Marie M. Riddle

Immunotoxicity of tributyltin oxide in rats exposed as adults or pre-weanlings

A comparison was made between adult and pre-weanling rats of the immunotoxic effects of subacute dosing with bis(tri-n-butyltin) oxide (TBTO). Adult (9 weeks old) male Fischer rats were dosed by oral gavage with TBTO for 10 consecutive days at 1.25-10 mg/kg per dose or 3 times/week for a total of 10 doses at 5-20 mg/kg per dose. Adult rats similarly dosed by oral gavage with 6 mg/kg per dose cyclophosphamide (CY) served as positive controls. Pre-weanling rats (3-24 days old) were dosed 3 times/week for a total of 10 doses at 2.5, 5 or 10 mg/kg per dose. At various times after dosing rats were evaluated for alterations in body and lymphoid organ weights, mitogen and mixed lymphocyte reaction (MLR) lymphoproliferative (LP) responses, natural killer (NK) cell activity, cytotoxic T lymphocyte (CTL) responses and primary antibody plaque-forming cell (PFC) responses. In adult rats given 10 daily doses of TBTO, thymic involution was observed at a dosage of 2.5 mg/kg and mitogen responses to Con A and PHA were suppressed at 5 mg/kg. The PFC response was enhanced in adult rats dosed daily at 2.5 mg/kg. A dosage of 5 mg/kg given intermittently (3 times/week) to adults or pre-weanlings resulted in thymic involution. Reductions in mitogen responses were observed in adults dosed intermittently at 10 and 20 mg/kg and in pre-weanlings at 5 and 10 mg/kg. The MLR response was suppressed in adult rats dosed intermittently at 20 mg/kg and in pre-weanling rats at 10 mg/kg. NK cell activity was suppressed only in pups dosed intermittently at 10 mg/kg. CTL responses were not affected in either age group. Within 3 weeks following the last exposure of adult rats to TBTO all parameters returned to normal. On the other hand, LP responses to mitogens were suppressed in 10-week-old rats that were dosed with 10 mg/kg TBTO as pre-weanlings. However, this exposure regimen in reductions in body weight that persisted for up to 13 weeks of age, which suggests that TBTO may be a developmental toxicant. These data indicate that while exposure of young rats to TBTO resulted in immune alterations at doses lower than those required to suppress responses in adults, the observed effects may also be influenced by the developmental toxicity of this compound.

Evaluation of the immunotoxicity of low level PCB exposure in the rat

Weanling male Fischer 344 rats were exposed daily by gastric intubation for up to 15 weeks to the polychlorinated biphenyl (PCB) Aroclor 1254 at 0.1, 1, 10, or 25 mg/kg body weight. At 5, 10 and 15 weeks groups of rats were killed and immune functions were evaluated. The immune parameters examined included the following: body and lymphoid organ weights, mitogen-stimulated lymphoproliferative (LP) responses, natural killer (NK) cell activity, mixed lymphocyte reaction (MLR), and cytotoxic T lymphocyte (CTL) response. After 15 weeks of dosing body weights were reduced in rats receiving 25 mg/kg PCB while thymus weights were decreased in rats receiving 10 and 25 mg/kg. NK cell activity was reduced in rats dosed for 15 weeks at 10 and 25 mg/kg. The LP response to phytohemagglutinin was enhanced in rats dosed for 15 weeks at 25 mg/kg PCB. Exposure of rats to PCB did not affect the MLR or CTL responses. Other groups of rats were exposed to cyclophosphamide (CY) and served as positive controls for the immune assays employed. CY induced alterations in all of the immune parameters measured, indicating that this is an appropriate battery of immune function tests which is capable of detecting immune alterations in the rat. Alterations in immune function induced by daily gastric intubation with PCB were accompanied by reductions in body weight and/or hepatomegaly. These results suggest that the observed immune alterations may be related to the overt toxicity of this PCB in the rat.

The effects of nickel on immune function in the rat.

The immunotoxic potential of NiCl2 was evaluated in Fischer 344 rats following a single intramuscular injection at doses ranging from 10 to 20 mg/kg. Twenty-four hours following treatment, selected cellular and humoral immune function parameters were examined. Significant (P less than 0.05) decreases in body weights were observed in rats injected with 15 and 20 mg/kg NiCl2 as were decreases in spleen weights of rats receiving 20 mg/kg. The lymphoproliferative responses of splenocytes to the T cell mitogens concanavalin A (Con A), phytohemagglutinin (PHA), the T and B cell mitogen pokeweed mitogen (PWM) and the B cell mitogen Salmonella typhimurium mitogen (STM) were not significantly different from controls. No significant differences were observed between control and Ni-treated rats in the primary antibody response to sheep red blood cells (SRBC). On the other hand, natural killer (NK) cell activity was significantly (P less than 0.05) suppressed in rats injected with 10, 15, or 20 mg/kg NiCl2. NK cell suppression was observed in both male and female rats and for both allogeneic W/Fu-G1 target cells as well as xenogeneic YAC-1 target cells. Ni-induced suppression of NK activity was transient, with levels returning to control values within three days following treatment. Ni-induced suppression of NK activity was also manifested by an increase in mortality of rats injected with MADB106 tumor cells. These results extend to a second species our earlier findings that Ni suppresses NK activity.

