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In 1981-1982, individuals in fourteen central and northwest provinces in Spain were affected by an illness that was eventually labeled toxic oil syndrome (TOS) by the World Health Organization. Thousands of individuals were diagnosed with, and 356 people eventually died from, the disease. The disease shares striking similarities with several autoimmune diseases, particularly eosinophilia-myalgia syndrome (EMS) and diffuse fasciitis with eosinophilia (DFE). As with many other autoimmune diseases, women were more severely affected than men and made up a significant portion of TOS-related deaths. While a number of etiologic agents were investigated, disease occurrence was found to be significantly associated with consumption of contaminated rapeseed oil produced by a particular refinery. Two compounds, 1,2-di-oleyl ester (DEPAP) and oleic anilide are considered to be biologically relevant contaminants that may contribute to disease development. Toxic oil syndrome was a three-phase disease with an initial non-necrotizing vasculitis in multiple organs. Suspected immune mechanisms in TOS include activation of T-cells, altered cytokine production, and several studies have associated disease severity with HLA-DR2 and polymorphisms in metabolism and immune response genes. While a number of animal models have been used to investigate the underlying immune mechanisms in TOS, only a few studies in rodents have demonstrated the classical symptoms of TOS. Biotransformation and oxidation of the parent compound(s) to reactive intermediates prior to induction of autoreactive pathways appears to be an important component of the disease process. These reactive intermediates could haptenate self-proteins and activate autoreactive T-cells, disrupt signal transduction, or induce apoptosis and necrosis to release abnormal forms of self-antigens. Although the TOS epidemic was limited to a discrete period of time, the origin of the contamination determined, and the spread of the disease halted by government intervention, the underlying immune mechanisms have yet to be elucidated.

Hypersensitivity reactions to systemically administered drugs cannot be predicted using available preclinical models. This research is a collaborative project to evaluate the ability of the Lymph Node Proliferation Assay (LNPA) to predict systemic hypersensitivity caused by pharmaceuticals. The assay design is a modification of the Local Lymph Node Assay with the major modification being injection of the test substance subcutaneously to achieve a known systemic exposure to the drug. Fourteen compounds were evaluated in the LNPA. These were two clinically negative drugs (Metformin, phenobarbital), an assay positive control (streptozotocin), eight human hypersensitivity positive drugs (sulfamethoxazole, procainamide, clonidine, ofloxacin, nevirapine, abacavir, lamotrigine, zomepirac), and 3 investigational drugs (CM40874, CM40954 and CM40420), one of which caused hypersensitivity in primates. Hypersensitivity-positive drugs were classified as such based on at least two of three independent data sources: U.S. FDA postmarketing database, drug labeling information, and clinical trial data. All drugs were tested in multiple laboratories for a total of 2-12 evaluations per compound. The pure drug substance was used for testing if it could be obtained commercially, otherwise the marketed drug formulation was used. Neither of the negative control drugs showed a positive reaction in the test system. Four of the eight hypersensitivity positive drugs showed a mixed or positive reaction. Two of the three investigational compounds gave a positive response. A smaller number of LNPAs were run concurrently using footpad injection and evaluation of the popliteal lymph node and gave generally comparable results. Additional development may increase the reproducibility of the assay and facilitate detection of drugs that require metabolic activation to become allergenic, or drugs for which there is dose-limiting toxicity. The data suggest that this method might be useful as a first-line screen to identify candidate drugs that are more likely to cause a high prevalence of human drug hypersensitivity.

Data increasingly implicate a possible role of immune and inflammatory responses to infection in sudden infant death syndrome (SIDS). We have previously described a dual challenge model that results in pathology, organ damage, vascular collapse and unexplained death similar to that seen in SIDS. In this study, we examined changes in inflammatory cytokine mRNA in the lung and liver and regulation of pathways associated with nitric oxide production. Our data suggest that priming of the immune system by mild viral infection disturbs normal inflammatory response to endotoxin. This results in an increased nitric oxide synthase production, most likely the cause of liver pathology and clotting abnormalities.

Previous studies in our laboratory indicate that arsenic alters secretion of growth promoting and inflammatory cytokines in the skin that can regulate the migration and maturation of Langerhans cells (LC) during allergic contact dermatitis. Therefore, we hypothesized that arsenic may modulate hypersensitivity responses to cutaneous sensitizing agents by altering cytokine production, LC migration, and T-cell proliferation. To investigate this hypothesis, we examined the induction and elicitation phases of dermal sensitization. Mice exposed to 50 mg/l arsenic in the drinking water for 4 weeks demonstrated a reduction in lymph node cell (LNC) proliferation and ear swelling following sensitization with 2,4-dinitrofluorobenzene (DNFB), compared to control mice. LC and T-cell populations in the draining lymph nodes of DNFB-sensitized mice were evaluated by fluorescence-activated cell sorting; activated LC were reduced in cervical lymph nodes, suggesting that LC migration may be altered following arsenic exposure. Lymphocytes from arsenic-treated animals sensitized with fluorescein isothiocyanate (FITC) exhibited reduced proliferative responses following T-cell mitogen stimulation in vitro; however, lymphocyte proliferation from nonsensitized, arsenic-treated mice was comparable to controls. Arsenic exposure also reduced the number of thioglycollate-induced peritoneal macrophages and circulating neutrophils. These studies demonstrate that repeated, prolonged exposure to nontoxic concentrations of sodium arsenite alters immune cell populations and results in functional changes in immune responses, specifically attenuation of contact hypersensitivity.

