Pyridostigmine bromide (PYR) alters immune function in B6C3F1 mice.

Pyridostigmine bromide (PYR) is an anticholinesterase drug indicated for the treatment of myasthenia gravis and neuromuscular blockade reversal. It acts as a reversible cholinesterase inhibitor and was used as a pretreatment for soldiers during Operation Desert Storm to protect against possible nerve gas attacks. Since that time, PYR has been implicated as a possible causative agent contributing to Gulf War Illness. PYR's mechanism of action has been well-delineated with regards to its effects on the nervous system, yet little is known regarding potential effects on immunological function. To evaluate the effects of PYR on immunological function, adult female B6C3F1 mice were gavaged daily for 14 days with PYR (0, 1, 5, 10, or 20 mg/kg/day). Immune parameters assessed were lymphoproliferation, natural killer cell activity, the SRBC-specific antibody plaque-forming cell (PFC) response, thymus and spleen weight and cellularity, and thymic and splenic CD4/CD8 lymphocyte subpopulations. Exposure to PYR did not alter splenic and thymus weight or splenic cellularity. However, 20 mg PYR/kg/day decreased thymic cellularity with decreases in both CD4+/CD8+ (20 mg/kg/day) and CD4-/CD8- (10 and 20 mg/kg/day) cell types. Functional immune assays indicated that lymphocyte proliferative responses and natural killer cell activity were normal; whereas exposure to PYR significantly decreased primary IgM antibody responses to a T-cell dependent antigen at the 1, 5, 10 and 20 mg/kg treatment levels for 14 days. This is the first study to examine the immunotoxicological effects of PYR and demonstrate that this compound selectively suppresses humoral antibody responses.

Subchronic exposure to ellagic acid impairs cytotoxic T-cell function and suppresses humoral immunity in mice.

Ellagic acid (EA) is present in a variety of foods such as grapes, strawberries, raspberries, and nuts. It is a dietary plant phenol that has been shown to inhibit oxidative stress and chemical carcinogenesis. Although several studies have examined the protective mechanisms of dietary EA including the induction of detoxifying enzymes, regulation of cell cycle, chelation of nickel, and prevention of DNA methylation, none have addressed the role of EA in immunological surveillance. This study investigates the status of immune function in B6C3F1 mice exposed continuously to EA in drinking water at 0.5, 1.0, or 2.0 mg/kg/day for 28 days. Although this range of exposure is above the estimated human daily intake (approximately 940 microg/day for 70 kg person or 13.4 microg/kg/day), these levels would not be unreasonable if EA were used as a dietary supplement or as a chemotherapeutic agent. Previous reports have demonstrated the anticarcinogenic effects of EA at levels 10- to 250-fold greater than those applied in this study. Immunological parameters assessed included natural killer (NK) cell activity, cytotoxic T lymphocyte (CTL) activity, IgM antibody plaque forming cell (PFC) response, thymus, spleen, kidney, and liver mass, and total cellularity for the thymus and spleen. Subchronic exposure to EA for 28 days in drinking water caused significant suppression of specific IgM antibody responses in the 2.0 mg/kg EA treatment group and suppressed cytotoxic T-cell function in the 0.5 and 1.0 mg/kg EA treatment groups. All other immunological parameters were within normal ranges. Kidney and liver mass were not altered after treatment with EA. The results from this study indicate that EA suppressed both IgM antibody responses and CTLs. These observations suggest important implications on human health should EA be prescribed as a chemotherapeutic agent or a preventative dietary supplement for cancer.

Toxicology of metam sodium.

Metam sodium is the third most commonly used agricultural pesticide (by weight) in the U.S. A spill of 19,000 gallons of metam sodium into the Sacramento River in 1991 clearly demonstrated that a major uncontrolled release can have adverse ecological and human health effects. Furthermore, this incident revealed that estimates of Reference Exposure Levels for the major breakdown product of metam sodium (methylisothiocyanate, MITC) were reasonable with regard to the induction of discomfort. In fact, the irritant properties of MITC seem to account for many of the most commonly reported symptoms in this incident. However, neurotoxicity may also account for some of these symptoms. There is evidence that metam sodium can act as a contact sensitizer in humans, inducing allergic dermatitis. It also may exacerbate or induce respiratory allergy (asthma). The ecological impact of routine use of metam sodium is not clear, but adverse effects on non-target plants have been inferred from modeling studies, and adverse effects on soil microbes have been observed. These issues deserve further study. Human health effects of occupational or routine environmental exposure to metam sodium are not known, but there is limited evidence for immunological (hypersensitivity) and developmental effects as well as irritation and associated symptoms. Animal studies suggest a potential for immunological, developmental, carcinogenic, and atherogenic effects. Metam sodium and some of its breakdown products have a wide variety of molecular and cellular actions that could explain the health effects noted here. However, further studies are needed to relate specific molecular or cellular actions to specific health effects.

An aryl hydrocarbon receptor independent mechanism of JP-8 jet fuel immunotoxicity in Ah-responsive and Ah-nonresponsive mice.

