Quantifying the relationship between multiple immunological parameters and host resistance: probing the limits of reductionism.

Although reductionist experimental designs are excellent for identifying cells, molecules, or functions involved in resistance to particular microbes or cancer cells, they do not provide an integrated, quantitative view of immune function. In the present study, mice were treated with either dexamethasone (DEX) or cyclosporin A (CyA), and immune function and host resistance were evaluated. Multivariate statistical methods were used to describe the relative importance of a broad range of immunological parameters for host resistance in mice treated with various dosages of DEX. Multiple regression and logistic regression analysis indicated that changes in 24 immunological parameters explained a substantial portion of the changes in resistance to B16F10 tumor cells or streptococcus group B. However, at least 40% of the change in host resistance remained unexplained. DEX at all dosages substantially suppressed numerous relevant immunological parameters, but significantly decreased resistance to Listeria monocytogenes only at the highest dosage. In contrast, CyA substantially decreased resistance to L. monocytogenes at dosages that caused relatively minor suppression of just a few immunological parameters (unfortunately, CyA data and host resistance data for L. monocytogenes were not suitable for multivariate analysis). These results illustrate that mathematical models can be used to explain changes in host resistance on the basis of changes in immune parameters, and that moderate changes in relevant immunological parameters may not produce the types of changes in host resistance expected on the basis of results from reductionist experimental designs.

Evaluation of the immunomodulatory effects of the disinfection by-product, sodium chlorite, in female B6C3F1 mice: a drinking water study.

Sodium chlorite is an inorganic by-product of chlorine dioxide formed during the chlorination of drinking water. Relatively little is known about the adverse health effects of exposure to sodium chlorite in drinking water. In this study, we evaluated sodium chlorite's immunomodulatory properties using female B6C3F1 mice and a panel of immune assays that were designed to evaluate potential changes in innate and acquired cellular and humoral immune responses. Female B6C3F1 mice were exposed to sodium chlorite in their drinking water (0, 0.1, 1, 5, 15, and 30 mg/L) for 28 days, and then evaluated for immunomodulation. Overall, minimal toxicological and immunological changes were observed after exposure to sodium chlorite. Increases in the percentages of blood reticulocytes, and the relative spleen weights were both observed at different sodium chlorite treatment levels; however, these increases were not dose-dependent. An increasing trend in the number of spleen antibody-forming cells was observed over the range of sodium chlorite concentrations. This increase was not, however, significant at any individual treatment level, and was not reflected by changes in serum IgM levels. A significant increase (26%) in the total number of splenic CD8+ cells was observed in mice treated with 30 mg/L of sodium chlorite, but not at the other concentrations. Splenic mixed leukocyte response and peritoneal macrophage activity were unaffected by sodium chlorite. Lastly, exposure to sodium chlorite did not affect natural killer cell activity, although a decrease in augmented natural killer cell activity (42%) was observed at the lowest sodium chlorite treatment level. These results suggest that sodium chlorite, within the range 0.1-30 mg/L, produces minimal immunotoxicity in mice.

Suppression of allergic immune responses to house dust mite (HDM) in rats exposed to 2,3,7,8-TCDD.