Nonylphenol (NP) has been identified at low levels in surface waters throughout North America. This industrial chemical is primarily used for the production of certain non-ionic surfactants, and has been reported to have weak estrogen-like activity. As estrogen has immunoregulatory properties and is crucial for normal fetal development, it was hypothesized that adult and developmental exposures to NP had the potential to adversely affect the immune system. Furthermore, developmental exposure to NP might also produce differential immunomodulation in F(1) male and female rats. Thus, a two-generation feeding study was conducted to evaluate the potential for NP to modulate certain immune parameters. Pregnant female Sprague-Dawley rats were exposed to NP (0, 25, 500, and 2000 ppm) in their feed for 65 days, beginning 7 days into gestation. The F(1) generation male and female offspring were exposed in utero at the respective treatment levels, commencing the 7th day of gestation, and continuing through to 64 days of age. Changes in splenic antibody-forming cell response, natural killer cell activity, and leukocyte numbers were used to evaluate NP immunotoxicity. The results from the present study indicate that dietary exposure to NP can increase splenic natural killer (NK) cell activity and splenocyte subpopulation numbers in the F(1) generation rats, without similar changes to the F(0) generation. The immunological changes that were observed in the F(1) generation also appeared to be gender-specific.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent and persistent environmental toxin that induces hepatotoxicity and increases endotoxin-induced liver injury. The objective of this study was to evaluate whether TCDD could modulate apoptosis and cytokine-controlled apoptotic signaling pathways following lipopolysaccharide (LPS) exposure in female B6C3F1 mice. The effects of TCDD treatment were most dramatic late in the time course (10-14 days posttreatment). Serum enzyme activities were elevated at day 10 (100 microg TCDD/40 microg LPS treatment) and day 14 (100 microg TCDD/saline treatment), indicating peak liver damage occurred at those times. Histological examination of perfused livers showed an increase in apoptotic cells at day 14 in animals treated with 10 microg TCDD. Caspase-1 activity was suppressed at 14 days in mice treated with 100 microg TCDD/40 microg LPS and 100 microg TCDD/4 microg LPS compared to the respective corn oil (CO)/LPS-treated controls. Caspase-3 activity was suppressed at 14 days in 100 microg TCDD/saline-100 microg TCDD/40 microg LPS- and 100 microg TCDD/4 microg LPS-treated mice compared to respective CO/saline- or CO/LPS-treated control mice. At 40 microg LPS, caspase activity was stimulated in TCDD (100 microg)-exposed mice at 3 and 7 days and then suppressed at 10 and 14 days. Western blot analysis, electrophoretic mobility shift assay, and ELISA did not show any effect by TCDD (100 microg) on IkappaB-beta and IkappaB-alpha protein expression or on DNA binding activity of the nuclear NFkappaB protein. These data indicate that TCDD induces apoptosis 14 days posttreatment; however, we found no evidence of suppression of the antiapoptotic transcription factor NFkappaB.

Human exposure to arsenic, a ubiquitous and toxic environmental pollutant, is associated with an increased incidence of skin cancer. However, the mechanism(s) associated with AsIII-mediated toxicity and carcinogenesis at low levels of exposure remains elusive. Aberrations in cell proliferation, oxidative damage, and DNA-repair fidelity have been implicated in sodium arsenite (AsIII)-mediated carcinogenicity and toxicity, but these events have been examined in isolation in the majority of biological models of arsenic exposure. We hypothesized that the simultaneous interaction of these effects may be important in arsenic-mediated neoplasia in the skin. To evaluate this, normal human epidermal keratinocytes (NHEK) were exposed to nontoxic doses (0.005-5 micro M) of AsIII and monitored for several physiological endpoints at the times when cells were harvested for gene expression measurements (1-24 h). Two-fluor cDNA microarray analyses indicated that AsIII treatment decreased the expression of genes associated with DNA repair (e.g., p53 and Damage-specific DNA-binding protein 2) and increased the expression of genes indicative of the cellular response to oxidative stress (e.g., Superoxide dismutase 1, NAD(P)H quinone oxidoreductase, and Serine/threonine kinase 25). AsIII also modulated the expression of certain transcripts associated with increased cell proliferation (e.g., Cyclin G1, Protein kinase C delta), oncogenes, and genes associated with cellular transformation (e.g., Gro-1 and V-yes). These observations correlated with measurements of cell proliferation and mitotic measurements as AsIII treatment resulted in a dose-dependent increase in cellular mitoses at 24 h and an increase in cell proliferation at 48 h of exposure. Data in this manuscript demonstrates that AsIII exposure simultaneously modulates DNA repair, cell proliferation, and redox-related gene expression in nontransformed, normal NHEK. It is anticipated that data in this report will serve as a foundation for furthering our knowledge of AsIII-regulated gene expression in skin and other tissues and contribute to a better understanding of arsenic toxicity and carcinogenesis.

Sudden infant death syndrome is the most common cause of postneonatal infant mortality in the developed world. It is a diagnosis of exclusion with peak age of incidence between 2 and 6 mo. Fifty to 63% of these infants have a preexisting upper respiratory tract infection before death. We hypothesized that the immature immune system may be altered by a primary infection, preventing a protective response after secondary challenge. To mimic dual infection, we used a nonlethal strain of a rat-adapted influenza A virus and a sublethal dose of endotoxin to establish a model that results in pathology and death in 12-d-old rat pups similar to that seen in infants dying of sudden infant death syndrome. Mortality only occurred when specific criteria such as timing between infectious insults and developmental age of the pup were met. Results suggest that mortality is caused by a rapid systemic shock event rather than lung-specific damage. Gross pathologic findings such as lung petechiae and liquid blood around the heart on necropsy were consistent with those seen in infants dying of sudden infant death syndrome. Histopathologic lesions including subendocardial hemorrhage and mild cortical thymocyte necrosis were found with greater severity and frequency in dually challenged animals. Macrophage subpopulation in rat-adapted influenza A virus-inoculated animals was significantly elevated in the spleen at the time of death. Our model suggests that the developing immune system can be primed to respond in an exaggerated way to a second immune challenge resulting in unexpected death.

This study reports the detection of protein free radicals using the specific free radical reactivity of nitrone spin traps in conjunction with nitrone-antibody specificity. Polyclonal antibodies were developed that bind to protein adducts of the nitrone spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The antibodies were used to detect DMPO protein adducts produced on horse myoglobin resulting from self-peroxidation. Western blot analysis demonstrates that myoglobin forms the predominant radical-derived nitrone adduct in rat heart supernatant.

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.

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.

There is strong evidence from epidemiologic studies of an association between chronic exposure to inorganic arsenic (iAs) and hyperpigmentation, hyperkeratosis, and neoplasia in the skin. Although it is generally accepted that methylation is a mechanism of arsenic detoxification, recent studies have suggested that methylated arsenicals also have deleterious biological effects. In these studies we compare the effects of inorganic arsenicals (arsenite (iAs(III)) and arsenate (iAs(V))) and trivalent and pentavalent methylated arsenicals (methylarsine oxide (MAs(III)O), complex of dimethylarsinous acid with glutathione (DMAs(III)GS), methylarsonic acid (MAs(V)), and dimethylarsinic acid (DMAs(V))) in human keratinocyte cultures. Viability testing showed that the relative toxicities of the arsenicals were as follows: iAs(III) > MAs(III)O > DMAs(III)GS > DMAs(V) > MAs(V) > iAs(V). Trivalent arsenicals induced an increase in cell proliferation at concentrations in the 0.001 to 0.01 microM range, while at high concentrations (>0.5 microM) cell proliferation was inhibited. Pentavalent arsenicals did not stimulate cell proliferation. As seen in the viability studies, the methylated forms of As(V) were more cytotoxic than iAs(V). Exposure to low doses of trivalent arsenicals stimulated secretion of the growth-promoting cytokines, granulocyte macrophage colony stimulating factor and tumor necrosis factor-alpha. DMAs(V) reduced cytokine secretion at concentrations at which proliferation and viability were not affected. These data suggest that methylated arsenicals, products of the metabolic conversion of inorganic arsenic, can significantly affect viability and proliferation of human keratinocytes and modify their secretion of inflammatory and growth-promoting cytokines., (Copyright 2001 Academic Press.)