JP-8 jet fuel is handled extensively by personnel in the military and commercial airlines, despite the paucity of information regarding its potential human health effects. JP-8 is a complex mixture primarily consisting of kerosene plus aliphatic and aromatic hydrocarbons. Recent reports indicate that acute JP-8 exposure via inhalation or dermal routes can overtly and persistently impair immune function in mice. Data from preliminary studies in this laboratory assessing the immunotoxicity of JP-8 indicated that oral JP-8 exposure caused an increase in liver weight, a decrease in thymus weight, and a decrease in the PFC response. As these results were similar to classic effects elicited by TCDD, a strong AhR ligand, it was hypothesized that JP-8 may exert immunosuppression via a similar mechanism. To test this hypothesis, an Ah-responsive mouse strain (B6C3F1) and a classically non-responsive mouse strain (DBA/2) bearing a lower affinity AhR were gavaged with JP-8 for 7 days. The results suggest that both mouse strains were equally sensitive to JP-8's toxicity at several endpoints including thymus weight and cellularity, liver weight, and specific IgM antibody responses. Furthermore, JP-8 did not induce CYP1A1 or promote down regulation of the AhR when evaluated by Western blot in either B6C3F1 or DBA/2 mice. In vitro studies corroborated these findings as JP-8 did not induce CYP1A1, promote down regulation of the AhR, or activate an XRE-driven reporter gene in murine Hepa-1 cells. These results suggest that JP-8 may exert its toxicity via an AhR-independent mechanism.

Evaluation of immunotoxicity induced by single or concurrent exposure to N,N-diethyl-m-toluamide (DEET), pyridostigmine bromide (PYR), and JP-8 jet fuel.

Approximately 5,000 to 80,000 of the US service personnel involved in the Persian Gulf War have complained of a variety of nonspecific symptoms since their return in 1991. These symptoms have been collectively labeled Gulf War Illness and include muscle fatigue, general malaise, myalgia, impaired cognition, ataxia, headaches, fever, joint pain, skin rash, gastrointestinal disturbances, sleep disturbances, and respiratory difficulties. Exposures of military and service personnel were diverse and included the prescribed anti-nerve gas agent pyridostigmine bromide (PYR), N.N-diethyl-m-toluamide (DEET) insect repellent, and environmental exposures to jet fuel. Thus, studies in our laboratory were undertaken to determine if concurrent exposure to these agents, singly or in combination, would contribute to significant alterations in immunological function and disease susceptibility. To assess immune status, eight-week old B6C3F1 female mice were exposed for 14 days to single compounds or tertiary mixtures of 15.5 mg/kg DEET, 2 mg/kg PYR, and 500 mg/kg JP-8 (termed low dose), or 31 mg/kg DEET, 5 mg/kg PYR, and 1,000 mg/kg JP-8 (termed high dose). Immunosuppression was assessed 24 h after the last exposure. No remarkable alterations were evident in hematological parameters, spleen and thymus organ weight and total cellularity, natural killer (NK) cell activity, cytotoxic T-cell activity, or mitogen-induced lymphocyte proliferation after exposure to either single or tertiary mixtures at low or high doses. A few changes in CD4/CD8 flow cytometric lymphocyte subpopulations were detected after exposure to the tertiary mixture at the high dose. Delayed type hypersensitivity (DTH) was decreased by 88% after exposure to the high-dose mixture, and suppression of antibody-specific IgM immune responses (plaque-forming cell, PFC) occurred after exposure to all single and tertiary mixtures at both dose levels. In the PFC response, antagonism was apparent in the mixture, while coexposure to these agents resulted in a synergistic effect in the DTH response. Susceptibility to B16F10 tumor or Listeria monocytogenes challenge was not affected after single or tertiary exposures. These data suggest that combined exposure to DEET, PYR, and JP-8 does not profoundly alter many immunological endpoints, but does selectively target functional endpoints such as the PFC and DTH response. This should be considered when assessing human health risks in the military environment.

Role of decomposition products in sodium methyldithiocarbamate-induced immunotoxicity.

Sodium methyldithiocarbamate (SMD) is a widely used agricultural agent that causes immunological changes in B6C3F1 mice. The most prominent effects of SMD include a decrease in thymus weight and percentage of CD4+CD8+ thymocytes, an increase in spleen weight, an increase in the percentage of neutrophils in the blood, a decrease in the percentage of lymphocytes in the blood, and a decrease in natural killer (NK) cell activity in the spleen. The mechanism by which SMD causes these changes is unknown, and the relative importance of the parent compound and its decomposition products is not known. In addition, it is not known if these effects are unique to mice, or if other mammals are affected similarly. This prompted the present investigation of the major decomposition product of SMD, methylisothiocyanate (MITC), and two minor products, methylamine and carbon disulfide, in mice. Equimolar dosages of methylamine and carbon disulfide caused minimal immunological changes, and these changes were not characteristic of those noted for SMD. In contrast, MITC significantly decreased thymus weight and cellularity and changed peripheral white blood cell populations in a manner similar to that noted for an equimolar dosage of SMD. However, MITC did not significantly affect NK cell activity or increase spleen weight. Thus, MITC is probably responsible for some of the immunological changes noted in SMD-treated mice. The remaining changes are not produced by MITC, methylamine, or carbon disulfide. Thus, it is likely that the parent compound or a synergistic action of the parent compound with one or more of the decomposition products is responsible for these remaining changes (increased spleen weight and decreased splenic NK cell activity). Data are also presented that indicate that SMD-induced thymic atrophy occurs in rats as well as mice and that the dosage required to decrease thymus weight by 50% is lower for rats than for mice. Investigations of other mammals are needed to indicate SMD's potential as a human immunotoxicant and to compare the role of MITC in the immunotoxic effects of SMD in different species.

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.