Exposure to various xenobiotics, including oxidant gases, diesel exhaust, and certain pesticides, has been reported to exacerbate pulmonary allergic hypersensitivity responses. Increased lymphocyte proliferative responses to parasite antigens or increased antibody responses to sheep erythrocyte have also been reported in rats exposed to TCDD before infection or immunization. As a result, these studies were conducted to test the hypothesis that TCDD exposure exacerbates the allergic response to house dust mite antigen. Brown Norway rats were injected, ip, with 0, 1, 10, or 30 microg TCDD/kg 7 days before intratracheal (it) sensitization to semipurified house dust mite allergen (HDM). Fourteen days later, rats were challenged with HDM and immediate bronchospasm was measured. At this time point, plus 2 and 7 days later, inflammatory cells in bronchoalveolar lavage fluid (BALF), HDM-specific IgE levels in serum, and HDM-driven cell proliferation in bronchial lymph nodes and spleen were evaluated. TCDD exposure decreased both immediate bronchoconstriction and specific IgE synthesis after the HDM challenge; 7 days later, HDM-specific IgE responses remained suppressed. Total serum IgE levels were similar in all groups. HDM challenge alone significantly increased cellular and biochemical indicators of lung injury, both of which were suppressed by TCDD exposure. The proliferative response of lymph node cells, but not of spleen cells, to HDM was also suppressed at the highest TCDD dose, although the splenic response to Concanavalin A was elevated. It appears that early events in the response to HDM are affected by TCDD exposure, since message for IL5 was dramatically reduced 2 days after sensitization, but not after challenge. We therefore conclude that TCDD exposure suppressed, rather than enhanced the development of allergic immune responses and the expression of immune-mediated lung disease.

Immunotoxicity of sodium bromate in female B6C3F1 mice: a 28-day drinking water study.

Bromate is one of the water disinfection by-products (DBPs) produced during the process of ozonation. The purpose of this study was to evaluate the immunotoxic potential of sodium bromate (SB) in female B6C3F1 mice. SB was administered in the drinking water for 28 days at doses of 80-800 mg/l. There was no difference in drinking water consumption between the animals exposed to SB and the tap water controls. Exposure to SB did not produce any signs of overt toxicity. Furthermore, no significant differences were observed in body weight, body weight gain, or the weights of thymus, liver, kidneys or lungs. No gross pathological lesions were observed in SB-treated animals. However, animals exposed to SB had a significant increase in absolute (28%) and relative (26%) spleen weights. The erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume (MCV), platelet count, total leukocyte count, and counts of differential leukocytes were unaffected by SB. A dose-related increase in reticulocytes was observed following exposure to SB with the greatest increase (78%) observed at the highest dose level. Overall, there were no changes in the absolute number of total T cells, CD4+CD8- T cells, CD4-CD8+ T cells, natural killer (NK) cells and macrophages. Exposure to SB did not affect the percentage of B cells, although a slight increase in absolute number of B cells at the dose of 600 mg/l was observed. There was no alteration in IgM antibody-forming cell (AFC) response, mixed leukocyte reaction (MLR) and NK cell activity after exposure to SB. When the activity of peritoneal macrophages, unstimulated or stimulated with IFN-gamma and LPS, was evaluated using the cytotoxic/cytostatic assay of B16F10 tumor cells, the suppressive effect of macrophages on the proliferation of B16F10 tumor cells was decreased after exposure to SB. In conclusion, SB, when administered in the drinking water at doses from 80 mg/l to 800 mg/l, produced minimal toxicological and immunotoxic effects in female B6C3F1 mice.

Aging and resistance to Trichinella spiralis infection following xenobiotic exposure.

Aging is accompanied by well-documented physiological changes, including alterations in the immune system that can lead to reduced resistance to a variety of infectious agents. We tested the hypothesis that immunosenescence exacerbates the immunosuppressive effect of xenobiotics. If proven true, a given dose of an immunosuppressive xenobiotic would cause greater suppression of host resistance in an aged population.

Evaluation of multivariate statistical methods for analysis and modeling of immunotoxicology data.