Background: Although the Murine Local Lymph Node Assay (LLNA) is efficient in identifying chemicals with sensitizing potential, there is increasing need for alternative end points. Cinnamaldehyde (CIN) was chosen for evaluation based on its moderate potency and extensive use in fragrance materials., Objectives: The purpose of the present studies is to incorporate some alternative end points, such as phenotypic analysis and cytokine production, into a modified LLNA/irritancy assay (IA) to evaluate the sensitization of female B6C3F1 mice to CIN., Methods: Several nontraditional end points, including the analysis of lymphocyte subpopulations, B7 costimulatory molecule and cytokine messenger RNA (mRNA) expression, and intracellular interferon-gamma (IFN-gamma) levels, were incorporated into a modified murine local lymph node (LLNA)/irritancy assay (IA) to evaluate the sensitization of female B6C3F1 mice to cinnamaldehyde (CIN)., Results: The alternate end points used in these studies support the classification of CIN as a moderately potent sensitizer. Dermal treatment with CIN resulted in an increase in the percentage of B cells in the auricular lymph nodes (ALNs) and expression of the costimulatory molecule, B7-2, on B cells. Lymph node cells also showed increased transforming growth factor-beta1, migration-inhibition factor, and mild increases in IFN-gamma and interleukin-2 cytokine mRNA expression. Although the increase in IFN-gamma mRNA expression did not translate into increased intracellular IFN-gamma levels, the absolute number of T cells producing IFN-gamma in the ALNs increased. Conversely, the MEST did not classify CIN as a contact allergen., Conclusion: The nontraditional end points used in the LLNA/IA were not as sensitive as the traditional radioisotope method used to assess cell proliferation. However, they may help identify compounds inappropriately classified as sensitizers or nonsensitizers by the LLNA and MEST., (Copyright 2001 by W.B. Saunders Company)

Gadolinium chloride (GdCl(3)) destroys large Kupffer cells and has been used extensively in mechanistic studies in a number of disease and toxicity processes; however, it cannot be used to study hepatocyte turnover since it increases cell proliferation itself. The mechanism by which GdCl(3) activates cell turnover in liver is unknown, but several possibilities exist. Here it was demonstrated that a direct mitogenic action on hepatocytes is unlikely since GdCl(3) did not stimulate the growth of primary rat hepatocyte in vitro. Therefore, it was hypothesized that GdCl(3) acts indirectly through mitogenic cytokines of nonparenchymal cell origin. Antibodies to tumor necrosis factor alpha (TNFalpha) were used to evaluate if TNFalpha is causally responsible for GdCl(3)-induced cell proliferation. GdCl(3) treatment of rats in vivo increased hepatocyte replication 5-fold in 24 h and 3-fold in 48 h. Pretreatment with specific anti-TNFalpha antibodies completely prevented these effects. However, when antibody treatment was delayed until 24 h after GdCl(3), increased cell proliferation was not prevented, suggesting that TNFalpha production during the first 24 h after treatment is responsible for activation of a signaling cascade involving other mitogens that sustain hepatocyte replication at 48 h. Twenty-four hours after treatment with GdCl(3), TNFalpha mRNA transcripts were increased 2-fold over control, an effect that was prevented by pretreatment with anti-TNFalpha antibody. NFkappaB, which is known to be involved in TNFalpha transcription, was activated by GdCl(3) about 4.5-fold over control 8 h after treatment in vivo, an increase not observed when antibodies to TNFalpha were present. When GdCl(3) was added to macrophages in culture, TNFalpha was nearly doubled 4 h after treatment. Additionally, conditioned media harvested from macrophages treated with GdCl(3) for 2 to 8 h stimulated the growth of HepG2 cells in culture about 2-fold, while antibodies to TNFalpha completely prevented this effect. Taken together, these data are consistent with the hypothesis that TNFalpha released from Kupffer cells at early time points prior to their destruction is causally responsible for triggering a cascade of events responsible for GdCl(3)-induced cell proliferation., (Copyright 2001 Academic Press.)

While less than 1% of the general population is sensitized to latex, the U.S. Occupational Safety and Health Administration estimates that 8-12% of health-care workers are sensitized. The major source of workplace exposure is powdered natural rubber latex (NRL) gloves. NRL is harvested from HEVEA: brasiliensis trees and ammoniated to prevent coagulation resulting in the hydrolysis of the latex proteins. Prior to use in manufacturing, the latex is formulated by the addition of multiple chemicals. Thus, human exposure is to a mixture of residual chemicals and hydrolyzed latex peptides. Clinical manifestations include irritant contact dermatitis, allergic contact dermatitis (type IV), and type I immediate hypersensitivity response. Type I (IgE-mediated) NRL allergy includes contact urticaria, systemic urticaria, angioedema, rhinitis, conjunctivitis, bronchospasm, and anaphylaxis. Taking an accurate history, including questions on atopic status, food allergy, and possible reactions to latex devices makes diagnosis of type-I latex allergy possible. To confirm a diagnosis, either in vivo skin prick testing (SPT) or in vitro assays for latex-specific IgE are performed. While the SPT is regarded as a primary confirmatory test for IgE-mediated disease, the absence of a U.S. Food and Drug Administration-licensed HEVEA: brasiliensis latex extract has restricted its use in diagnosis. Serological tests have, therefore, become critically important as alternative diagnostic tests. Three manufacturers currently have FDA clearance for in vitro tests, to detect NRL-specific IgE. The commercially available assays may disagree on the antibody status of an individual serum, which may be due to the assay's detecting anti-NRL IgEs to different allergenic NRL proteins. Sensitized individuals produce specific IgE antibody to at least 10 potent HEVEA: allergens, Hev b 1-Hev b 10, each of which differs in its structure, size, and net charge. The relative content and ratios of Hevs in the final allergen preparation most probably could effect diagnostic accuracy. The Hev proteins have been cloned and expressed as recombinant proteins. Sequencing demonstrates both unique epitopes and sequences commonly found in other plant proteins. Sequence homology helps to explain the cross reactivity to a variety of foods experienced by latex allergic individuals. The development of recombinant allergens provides reagents that should improve the diagnostic accuracy of tests for latex allergy. Although clinical and exposure data have been gathered on the factors affecting response in latex-allergic individuals, less is known regarding the development of sensitization. Coupled with in vitro dermal penetration studies, murine models have been established to investigate the route of exposure in the development of latex sensitization. Time-course and dose-response studies have shown subcutaneous, intratracheal, or topical administrations of non-ammoniated latex proteins to induce IgE production. Both in vitro penetration and in vivo studies highlight the importance of skin condition in the development of latex allergy, with enhanced penetration and earlier onset of IgE production seen with experimentally abraded skin. The diagnosis of latex allergy is complicated by these variables, which in turn hinder the development of intervention strategies. Further epidemiological assessment is needed to more explicitly define the scope, trends, and demographics of latex allergy. Diagnostic accuracy can be improved through greater knowledge of proteins involved in the development of latex allergy, and better documentation of the presently available diagnostic tests. In vivo and in vitro models can elucidate mechanisms of sensitization and provide an understanding of the role of the exposure route in latex allergy-associated diseases. Together, these efforts can lead to intervention strategies for reducing latex allergy in the workplace.