Manganese chloride enhances natural cell-mediated immune effector cell function: Effects on macrophages

Abstract A single intramuscular injection of MnCl2 in mice caused an increase in macrophage functional activity. Spleen cell antibody-dependent cell-mediated cytotoxicity against both chicken erythrocytes and P815 tumor cell targets was enhanced 24 h following a single injection of MnCl2. Enhanced antibody-dependent cell-mediated cytotoxicity activity following MnCl2 treatment was not associated with a change in spleen cellularities compared with saline-injected mice. Resident peritoneal macrophages from mice injected intramuscularly with MnCl2 displayed enhanced phagocytic activity for chicken erythrocytes in the presence or absence of opsonizing antibody. Enhanced cytolytic activity against P815 mastocytoma target cells and enhanced cytostatic activity against MBL-2 lymphoma target cells was also observed for nonelicited resident peritoneal macrophages from mice injected intramuscularly with MnCl2. There were no differences in the cellularity or relative number of adherent cells obtained from the peritoneal cavity of saline of MNCl2-injected mice. These enhanced macrophage functions were associated with the induction of increased interferon levels in mice injected with MnCl2.

Immune alterations in rats following subacute exposure to tributyltin oxide

Adult male Fischer 344 rats were dosed by oral gavage with bis(tri-n-butyltin)oxide (TBTO) in peanut oil for 10 consecutive days, at dosages ranging from 1.25 to 15 mg/kg/day. Other groups of rats were dosed daily for 10 days by oral gavage with cyclophosphamide (CY) at dosages ranging from 0.75 to 6 mg/kg/day. These rats served as positive controls for the immune assays employed. The immune function parameters examined included the following: delayed-type hypersensitivity (DTH) and antibody responses to bovine serum albumin (BSA), primary antibody responses to sheep red blood cells (SRBC) and trinitrophenyl lipopolysaccharide (TNP-LPS) and enumeration of splenic lymphocyte populations. The DTH and antibody responses to BSA were not affected by TBTO exposure; however these responses were suppressed in rats dosed with CY at 6 mg/kg/day. The plaque forming cell (PFC) response to the T cell-dependent antigen SRBC was enhanced in rats dosed with TBTO at from 5 to 15 mg/kg/day. On the other hand, the PFC response to the T cell-independent antigen TNP-LPS was unaffected by TBTO exposure. Rats dosed with CY had suppressed PFC responses to SRBC and TNP-LPS at dosages of 3 and 6 mg/kg/day, respectively. Enumeration of splenic lymphocyte populations from TBTO-exposed rats revealed a reduction in OX8- but not W3/25- or IgG-positive cells. These results, as well as results from an earlier study from this lab, suggest that T lymphocytes are a primary target for TBTO-induced immune alterations and that the enhancement of the PFC response to SRBC in TBTO-exposed rats may be mediated by alterations in the suppressor (OX8-positive) T lymphocyte population.

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.

Species and strain comparisons of immunosuppression by 2-methoxyethanol and 2-methoxyacetic acid.

2-Methoxyethanol (ME) and its principal metabolite 2-methoxyacetic acid (MAA) have been shown in our laboratory to be immunosuppressive in male Fischer 344 rats. In this study several strains of 12-week-old female rats and mice were used to compare the immunosuppressive activity of equimolar concentrations of ME and MAA on the trinitrophenyl-lipopolysaccharide (TNP-LPS) antibody plaque-forming cell (PFC) response, which we previously demonstrated to be a sensitive end point. Female inbred Lewis, Fischer 344 and Wistar/Furth, and outbred Sprague-Dawley rats were dosed by gavage with either ME or MAA at dosages of 0.33 to 2.64 mmol/kg/day for 10 consecutive days. Female inbred C3H and C57BL/6J, hybrid B6C3F1, and outbred CD-1 mice were similarly dosed with equimolar dosages of 0.66 to 5.28 mmol/kg/day ME or MAA. All animals were immunized on day 9 of dosing and PFC responses evaluated 3 days later. Suppression of the PFC response was observed in all strains of rats at 2.64 mmol/kg/day ME or MAA. Lewis and Wistar/Furth rats were found to be the most sensitive strains with suppression at levels as low as 0.66 mmol/kg/day ME or MAA. While ME and MAA dosing resulted in suppression of the TNP PFC response in all the rat strains tested, such treatment did not suppress this PFC response in any of the mouse strains examined. These results indicate that under the conditions of this study rats, but not mice, are immunosuppressed by ME and MAA exposure, and that the susceptibility to immunosuppression differs among rat strains.