In immunotoxicology, the critical functions of the immune system (host resistance to infection and neoplasia) cannot be measured directly in humans. It is theoretically possible to predict changes in host resistance based on changes in immunological functions known to mediate host resistance. However, quantitative predictive models of this type have not yet been achieved in humans or in animal models. Multivariate statistical methods were developed for analysis and modeling of the effects of several explanatory variables on a dependent variable, and they seem well suited for attempts to predict host resistance changes caused by changes in immunological parameters. However, these methods were developed with the assumption that all variables can be measured for each experimental subject. For a number of reasons, this generally cannot be done in comprehensive immunotoxicology evaluations. In the present study, the suitability of multivariate methods for analysis of variables measured in different experiments was examined, using a limited data set consisting of immunological parameters that could all be measured for each mouse. Analysis was done on the original data set and test data sets produced by randomizing data within dosage groups. This was done to simulate the random pairing of data that would occur if measurements were obtained from different sets of mice in different experiments. Statistical theory indicates that randomization will disrupt the correlation matrices that are central in multivariate analyses. However, the present results demonstrate empirically that for at least one immunotoxicant (dexamethasone), remarkably similar multivariate models were obtained for the original and 109 randomized data sets. In contrast, the randomized data sets produced substantially different multivariate models when data obtained with a different immunotoxicant (cyclosporin A) were analyzed. The major difference between the two data sets was that dexamethasone strongly and dose-responsively suppressed many more parameters than did cyclosporin A. Additional work is needed to determine whether there are consistent criteria that could be used to identify immunotoxicology data sets, which would be amenable to multivariate analysis.

Effects of aging on resistance to Trichinella spiralis infection in rodents exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Immune function, including resistance to infection, decreases as humans and rodents age. We have shown that preinfection exposure of young (9-11 weeks) mice or rats to TCDD decreased resistance to Trichinella spiralis (Ts) infection, expressed as delayed onset or completion of parasite elimination and as increased muscle burdens of larvae. It has also been shown that aged mice express lower constitutive levels of resistance to Ts infection, compared to young adult animals. This study tested the hypothesis that the age-related decrease in constitutive levels of resistance to Ts infection exacerbates the decreased resistance to infection that follows TCDD exposure. This hypothesis addresses the concern that TCDD may pose a greater threat to the elderly than to the population at large. Animals were given a single oral dose of 1, 10, or 30 microg TCDD/kg, 7 days before infection. Eleven days later, young (approximately 10 weeks) control rodents had eliminated a greater proportion of the original parasite burden from the intestine than aged control animals. Nevertheless, parasite elimination was decreased by TCDD exposure only in young rodents. The effect of TCDD exposure on numbers of encysted larvae was evaluated only in rats. Increased larvae burdens occurred in young rats at 30 microg TCDD/kg and at 10 or 30 microg TCDD/kg in aged rats. Parasite-specific splenocyte and lymph node cell proliferation was suppressed following dioxin exposure in young mice; cells from aged mice were markedly less responsive to stimulation, yet less sensitive to TCDD exposure. The response to parasite antigens was not affected in aged rats exposed to TCDD, although elevated mitogen-driven B-cell proliferation was observed. These results indicate that age-related constitutive immunosuppression did not exacerbate TCDD-induced suppression of T-cell mediated adult parasite expulsion; rather, advanced age provided some degree of protection. On the other hand, a lower dose of TCDD was required in aged rats to suppress the combined humoral and cellular responses that limit the burden of encysted larvae, compared to young rats. These model-dependent results preclude acceptance or rejection of the tested hypothesis.

Evaluation of the potential immunotoxicity of bromodichloromethane in rats and mice.

In the past two decades, concern has been expressed over the potential carcinogenicity of disinfection by-products (DBPs) found in chlorinated drinking water. More recently, research efforts have expanded to include noncancer endpoints as well. The objective of the present studies was to evaluate the potential of bromodichloromethane (BDCM), one of the most prevalent DBPs, to adversely affect immune function in mice and rats following drinking water or gavage exposure. Antigen-specific immunity was assessed as the antibody response to sheep erythrocytes; responses to T- and B-cell mitogens were evaluated as a non-antigen-specific measure of the proliferative potential of splenic and mesenteric lymph node lymphocytes. In consideration of an exposure route relevant to humans, C57BL/6 mice received 0.05, 0.25, or 0.5 g BDCM/L and F344 rats received 0.07 or 0.7 g BDCM/L via drinking water. In order to evaluate the effects of higher doses, animals were administered 50, 125, or 250 mg BDCM/kg/d (mice) or 75, 150, or 300 mg BDCM/kg/d (rats) via gavage. Under the conditions of these studies, no significant adverse effects on immune function were observed in mice. Despite some changes that were observed in non-antigen-specific immunity in rats, these experiments suggest that the immune system is not a sensitive target organ for BDCM toxicity.