Biomethylation is considered a major detoxification pathway for inorganic arsenicals (iAs). According to the postulated metabolic scheme, the methylation of iAs yields methylated metabolites in which arsenic is present in both pentavalent and trivalent forms. Pentavalent mono- and dimethylated arsenicals are less acutely toxic than iAs. However, little is known about the toxicity of trivalent methylated species. In the work reported here the toxicities of iAs and trivalent and pentavalent methylated arsenicals were examined in cultured human cells derived from tissues that are considered a major site for iAs methylation (liver) or targets for carcinogenic effects associated with exposure to iAs (skin, urinary bladder, and lung). To characterize the role of methylation in the protection against toxicity of arsenicals, the capacities of cells to produce methylated metabolites were also examined. In addition to human cells, primary rat hepatocytes were used as methylating controls. Among the arsenicals examined, trivalent monomethylated species were the most cytotoxic in all cell types. Trivalent dimethylated arsenicals were at least as cytotoxic as trivalent iAs (arsenite) for most cell types. Pentavalent arsenicals were significantly less cytotoxic than their trivalent analogs. Among the cell types examined, primary rat hepatocytes exhibited the greatest methylation capacity for iAs followed by primary human hepatocytes, epidermal keratinocytes, and bronchial epithelial cells. Cells derived from human bladder did not methylate iAs. There was no apparent correlation between susceptibility of cells to arsenic toxicity and their capacity to methylate iAs. These results suggest that (1) trivalent methylated arsenicals, intermediary products of arsenic methylation, may significantly contribute to the adverse effects associated with exposure to iAs, and (2) high methylation capacity does not protect cells from the acute toxicity of trivalent arsenicals.

Thalidomide has been shown to have antiinflammatory and, more recently, immunomodulating properties, which are beneficial for the treatment of an ever-increasing list of immune related diseases. Although considerable knowledge regarding thalidomide s antiinflammatory properties has been acquired, relatively little is known about its immunomodulating properties in vivo. In this paper, a panel of immune assays was used to evaluate immunomodulation in female B6C3F1 mice treated intraperitoneally for 28 days with thalidomide (30, 100, or 150 mg/kg/day). Spleen antibody forming cell response was significantly enhanced by 37% in mice treated with 150 mg/kg/day, despite an 8% decrease in the percentage of Ig+ B cells. A significant stimulatory trend was observed for the cytotoxic T cell response across thalidomide treatment groups. An evaluation of the spleen leukocyte subpopulations revealed a 23% increase in the absolute number of CD8+ T cells in the 150 mg/kg treatment group and a 9 and 11% decrease in the absolute number of NK cells in both the 100 and 150 mg/kg thalidomide treatment groups, respectively. These findings demonstrate that, in addition to modulating spleen leukocyte numbers, thalidomide also stimulates murine humoral and cellular immune responses in vivo.

Extrahepatic factors, such as increased gut permeability and bacteria from the gut, have been shown to play a role in D-galactosamine toxicity in rats. Because bacterial endotoxin activates Kupffer cells, the purpose of this study was to clarify the role of Kupffer cells in the mechanism of D-galactosamine hepatotoxicity in rats and determine whether uridine, a compound that rescues animals from D-galactosamine toxicity, affects Kupffer cells. Rats were fed control or glycine (5%) containing diets to prevent Kupffer cell activation or treated with gadolinium chloride (GdCl3, 20 mg/kg) to destroy Kupffer cells selectively before injection of D-galactosamine (500 mg/kg, intraperitoneally). D-galactosamine caused panlobular focal hepatocellular necrosis, polymorphonuclear cell infiltration, and increased serum transaminases significantly at 24 hours. Dietary glycine or pretreatment with GdCl3 prevented these effects. D-galactosamine caused a transient increase in circulating endotoxin that was maximal at 1 hour and was blunted significantly by dietary glycine. Additionally, antisera to tumor necrosis factor-alpha (TNF-alpha) prevented hepatotoxicity caused by D-galactosamine. Moreover, apoptosis in hepatocytes caused by D-galactosamine occurred before necrosis (6 hours) and was prevented by glycine, GdCl3, TNF-alpha antiserum, and uridine. Thus, it was hypothesized that TNF-alpha from Kupffer cells causes apoptosis after D-galactosamine administration in the rat. Indeed, increases in TNF-alpha messenger RNA (mRNA) were detected as early as 2.5 hours after D-galactosamine treatment. Previous work proposed that uridine blocks D-galactosamine toxicity by preventing inhibition of mRNA synthesis. In view of these results, the possibility that uridine might affect Kupffer cells was investigated. Uridine significantly blunted the increase in [Ca2+]i and release of TNF-alpha caused by endotoxin in isolated Kupffer cells and prevented apoptosis caused by D-galactosamine treatment in vivo. These data support the hypothesis that uridine prevents D-galactosamine hepatotoxicity not only by rescuing the hepatocyte in the late phases of the injury but also preventing TNF-alpha release from Kupffer cells thereby blocking apoptosis that occurs early after D-galactosamine treatment. Taken together, these data strongly support the role of Kupffer cell activation by endotoxin early after D-galactosamine treatment as an important event in the mechanism of hepatotoxicity in the rat.