Immunological effects of 2-methoxyethanol administered dermally or orally to Fischer 344 rats☆

Exposure of rats to 2-methoxyethanol (ME) by gavage for 10 consecutive days results in immunotoxicity. To determine whether dermal exposure to ME also induces immunotoxicity, undiluted ME was applied to Fisher 344 male rats at dose levels of 150, 300, 600, 900 or 1200 mg/kg/day on shaved occluded test sites for 4 consecutive days. Decreased thymus weights were produced by all doses of ME, while reductions in spleen weight were observed at doses of 900 mg/kg/day ME or greater. The alterations in these lymphoid organ weights were produced in the absence of loss in body weight. The lymphoproliferative (LP) responses to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) were enhanced at 1200 mg/kg/day ME compared with water controls. Separate groups of rats, employed for the antibody plaque-forming cell (PFC) response to either trinitrophenyl-lipopolysaccharide (TNP-LPS) or sheep red blood cells (SRBC), were exposed dermally to 150, 300 or 600 mg/kg/day ME for 4 consecutive days. A reduction in the PFC response to TNP was observed at 600 mg/kg/day ME, whereas decreases in the PFC response to SRBC were observed at dosages of 300 and 600 mg/kg/day ME. To compare the immunotoxic effects of dermally applied ME to those effects caused by ME administered orally, rats were dosed by gavage with 25, 50, 100 or 200 mg/kg/day ME in distilled water for 4 consecutive days. Reductions in thymus weights were observed at oral dosages ranging from 50-200 mg/kg/day, while spleen weights were reduced in rats dosed at 200 mg/kg/day ME. LP responses to PHA, PWM and Salmonella typhimurium were increased at the 200 mg/kg/day ME dose level. PFC responses to TNP-LPS and SRBC were suppressed at the 50, 100 and 200 mg/kg/day ME dosages. These results indicate that, like oral exposure, dermal exposure to ME compromises the ability of the immune system to mount an effective humoral immune response.

Immune function in adult C57BL/6J mice following exposure to urethan pre- or postnatally.

Administration of urethan (URE or ethyl carbamate) to mice results in the development of a variety of tumors, and, in certain strains of mice, marked suppression of the immune response. Perinatal exposure of mice to URE has been found to result in increased tumor induction compared to exposure of adult animals. In the present study, the effects of perinatal exposure to URE on the development of immunocompetence was investigated. Pregnant mice were injected with total doses of either 0.5 or 1.0 mg URE/g of body weight over days 7-16 of gestation or pups of nontreated dams were administered a total dose of 2.0 mg URE/g of body weight over postpartum days 5-14. Postnatal exposure to URE suppressed NK (natural killer) cell activity but left intact other measured parameters of the host defense system. Prenatal exposure, on the other hand, resulted in elevated leukocyte counts and a trend toward increased spleen and thymus size in offspring of treated mothers. Humoral immune function, as measured by the IgM response to sheep erythrocytes, was suppressed in pups from dams injected with a total of 1.0 mg/g URE. These results indicate that marked differences in immunopharmacologic effects may be observed if chemical exposure occurs at different times during the ontogeny of the immune system.

Manganese Chloride Enhances Murine Cell-Mediated Cytotoxicity: Effects on Natural Killer Cells

Natural killer (NK) cell activity of mice given a single injection of manganese chloride (MnCl2) was significantly enhanced as measured in a 4-hr in vitro 51Cr release assay. Enhanced activity persisted for several days after injection. This cytotoxic activity was associated with nonadherent spleen cells and was completely eliminated by injecting MnCl2-treated mice with anti-asialo GM1 serum. Manganese-enhanced natural cytotoxicity was observed in several mouse strains with differing NK cell reactivity (CBA/J, C57BL/6, A/J, C3H/HeJ, and C57BL/6 beige mice) and with several tumor target cells with differing sensitivity to NK cytolysis (YAC-1, RBL-5, EL-4, and P815). The growth of B16-F10 melanoma lung tumors was inhibited in mice injected with MnCl2 one day before tumor challenge. Manganese chloride enhancement of NK cell activity appeared to be mediated by interferon (IFN). Low levels of IFN were detected in the serum of mice as early as 4 hr after MnCl2 injection. Rabbit anti-mouse IFN alpha, beta but not anti-mouse IFN gamma completely eliminated the MnCl2-enhanced NK cell activity in the spleens of mice. The observed enhancement of NK cell activity by MnCl2 is similar to that reported for more complex molecules that act by inducing IFN production.