Evaluation of the potential immunotoxicity of chlorinated drinking water in mice.

Recent epidemiological studies have reported associations between the consumption of chlorinated drinking water and various types of human cancer; in addition, exposure to chlorine (Cl-) in drinking water has been reported to suppress certain immune functions in laboratory animals. The current studies were conducted to extend our knowledge of the effects of drinking water exposure to Cl-. Female C57BL/6 mice were administered hyperchlorinated drinking water (7.5, 15, or 30 ppm Cl-) for 2 weeks prior to sacrifice for evaluation of spleen and thymus weights, the plaque-forming cell (PFC) response, hemagglutination (HA) titer, and lymphocyte proliferation (LP). Significant reductions in organ weights and immune response were observed in the positive control groups (i.e. dexamethasone- or cyclophosphamide-exposed mice). No consistent differences were observed between the Cl--exposed animals and vehicle control mice for the evaluated parameters. Thus, under the conditions of these experiments, 2 weeks of exposure to hyperchlorinated drinking water had no apparent adverse effects on immune function.

Aquatic pollution-induced immunotoxicity in wildlife species.

The potential for chemicals to adversely affect human immunologic health has traditionally been evaluated in rodents, under laboratory conditions. These laboratory studies have generated valuable hazard identification and immunotoxicologic mechanism data; however, genetically diverse populations exposed in the wild may better reflect both human exposure conditions and may provide insight into potential immunotoxic effects in humans. In addition, comparative studies of species occupying reference and impacted sites provide important information on the effects of environmental pollution on the immunologic health of wildlife populations. In this symposium overview, Peter Hodson describes physiological changes in fish collected above or below the outflows of paper mills discharging effluent from the bleaching process (BKME). Effects attributable to BKME were identified, as were physiological changes attributable to other environmental factors. In this context, he discussed the problems of identifying true cause and effect relationships in field studies. Mohamed Faisal described changes in immune function of fish collected from areas with high levels of polyaromatic hydrocarbon contamination. His studies identified a contaminant-related decreases in the ability of anterior kidney leukocytes to bind to and kill tumor cell line targets, as well as changes in lymphocyte proliferation in response to mitogens. Altered proliferative responses of fish from the contaminated site were partially reversed by maintaining fish in water from the reference site. Peter Ross described studies in which harbor seals were fed herring obtained from relatively clean (Atlantic Ocean) and contaminated (Baltic Sea) waters. Decreased natural killer cell activity and lymphoproliferative responses to T and B cell mitogens, as well as depressed antibody and delayed hypersensitivity responses to injected antigens, were identified in seals fed contaminated herring. In laboratory studies, it was determined that rats fed freeze-dried Baltic Sea herring had higher virus titers after challenge with rat cytomegalovirus (RCMV) than rats fed Atlantic Ocean herring; perinatal exposure of rats to oil extracted from Baltic herring also reduced the response to challenge with RCMV. Keith Grassman reported an association between exposure to polyhalogenated aryl hydrocarbons and decreased T cell immunity in the offspring of fish-eating birds (herring gulls and Capsian terns) at highly contaminated sites in the Great Lakes. The greatest suppression of skin test responses to phytohemagglutinin injection (an indicator of T cell immunity) was consistently found at sites with the highest contaminant concentrations. Judith Zelikoff addressed the applicability of immunotoxicity studies developed in laboratory-reared fish for detecting altered immune function in wild populations. She presented data from studies done in her laboratory with environmentally relevant concentrations of metals as examples. Although the necessity of proceeding with caution when extrapolating across species was emphasized, she concluded that published data, and results presented by the other Symposium participants, demonstrate that assays similar to those developed for use in laboratory rodents may be useful for detecting immune system defects in wildlife species directly exposed to toxicants present in the environment.