Immunoassays are techniques for measuring the concentration or activity of a substance using immunological reactions. Several types of immunoassay are commonly used, including precipitation assays using antibody/antigen complexes, agglutination assays using coated erythrocytes or other particles, and radio-(RIA) and enzyme immunoassays. Multiple factors determine the best assay for a particular use, including sample concentration, technical difficulty, required precision, and availability of specialized equipment. The most sensitive assays are the radio- and enzyme immunoassays, however safety and environmental issues regarding the use of radioisotopes have limited the application of RIAs, and thus enzyme-linked immunosorbent assays (ELISA) tend to be the method of choice when sample concentration is a critical issue. The potential for robotic automation and the availability of inexpensive microplate dilutors, pipetors, and readers has allowed for the generation of highly reproducible results for large numbers of samples in an short time.

Although numerous epidemiological studies have shown that inorganic arsenicals cause skin cancers and hyperkeratoses in humans, there are currently no established mechanisms for their action or animal models. Previous studies in our laboratory using primary human keratinocyte cultures demonstrated that micromolar concentrations of inorganic arsenite increased cell proliferation via the production of keratinocyte-derived growth factors. As recent reports demonstrate that overexpression of keratinocyte-derived growth factors, such as transforming growth factor (TGF)-alpha, promote the formation of skin tumors, we hypothesized that similar events may be responsible for those associated with arsenic skin diseases. Thus, the influence of arsenic in humans with arsenic skin disease and on mouse skin tumor development in transgenic mice was studied. After low-dose application of tetradecanoyl phorbol acetate (TPA), a marked increase in the number of skin papillomas occurred in Tg.AC mice, which carry the v-Ha-ras oncogene, that received arsenic in the drinking water as compared with control drinking water, whereas no papillomas developed in arsenic-treated transgenic mice that did not receive TPA or arsenic/TPA-treated wild-type FVB/N mice. Consistent with earlier in vitro findings, increases in granulocyte/macrophage colony-stimulating factor (GM-CSF) and TGF-alpha mRNA transcripts were found in the epidermis at clinically normal sites within 10 weeks after arsenic treatment. Immunohistochemical staining localized TGF-alpha overexpression to the hair follicles. Injection of neutralizing antibodies to GM-CSF after TPA application reduced the number of papillomas in Tg.AC mice. Analysis of gene expression in samples of skin lesions obtained from humans chronically exposed to arsenic via their drinking water also showed similar alterations in growth factor expression. Although confirmation will be required in nontransgenic mice, these results suggest that arsenic enhances development of skin neoplasias via the chronic stimulation of keratinocyte-derived growth factors and may be a rare example of a chemical carcinogen that acts as a co-promoter.

Anthralin is the most common therapeutic agent among a small number of pro-oxidant, 9-anthrones effective in the topical treatment of psoriasis. However, the usefulness of this drug is diminished by toxic side effects, including skin irritation and inflammation. The activities of anthralin are believed to be mediated by the generation of reactive oxygen intermediates and anthrone radicals produced in the skin. In this study, the dermal inflammatory response to anthralin was determined using a mouse ear swelling test. Maximum ear swelling induced by anthralin coincided with the elevation of cytokine mRNA expression in the skin, including interleukin-6, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein-2, and tumor necrosis factor alpha at 24 h post challenge. The role of free radical generation in ear swelling and cytokine modulation were examined by systemic administration of cell permeable and impermeable antioxidants before anthralin challenge. Superoxide dismutase and alpha-tocopherol acetate, but not the glutathione precursor N-acetyl cysteine, were effective inhibitors of anthralin-induced ear swelling and cytokine elevation. Maximum inflammatory cell infiltration occurred 72-96 h post anthralin challenge and was also reduced by antioxidants. These data suggest that oxidative stress, generated at the site of anthralin treatment, alters the expression of dermal chemokines and other cytokines resulting in the recruitment of inflammatory cells. Systemic antioxidant administration may provide opportunities for therapeutic intervention against anthralin-associated toxicities.

Although numerous epidemiological studies have shown that inorganic arsenicals are human skin carcinogens, there is currently no accepted mechanism for its action or an established animal model for its study. We observed increased mRNA transcripts and secretion of keratinocyte growth factors, including granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-alpha (TGF-alpha) and the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in primary human epidermal keratinocytes cultured in the presence of low micromolar concentrations of sodium arsenite. Total cell numbers, as well as c-myc expression and incorporation of [3H]thymidine, both indicators of cell proliferation, were also elevated in keratinocyte cultures treated with sodium arsenite. As an in vivo model, the influence of arsenic on mouse skin tumor development was studied in transgenic TG.AC mice which carry the v-Ha-ras oncogene, and can serve as a genetically initiated model for skin carcinogenesis. Following low-dose application of 12-O-tetradecanoyl phorbol-13-acetate (TPA), a marked increase in the number of skin papillomas occurred in transgenic mice receiving arsenic in the drinking water as compared to control drinking water. Papillomas did not develop in arsenic-treated transgenic mice that had not received TPA or arsenic-treated wild-type FVB/N mice, suggesting that arsenic is neither a tumor initiator or promoter but rather an enhancer. Injection of anti-GM-CSF antibodies following application of TPA in transgenic mice reduced the number of papillomas. Consistent with that observed in human keratinocyte cultures, increases in GM-CSF and TGF-alpha mRNA transcripts were found within the epidermis of arsenic-treated mice when compared to controls within 6 weeks of treatment. These results suggest that arsenic enhances papilloma development via the chronic stimulation of keratinocyte-derived growth factors and represents the first example of a chemical carcinogen that acts in this manner. These studies suggest that in vitro studies with human keratinocyte cultures examined in conjunction with TG.AC transgenic mice can provide a useful model for examining the tumor enhancing properties of environmental chemicals.

We and others have shown that tumor necrosis factor alpha (TNF-alpha) expression is increased in the livers of experimental animals following exposure to the chemical hepatotoxin, carbon tetrachloride (CCl4). Because TNF-alpha is involved in mediating inflammatory responses, its elevated expression is presumed to be associated with potentiating hepatotoxicity and/or aiding in liver repair processes. To study the role of TNF-alpha in chemical-induced hepatotoxicity, mice were administered neutralizing antibodies to TNF-alpha before administration of low, but hepatotoxic, doses of CCl4. Antibody treatment prevented CCl4-mediated increases in early-immediate gene expression associated with liver regeneration, including expression of c-jun and c-fos proto-oncogenes, as well as DNA binding of the activator protein-1 (AP-1) nuclear transcription factor. Hepatocyte proliferation following CCl4 treatment was also reduced in anti-TNF-alpha antibody-treated mice, as evidenced by a lack of proliferating cell nuclear antigen (PCNA) staining. Antibody treatment slightly delayed liver repair processes, as evidenced by extending the period in which plasma liver enzyme levels were increased and hepatocellular necrosis could be observed. Consistent with the above observations, injection of recombinant TNF-alpha into control mice induced rapid expression of c-jun and c-fos proto-oncogenes. Taken together, these results indicate that TNF-alpha positively modulates liver recovery following CCl4 exposure presumably by stimulating early-immediate genes involved in hepatic mitogenesis, a phenomenon also observed following partial hepatectomy.

