Ralph J. Smialowicz

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.

Immunomodulation by Metals

A symposium entitled Immunomodulation by Metals was held at the 32nd Annual Meeting of the Society of Toxicology (SOT) in New Orleans, Louisiana. The symposium was co-sponsored by the Immunotoxicology and Metals Specialty Sections of SOT and was designed to describe the types of adverse immunological reactions which occur in response to environmental and/or occupational exposure to metals. Epidemiological evidence and underlying mechanisms responsible for the observed alterations were also discussed. The following is a summary of each of the individual presentations.

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.

Immunotoxicologic Assessment of Subacute Exposure of Rats to Carbon Tetrachloride with Comparison to Hepatotoxicity and Nephrotoxicity

The immunotoxicity, hepatotoxicity, and nephrotoxicity of subacute exposure to carbon tetrachloride (CCl4) were evaluated in young adult (8-9 weeks old) male Fischer 344 rats dosed by gavage with CCl4 for 10 consecutive days at 0, 5, 10, 20 or 40 mg/kg/day. Two days following the last treatment rats were evaluated for alterations in immune function by monitoring the following: body and lymphoid organ weights; mitogen and mixed leukocyte reaction lymphoproliferative responses; natural killer cell activity; and cytotoxic T lymphocyte responses. A separate group of similarly dosed rats was immunized with sheep red blood cells (SRBC) on Day 9 of dosing, and the primary antibody response was assessed 4 days later. Hepatic and renal toxicity were assessed 2 days after the last treatment by monitoring organ weights, serum indicators of hepatic and renal damage, and hepatic cytochrome P450 levels, as well as by histological evaluation. Significant increases in relative liver weights were observed in rats dosed at 40 mg/kg/day. Histologically, these livers displayed mild to moderate vacuolar degeneration and minimal to mild hepatocellular necrosis. In addition, serum levels of aspartate aminotransferase and alanine aminotransferase were elevated at this dosage, as well as at 20 mg/kg/day. There were no renal effects observed at these dosages of CCl4. In addition, no consistent alterations were observed in the immune parameters examined in these same animals nor in the rats immunized with SRBC. Furthermore, there was no difference in the antibody response to SRBC in another set of rats dosed at 40, 80 or 160 mg/kg/day CCl4. These results indicate that CCl4 is not immunotoxic in the rat at dosages that produce overt hepatotoxicity.

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.

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.

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.

Exposure of Rats to 425-MHz (CW) Radiofrequency Radiation: Effects on Lymphocytes

Four experiments were performed in which six pregnant rats were exposed from day 12 of pregnancy to parturition, for 4 hours a day in a temperature-controlled environment, to 425-MHz (CW) radiation, using a multimode rectangular strip transmission line. Four male pups born to each dam were subsequently irradiated under the same RF exposure condition for 20-21 days of age (2 pups) and 40-41 days of age (2 pups). Specific absorption rates (SARs) for rats of different ages were determined by twin-well calorimetry as well as from calculations of power measurements of incident, reflected, and transmitted energy. Values of SARs between 3.1 and 6.7 mW/g were obtained for rats so exposed at 425 MHz. At selected times, rats were weighed to determine if the irradiation affected growth. Two rats from each litter (4 pups) were euthanized at 20-21 and two at 40-41 days of age and blood was obtained for complete blood counts. The in vitro blastogenic response of blood and lymph-node lymphocytes was measured by 3H-thymidine incorporation into DNA following stimulation of cells with T- or B-lymphocyte mitogens. No difference was observed in the weights of irradiated compared with sham-irradiated rats. No consistent change in the peripheral blood picture was observed between irradiated and sham-irradiated rats. Significant increases in the response of lymph-node but not of blood lymphocytes from irradiated rats following stimulation with mitogens was observed in two of four experiments. These changes were observed for both T- and B-lymphocytes. In another experiment at the same frequency, six pregnant rats were irradiated for 16 hours daily from day 6 through day 19 of pregnancy. The pups born to these dams were not subsequently irradiated. These rats, born to irradiated dams, showed a similar increased response of node but not of blood lymphocytes to T-cell mitogens at 42 days of age. These results indicate that exposure to 425-MHz microwave radiation, under the conditions described, may lead to increased responsiveness of node lymphocytes to in vitro stimulation by mitogen.