Differences in the effects of dexamethasone on macrophage nitrite production: dependence on exposure regimen (in vivo or in vitro) and activation stimuli.

Exposure to glucocorticoids in vitro is known to suppress the production of reactive nitrogen intermediates (RNI) by macrophages, and it has been suggested that this contributes to the anti-inflammatory action of glucocorticoids in vivo. However, the effects of glucocorticoid administration in vivo on subsequent RNI production as measured in vitro are not known. In the present study, dexamethasone was administered in vivo and was also used to treat macrophages in vitro prior to, and during, stimulation of nitrite production by interferon-gamma (IFN-gamma) and/or bacterial lipopolysaccharide (LPS). Macrophages were isolated 24 h after daily administration of dexamethasone (0.1-30 mg/kg/day) to female B6C3F1 mice for 3, 6, or 16 days. In most cases, these cells produced an equal or greater concentration of nitrite in response to IFN-gamma, LPS, or IFN-gamma plus LPS, than cells from vehicle control mice. In contrast, continuous exposure of macrophages to dexamethasone during stimulation in vitro caused dose-dependent inhibition of nitrite production. However, the inhibition was much less pronounced when LPS or IFN-gamma together were used to stimulate the macrophages than when either was used separately. Similar results were noted when macrophages were exposed to dexamethasone for 24 or 72 h in vitro followed by a 0-24 h recovery period after removal of dexamethasone. Thus, immunosuppressive doses of dexamethasone in vivo do not decrease the induction of nitrite production 24 h after the last dose, whereas significant decreases are noted 24 h after termination of dexamethasone exposure in vitro. The basis for this difference is not clear, but there was no indication that administration of dexamethasone in vivo selects for a "glucocorticoid resistant" population of macrophages. These observations have implications with regard to the mechanisms of glucocorticoid-mediated anti-inflammatory and immunosuppressive action in vivo.

Dose-response relationships of tissue distribution and induction of CYP1A1 and CYP1A2 enzymatic activities following acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice.

Tissue disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to be dose-dependent in rats. However, no reported studies in mice have demonstrated dose- and time-dependent distribution of TCDD and the potential sensitivities of target tissues to enzyme induction. The objectives of this study were to determine in mice the effects of dose (0, 0.1, 1, or 10 micrograms [3H]TCDD/kg) and time (7, 14, 21, and 35 days posttreatment) on tissue distribution (18 tissues) and enzyme induction (CYP1A1 in liver, skin, and lung and CYP1A2 in liver). Distribution of TCDD-derived radioactivity in all tissues was dose- and time-dependent with nonlinear distribution. Liver-to-adipose tissue concentration ratios range from 0.6 to 3.1 (low to high dose at Day 7) demonstrating a dose-dependent shift in the disposition of TCDD. In contrast to liver, relative concentrations of percentage dose/g and percentage dose/total tissue decreased with increasing doses in all other tissues. At Day 7 and lowest dose, all tissues contained < 3% dose/g except for thyroid, adrenals, skin, liver, and adipose tissue which had 3, 6, 6, 15, and 24% dose/g, respectively. Induction of EROD activity, a marker for CYP1A1, was dose-dependent in liver, lung, and skin but did not parallel tissue concentrations of TCDD. At the highest dose, fold induction of EROD activity was two times greater in lung than liver, while the concentration in liver was 100 times greater than that in lung. Fold inductions of EROD activity in liver and skin were similar but the concentration was 20 times greater in liver than that in skin. Induction of hepatic acetanilide-4-hydroxylase (ACOH) activity, a CYP1A2 marker, was dose-dependent. Results of the present study demonstrated dose and time dependency in tissue distribution and induction of CYP1A1 and CYP1A2 as well as tissue sensitivities for enzyme induction in the female B6C3F1 mouse. These results provide important considerations for high- to low-dose extrapolations in risk assessments and use of sensitive markers of enzyme induction as surrogates for estimating exposure and in predicting risk.