Although epidemiological studies have shown that inorganic arsenicals are human skin carcinogens and induce hyperproliferation and hyperkeratosis, there is currently no known mechanism for their action or an established animal model for its study. We observed increased mRNA transcripts and secretion of keratinocyte growth factors, including granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-alpha (TGF alpha) and the proinflammatory cytokine tumor necrosis factor-alpha in primary human epidermal keratinocytes cultured in the presence of low micromolar concentrations of sodium arsenite. Treatment with sodium arsenite resulted in a significant increase in cell proliferation, as indicated by increases in cell numbers, c-myc gene expression, and incorporation of [3H]thymidine into cellular DNA. Studies of transcriptional regulation indicate that the rate of GM-CSF mRNA transcription is increased, while the elevated TGF alpha is likely the results of message stabilization. While a number of cytokine regulatory networks exist in the skin, studies utilizing neutralizing antibodies against the growth factors of interest indicate that inhibition of the arsenic-induced increase in TGF alpha results in a corresponding decrease in the gene expression and secretion of GM-CSF. The present studies demonstrate that growth-promoting cytokines and growth factors are induced in keratinocytes following treatment with arsenic and could play a significant role in arsenic-induced skin cancer.

In many organ/tissues rapid and transient increases in early-immediate gene responses, such as those that encode for the AP-1 family of transcription factors, occur in response to exogenous stimuli. Activation of AP-1, in turn, helps regulate the expression of genes involved in cell growth, inflammatory responses, and repair processes. In the present studies, we demonstrate that increases in AP-1 DNA binding activity, as well as c-jun and c-fos mRNA levels, occur in human keratinocytes in response to diverse dermatotoxic chemicals, including phenol and arsenic as well as phorbol ester, the latter employed as a positive control. The AP-1 DNA binding complex has affinity for the consensus AP-1 sequence but not the CRE2 binding sequence of the proenkephalin promoter or the NF kappa B consensus sequence indicating that the response is relatively specific. The binding complex is composed of Jun:Fos heterodimers, including Jun B and Jun D. Evidence is provided suggesting that AP-1 binding is associated with an increase in IL-1 alpha expression, an early mediator of toxic response in the skin.

The AP-1 transcription factor family, which is involved in early response genes, consists of two groups of proteins, Fos-related antigens (fra) and Jun proteins. AP-1 is usually expressed at low basal cellular levels, but can be up-regulated by a variety of exogenous stimuli which results in synthesis of Fos and Jun proteins and increased AP-1 DNA binding activity. Changes in early immediate gene responses are associated with liver necrosis, inflammation and repair, although investigations into their role in drug-induced hepatotoxicity have not been actively examined. In the present studies, we determined that exposure to necrogenic doses of acetaminophen (APAP) was associated with increased AP-1 DNA binding activity in mouse liver. The APAP-induced hepatic AP-1 DNA binding complex had affinity for both the consensus AP-1 and CRE sequences. Furthermore, c-jun, but not c-fos, mRNA transcripts were transiently increased following exposure to hepatotoxic doses of APAP. When endotoxin was administered to mice in order to elicit a hepatic inflammatory response without necrosis, increases in c-jun expression occurred without accompanying changes in AP-1 activity, indicating a different mechanism of action. When compared to conventional indicators of hepatotoxicity, such as plasma levels of liver-associated enzymes, changes in gene expression occurred much earlier and, at least with AP-1 activity, remained activated following normalization of liver enzyme levels. These studies suggest that the AP-1 transcription factor and associated genes are associated in the hepatotoxic response of liver to APAP and may serve as useful molecular biomarkers for chemical-induced hepatotoxicity.

The immune system is a primary target for toxic insult by a number of drugs and environmental chemicals, many of which require activation to toxic metabolites by drug-metabolizing enzymes. We compared the induction of drug-metabolizing enzymes, including cytochrome P450 1A1 (CYP1A1) and aldehyde dehydrogenase (ALDH), in lymphoid tissues of F344 rats following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). ALDH was induced in both the spleen and the thymus after TCDD treatment, with maximal expression at 9 and 15 days, respectively. Thymic microsomal preparations from TCDD-treated animals expressed elevated levels of inducible CYP1A1 as compared to microsomes from the spleens of treated animals or tissues from control rats. TCDD treatment also resulted in increased ethoxyresorufin-O-deethylase (EROD) activity in the thymus. There were no detectable mRNA transcripts for CYP1A1 in peripheral blood or splenic lymphocytes from treated animals; however, CYP1A1 transcripts were induced in isolated thymocytes, whole spleen, and whole thymus. In vitro exposure to TCDD did not result in induction of immunoreactive CYP1A1 in thymocytes unless simultaneously activated with the mitogen, phytohemagglutinin (PHA). Immunohistochemical localization of CYP1A1 in immune tissues indicated that cells other than the lymphoid populations are responsible for the increased CYP1A1 expression. The pattern of CYP1A1 induction was related to the expression of the Ah receptor (AhR) in immune tissues. Western blot analyses demonstrated less AhR present in peripheral blood lymphoid cells and spleen, as compared to whole tissues. These studies indicate that while drug-metabolizing enzymes are present in immune tissues, the induction of enzymes is selective in different lymphoid cells.

Nuclear transcription factors, such as NF-kB and NF-IL6, are believed to play an important role in regulating the expression of genes that encode for products involved in tissue damage and inflammation and, thus, may represent early biomarkers for chemical toxicities. In the present study changes in DNA binding activity of these factors were examined in livers of mice administered hepatotoxic doses of acetaminophen (APAP). NF-kB and NF-IL6 DNA binding occurred constitutively in control mouse liver. However, within 4 hr following administration of hepatotoxic doses of APAP, their binding activities were transiently lost and is in contrast to AP-1 transcription factor where activation occurs under similar conditions. These changes corresponded with increased release of inflammatory mediators (IL-6, serum amyloid A) and increased levels of enzymatic markers of hepatocyte damage. Similarly, treatment of mice with gadolinium chloride, an inhibitor of Kupffer cell activation and known to protect against APAP-induced hepatotoxicity, reduced the observed pathophysiological response in the liver while altering the APAP-associated changes in NF-kB DNA binding activity. NF-kB was found predominantly in parenchymal and endothelial cells and was composed primarily of relatively inactive p50 homodimer subunits in control liver. Taken together, these studies suggest that hepatotoxicity is associated with early and complex changes in DNA binding activities of specific transcription factors. In particular, NF-kB and NF-IL6 may serve as negative regulators of hepatocyte-derived inflammatory mediators and is analogous to that previously observed in certain other cell systems such as B lymphocytes.

