarose wells and also contained an activity(s) that stimulated macrophage migration through Nuclepore filters. Fractionated supernatants of mouse spleen cells and peritoneal cells harvested from uninfected mice incubated with and without brucella antigen, as well as of cells harvested from infected mice and not incubated with antigen, did not contain detectable lymphocyte activation products.