Host resistance to Trichinella spiralis infection in rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

We have previously shown decreased resistance to Trichinella spiralis (Ts) infection and reduced parasite antigen-specific responses in B6C3F1 mice exposed to TCDD before infection. The current study was done to characterize the effects of preinfection administration of 1, 10, or 30 micrograms TCDD/kg on host resistance of female F344 rats to Ts infection and to examine parasite antigen-specific responses in the spleen and mesenteric lymph nodes of infected animals. TCDD exposure did not affect adult parasite elimination from the small intestine or the numbers of encysted larvae in the muscle, although host control of newborn larvae production in female parasites isolated from the highest dose group was compromised. Proliferative responses of lymphocytes cultured with parasite antigen were enhanced in groups of rats exposed to 30 micrograms TCDD/kg. These results, which are in marked contrast to the effects obtained in B6C3F1 mice, demonstrate a clear species difference in the effects of TCDD on immune function in rodents and underscore the need to determine which species more closely reflects the potential outcome of human exposure to TCDD.

Assessment of host resistance to Trichinella spiralis in mice following preinfection exposure to 2,3,7,8-TCDD.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been reported to decrease host resistance to a variety of infectious agents when exposure occurs prior to infection. Resistance to viral infection has been observed at doses as low as 0.1 microgram TCDD/kg body wt, well below the thymolytic dose in mice. In the present study, female B6C3F1 mice were exposed to a single intraperitoneal injection of 0, 0.1, 1.0, 10.0, or 30.0 micrograms TCDD/kg 7 days prior to infection to determine the effects of TCDD exposure on resistance to the nematode parasite Trichinella spiralis. Exposure to 10 or 30 micrograms TCDD/kg delayed adult parasite elimination from the small intestine. Significantly more larvae were released by female parasites and greater numbers of encysted larvae were recovered from the muscle of mice exposed to TCDD. Proliferative responses of splenocytes and mesenteric lymph node cells stimulated with T. spiralis antigen were significantly suppressed at exposure levels of TCDD > or = 1.0 microgram/kg 7 days after infection and in splenocytes only at 14 days after infection, demonstrating the greater sensitivity of proliferative responses to TCDD exposure than actual host resistance to Ts infection. Suppressed proliferation was observed at doses which produced TCDD concentrations > or = 0.2 pmol/g of lymphoid tissue on Day 7 of infection. In addition, it was determined that infected mice had higher TCDD levels than noninfected mice given the same dose. These results suggest an interaction between TCDD exposure and infection, i.e., that exposure to TCDD altered the host response to infection, while infection delayed elimination of TCDD from the host.

Repeated exposure to the polychlorinated biphenyl (Aroclor 1254) elevates the basal serum levels of corticosterone but does not affect the stress-induced rise.

Previous studies indicate that repeated exposure of weanling male Fischer 344 rats to Aroclor can cause immune system alterations but the pattern of effects suggested the release of corticosteroids may have played a role. Rats were exposed daily by gastric intubation to the polychlorinated biphenyl (PCB) Aroclor 1254 at 0.1, 1.0, 10, or 25 mg/kg for exposure durations of 5, 10 or 15 weeks. By the 15th week of dosing all groups displayed an elevation in the basal level of serum corticosterone but no change in adrenal weight. Further, rats exposed to Aroclor 1254 for 15 weeks and subjected to stress prior to serum collection displayed elevations in corticosterone levels equivalent to stressed control rats. The failure to observe altered adrenal structure indicative of hyperactivity in the presence of increased serum levels of corticosterone suggest these basal increases may be indirect rather than direct effects of Aroclor 1254.