We have previously reported on the design and content of a screening battery involving a 'tier' approach for detecting potential immunosuppressive compounds in mice. This battery has been used to examine a variety of compounds, and the database generated from these studies, which consists of over 50 compounds, has been collected and analyzed in an attempt to improve the accuracy and efficiency of screening chemicals for immunosuppression and to identify better those tests that predict experimentally-induced, immune-mediated diseases. Specifically, these analyses attempted to develop an improved testing configuration for the accurate prediction of immunotoxic agents and to provide insight into the qualitative and quantitative relationships between a number of immune and host resistance assays commonly employed to examine potential immunotoxic chemicals in experimental animals. While a number of limitations existed in the analyses, several conclusions were drawn from the results which will be discussed.

Peptide mediators, including tumor necrosis factor-alpha, interleukin 1 and interleukin-6, are associated with many chronic inflammatory diseases and septic shock. As such, considerable information has been collected by means of study of cytokine secretion from isolated cells or plasma cytokines during septic shock or inflammatory disorders. In this investigation, we used semiquantitative polymerase chain reaction analysis and a recently developed liver slice model to examine the characteristics of cytokine profiles that occur in the liver, the main organ involved in endotoxemia, after lipopolysaccharide challenge. Tumor necrosis factor-alpha, interleukin-1 alpha and interleukin-6 were rapidly secreted after in vivo LPS exposure or when added in vitro to rodent or human liver slice samples. This increase was associated with increased cytokine-specific mRNA transcripts. Kinetic analysis revealed that most tumor necrosis factor-alpha is released from the liver within 1 hr of lipopolysaccharide challenge, whereas interleukin-1 alpha and interleukin-6 continued to be produced for the entire culture period. Addition of monoclonal antibodies against tumor necrosis factor-alpha or interleukin-1 alpha to the culture partly inhibited interleukin-6 secretion, indicating that interleukin-1 alpha and tumor necrosis factor-alpha help mediate and sustain interleukin-6 synthesis. Depletion of hepatic sinusoidal macrophages (Kupffer cells) by a liposome-mediated macrophage "suicide" technique indicated that almost all of the secreted interleukin-1 alpha and tumor necrosis factor-alpha originate from these cells, whereas interleukin-6 secretion might also include other cell types. This study supports and extends previous findings and allows for a more rational approach to developing effective therapies against chronic inflammatory diseases and septic shock.

Mice with the severe combined immunodeficient (SCID) and triple-deficient (bg/nu/xid) mutations lack select populations of functional immune cells. Studies by several laboratories have demonstrated the ability to restore certain missing immune components in these mice by reconstituting with various lymphoid tissues including peripheral blood lymphocytes (PBL) from mice and humans. Such a model could provide an opportunity to examine human lymphoid cells in an in vivo environment for immunotoxicity assessment. In the present studies, bg/nu/xid and SCID mice were reconstituted by intraperitoneal or intravenous injection with either tetanus-immunized syngeneic mouse splenocytes (mo-SPL) or tetanus-immunized human PBLs (hu-PBL) under various test conditions. Hu-PBL-SCID mice from the C.B-17 strain produced more successful human engraftments than mice from the bg/nu/xid or C3H-SCID strains. Using optimal conditions, mo-SPL-SCID and hu-PBL-SCID mice were engrafted and administered either 2,3,7,8-tetrachlorodibenzo-p-dioxin or cyclosporin A (Cys A) and periodically bled to measure tetanus-specific antibody and class-specific immunoglobulin concentrations. Comparison of the chemical-related changes in immunoglobulin and tetanus antibody concentrations revealed some similarities between control mice and mo-SPL-SCID or hu-PBL-SCID mice, particularly with Cys A groups. However, under the various conditions examined, hu-PBL-SCID mice demonstrated considerable variability in their ability to provide consistent reconstitution, thus, limiting the ability to determine whether human cells are more or less susceptible than mouse cells to the test agents. Provided that this system can be refined to provide consistent reconstitution, hu-PBL-SCID mice may be a promising in vivo model for assessment of potential immunotoxic agents.

We have reported on the design and content of a screening battery using a "tier" approach for detecting potential immunotoxic compounds in mice (Luster et al., Fundam. Appl. Toxicol., 10, 2-19, 1988). The data base generated from these studies, which consists of over 50 selected compounds, has been collected and analyzed in an attempt to improve future testing strategies and provide information to aid in developing future quantitative risk assessment for immunotoxicity. In a recent study it was shown that as few as two or three immune parameters were needed to predict immunotoxicants in mice (Luster et al., Fundam. Appl. Toxicol., 18, 200-210, 1992). In particular, enumeration of lymphocyte populations and quantitation of the T-dependent antibody response were particularly beneficial. Furthermore, commonly employed apical measures (e.g., leukocyte counts, lymphoid organ weights) were fairly insensitive. The present analyses focus on the use of this data base to develop statistical models that examine the qualitative and quantitative relationship(s) between the immune function and host resistance tests. The conclusion derived from these analyses are: (1) A good correlation exists between changes in the immune tests and altered host resistance in that there were no instances where host resistance was altered without affecting an immune test(s). However, in some instances immune changes occurred without corresponding changes in host resistance. (2) No single immune test could be identified which was fully predictive for altered host resistance, although most assays were relatively good indicators (i.e., > 70%). Several others, such as proliferative response to lipopolysaccharide and leukocyte counts, were found to be relatively poor indicators for host resistance changes. (3) The ability to resist infectious agent challenge is dependent upon the degrees of immunosuppression and the quantity of infectious agent administered. (4) Logistic and standard regression modeling using one extensive chemical data set from the immunosuppressive agent, cyclophosphamide, indicated that most immune function-host resistance relationships followed linear rather than linear-quadratic (threshold-like) models. For most of the relationships this could not be confirmed using a large chemical data set and, thus, a more mechanistically based approach for modeling will need to be developed. (5) Using this limited data set, methods were developed for modeling the precise quantitative relationships between changes in selected immune tests and host resistance tests.