Assessment of the immunotoxic potential of the fungicide dinocap in mice.

The immunotoxic potential of dinocap was evaluated in female C57BL/6J mice following in vivo and in vitro exposure to this fungicide. In in vivo studies, groups of mice were dosed by gavage with technical grade dinocap at dosages ranging from 12.5 to 50 mg/kg per day for 7 or 12 days and selected immune functions examined. Mice dosed at 50 mg/kg per day dinocap died after 4 days of dosing. Twelve days of dosing with dinocap at 25 mg/kg per day resulted in decreased thymus weights and cellularity, and increased spleen weights. No changes were observed in body weight, absolute differential peripheral leukocyte counts, the lymphoproliferative responses to B- or T-cell mitogens, the mixed lymphocyte reaction, or natural killer (NK) cell activity of spleen cells from mice exposed to dinocap. Lymphoproliferative responses to concanavalin A (Con A) and phytohemagglutinin (PHA), however, were reduced in thymocytes from mice dosed at 25 mg/kg per day dinocap. The cytotoxic T lymphocyte (CTL) response to P815 mastocytoma cells was enhanced in mice exposed for 7 days to 25 mg/kg per day dinocap. Exposure of mice for 7 days to 25 mg/kg per day dinocap also caused a significant reduction in the IgM and IgG plaque-forming cell (PFC) response to sheep red blood cells (SRBC). A time-course study indicated that dinocap-induced suppression of the IgM PFC response was due to a delay in the peak PFC response to SRBC. In vitro studies using murine thymocytes cultured with dinocap (10 micrograms/ml for 72 h) resulted in suppression of the proliferative response to Con A and PHA. Exposure of thymocytes to dinocap in vitro for as little as 30 min resulted in suppression of the mitogen-stimulated response in the absence of any apparent direct cytotoxic effect. These results suggest that dinocap alters the immune system of the mouse, however, these effects are relatively modest in terms of adverse immune function and are only seen at relatively high exposure levels.

Differences between rats and mice in the immunosuppressive activity of 2-methoxyethanol and 2-methoxyacetic acid.

Previous studies from this laboratory have demonstrated that 2-methoxyethanol (ME) and its principal metabolite 2-methoxyacetic acid (MAA) are immunosuppressive in young adult male Fischer 344 rats. In the present study, the immunosuppressive potential of ME and MAA was evaluated in young adult female Fischer 344 rats and C57BL/6J mice. Rats and mice were dosed by gavage with either ME or MAA in water, at dosages ranging from 50-400 mg/kg/day, for 10 consecutive days. Rats and mice were examined for alterations in body, spleen and thymus weights and mitogen-induced proliferation of splenic lymphocytes in vitro; separate groups were employed for the antibody plaque-forming cell (PFC) response to trinitrophenyl-lipopolysaccharide (TNP-LPS). Rats dosed at 100-400 mg/kg/day ME and rats dosed at 50-400 mg/kg/day MAA had decreased thymus weights in the absence of decreased body or spleen weights. Lymphoproliferative (LP) responses to concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and Salmonella typhimurium mitogen (STM) were all reduced in rats treated with all dosages of ME. Rats treated with MAA displayed similar reductions in these LP responses except that the responses to PWM and STM in rats dosed at 50 mg/kg/day were not reduced. In contrast to the effects of ME and MAA on these end points in the rat, no thymic involution or suppression of LP responses were observed in mice dosed at 50-400 mg/kg/day. The PFC response to TNP-LPS was suppressed in rats dosed with either ME or MAA at dosages of 100-400 mg/kg/day. ME and MAA, however, failed to suppress the PFC response in mice immunized with TNP-LPS. These results indicate that unlike Fischer 344 rats, C57BL/6J mice are insensitive to the immunosuppressive effects of ME and MAA at the dosages employed in this study. Whether the different sensitivities of these two rodent species to ME- and MAA-induced immunosuppression are due to immunologic, pharmacokinetic or metabolic differences within each species remains to be determined.

Comparative immunosuppression of various glycol ethers orally administered to Fischer 344 rats.

Oral dosing of adult male F344 rats with the glycol ether 2-methoxyethanol (ME) or its principal metabolite 2-methoxyacetic acid (MAA) results in the suppression of the primary plaque-forming cell (PFC) response to trinitrophenyl-lipopolysaccharide (TNP-LPS). In the present study, the PFC response to TNP-LPS was used to evaluate the immunotoxic potential of ethylene glycol (EG) as well as the glycol ethers 2-methoxyethyl acetate (MEA), 2-(2-methoxyethoxy) ethanol, bis(2-methoxyethyl) ether, 2-ethoxyethanol and its principal metabolite 2-ethoxyacetic acid, 2-ethoxyethyl acetate, and 2-butoxyethanol relative to ME and MAA. Rats were immunized with TNP-LPS and then exposed 4 and 28 hr later to 50, 100, 200, or 400 mg/kg of glycol ether or EG. Three days following immunization, the PFC response to TNP-LPS was determined. In addition to ME and MAA, only MEA, which was as effective as ME, suppressed the PFC response to TNP-LPS. Concomitant administration of the alcohol dehydrogenase inhibitor 4-methylpyrazole with ME or MEA prevented suppression of the PFC response by these glycol ethers. These results indicate that of the chemicals tested only ME, MEA, and MAA are immunosuppressive, and that oxidative metabolism via alcohol dehydrogenase is necessary for ME- and MEA-suppression of the response to TNP-LPS.

Host resistance to Trichinella spiralis infection in rats and mice: species-dependent effects of cyclophosphamide exposure.

Host resistance to Trichinella spiralis infection was compared in male rats (F344) and female mice (C57BL/6J) following various cyclophosphamide (CY) treatment schedules. Doses of CY given to mice were adjusted by body surface area to be comparable to rat doses. Adult parasite elimination was not affected by oral administration of 1.5, 3 or 6 mg CY/kg per day to rats or 1.05, 2.1 or 4.2 mg CY/kg per day to mice for 10 days. In rats, resistance was suppressed by a single oral dose of 80 mg/kg given the day prior to infection, but was not affected at 20 or 40 mg/kg. A single oral dose of 14, 28 or 56 mg CY/kg did not affect parasite expulsion in mice. Rats were also given four daily intraperitoneal (i.p.) injections of 20, 40 or 80 mg CY/kg per day and mice received 14, 28 or 56 mg CY/kg per day. Infected rats did not survive at the two higher dose levels and parasite expulsion was suppressed at 20 mg/kg per day; parasite expulsion was suppressed in mice by four i.p. injections of 56 mg CY/kg per day, but not by lower doses. In rats, doses of CY which suppressed adult parasite expulsion also severely suppressed the proliferative response of mesenteric lymph node cells (MLNC) to an extract of T. spiralis (TsE). However, significant suppression of TsE-driven blastogenesis occurred at a dose of CY which did not affect parasite expulsion, indicating that the proliferative response in rats was more sensitive to suppression than actual parasite elimination. In contrast, the proliferative response to the T cell mitogen concanavalin A was elevated in the MLNC of CY-exposed rats. This was determined to be related to the interval between CY dosing and the day of assay rather than to an effect of infection with T. spiralis. Mouse MLNC proliferative responses to TsE were not suppressed by CY treatment, even at levels of CY which suppressed adult parasite expulsion. Mice differed from rats in that CY exposure did not affect the proliferative response to concanavalin A in infected animals. The species-dependent differences observed in these studies may have been secondary to the greater sensitivity of rats to CY. Nonetheless, these results highlight the potential for species-specific responses to chemical exposure and underscore the need for additional comparative studies of host resistance in rats and mice.