Male Fischer 344 rats and female B6C3F1 mice were each exposed through their drinking water to a mixture of pesticides and ammonium nitrate that simulated contaminated groundwater in California (California Chemical Mixture [CCM]). Exposures were for 71 or 91 days, respectively. In addition, B6C3F1 female mice were exposed for 91 days to another pesticide and ammonium nitrate mixture (Iowa Chemical Mixture [ICM]) through their drinking water. The spleens were removed from the animals, and the splenocytes were cultured for analyses of sister-chromatid exchange (SCE), chromosome aberrations (CA), and micronuclei (MN) in cytochalasin B-induced binucleate cells. A concentration-related increase in SCEs was found in the splenocytes of the rat at the 1x, 10x and 100x levels of the CCM and at the 100x concentration of the CCM in the mouse. There were no other consistent cytogenetic effects observed with the CCM, and no statistically significant cytogenetic damage was observed in mice exposed to the ICM. Evidence from the literature is discussed in order to infer which chemical or chemicals in the CCM might be responsible for the observed SCE response.

We have previously reported on the design and content of a screening battery using a "tier" approach for detecting potential immunosuppressive compounds in mice [1]. This battery was composed of various immune function, immunopathology and host resistance tests, the results of which could help establish the potential of chemical and biological agents to cause immunosuppression. The data from these studies, which now encompass over 50 compounds, have been analyzed in an attempt to improve future testing strategies and provide information to aid in the risk assessment process. Specifically, the following two issues will be addressed; what are the likelihood(s) for each of the individual tests and testing configurations to accurately identify immunotoxic compounds? and what are the quantitative and qualitative relationships between the immune tests and host resistance assays?

Adverse effects stemming from the therapeutic use of dideoxynucleoside derivatives continue to occur in patients with AIDS or AIDS-related complex. For example, the continued use of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-didoxycytidine (ddC), both of which confer clinical benefits in AIDS patients, may be complicated by anemia, neutropenia and thrombocytopenia and ddC also causes peripheral neuropathy. Subsequently, the National Toxicology Program (NTP) has undertaken efforts to define and characterize the toxicities associated with currently employed and potential AIDS therapeutics in experimental animals, with particular emphasis on the hematopoietic and immune systems. In addition to AZT and ddC, 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyinosine (ddI) and 2',3'-didehydrodideoxythymidine (d4T) have been examined. The present studies describe: (1) the development of a poorly regenerative macrocytic anemia in mice exposed to AZT or ddC. This anemia demonstrates a rapid and progressive time course of toxicity and reversibility after cessation of treatment; (2) the selective suppression of erythroid progenitor cells in mice exposed to d4T without concomitant effects on myeloid stem cells. Myelotoxicity appears to show metabolism-dependent strain-specificity and is more evident following in vitro exposure than in vivo exposure; and (3) the immunosuppressive effects following subchronic (30-day) exposure. Of the nucleoside derivatives studied, only ddA and ddI altered immune function and these changes were confined to suppression of antibody responses. It can be concluded that the overall similarities in the hematopoietic and immune system effects between rodents and humans indicate that such animal toxicology studies provide important information relevant to the toxicity of these drugs.

The present studies examine whether immune-associated toxicities develop in rodents exposed to recombinant human interferon-alpha A/D (rHuIFN-alpha) alone or in combination with anti-retroviral nucleoside analogs. Four findings have emerged from these studies: (1) lymphocyte cell number and functional activity are suppressed after subchronic (1, 8 or 10 day) in vivo exposure to therapeutic doses of rHuIFN-alpha; (2) lymphocyte-associated toxicities lessen as in vivo exposure time to rHuIFN-alpha is extended; (3) T-cell-dependent antibody production is decreased after in vitro exposure of both antigen-specific T- and B-cells to rHuIFN-alpha; and (4) in vivo-induced leukocyte toxicities associated with either rHuIFN-alpha or the nucleoside analogs alone do not synergize when the therapies are combined. These data suggest that certain key immune parameters should be monitored carefully in the early stages of IFN-alpha therapy.

Our need to understand the potential adverse human health effects of environmental chemical exposure has coincided with an increased understanding of the immune system and an appreciation of its complex regulatory network. This has spawned a broad interest in the area of immunotoxicology within the scientific community as well as certain concerns in the public sector regarding chemical-induced hypersensitivity and immunosuppression. The incidence of alleged human sensitization to chemicals has increased, in part, due to the fact that chemical companies are moving to larger and/or different markets. It has been estimated that 35 million Americans suffer from allergic disease of which 2% to 5% are from occupational exposure. Although there is not yet a clear understanding of dose-response relationships or disease predisposition, there are many well-defined examples (isocyanates, anhydrides) of chemical sensitizers in humans and experimental animals. Evidence that chemicals suppress immune responses in humans is considerably less well established, although there is a public perception that chemicals generally cause immunosuppression. This perception has been fueled by highly publicized legal cases (ie, W.R. Grace and Agent Orange) and scientific controversies within the academic and industrial communities (Aldicarb). As a consequence of public pressure, the regulatory agencies are considering immunotoxicity testing guidelines. At the present, however, there are limitations on adequate human methodology and data that allow the extrapolation of animal data to assess accurately human risk.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon-alpha (IFN-alpha) is a naturally occurring cytokine that mediates numerous biological activities and has demonstrated therapeutic potential in a variety of malignancies. Encouraging activity against HIV-1 replication has also been observed with IFN-alpha in the treatment of AIDS, although hematotoxicity has been a frequently observed side effect. In addition, in vitro studies have suggested that IFN-alpha may function as a down-regulator of myelopoiesis. A recombinant hybrid of subtypes of human IFN-alpha, rHuIFN-alpha A/D, has antiviral activity in murine cells in vitro and in vivo. This study examines the effect of acute and subchronic exposure to rHuIFN-alpha A/D on hemopoietic and immune parameters in C57Bl/6 mice. IFN-alpha was administered ip at 0, 1000, 10,000, and 100,000 units/day for either 1 or 10 consecutive days. Many of the known effects of IFN-alpha in humans such as anemia, leukopenia, and thrombocytopenia were observed in mice following subchronic exposure, with the latter two effects also manifested following acute exposure. Further analysis showed that this leukopenia was not selective. Both splenic and bone marrow cells were examined following 10 days of dosing with the high dose of IFN-alpha. Lymphocytes were reduced in both compartments, while granulocytes were increased in both compartments. Bone marrow cells programmed to differentiate into granulocytes (CFU-G) were suppressed, while macrophage progenitors (CFU-M) were stimulated. Erythroid cells decreased in the marrow but increased in the spleen, suggesting that the microenvironment may play a significant role in the effect of IFN-alpha. The proliferative capacity of both B and T splenic lymphocytes was significantly suppressed in a dose-related fashion following multiple exposure to IFN-alpha. Clinically, IFN-alpha is most often given in multiple doses and the present data suggest that such a regimen is toxic to both erythroid and myeloid cells, as well as being immunotoxic to splenic B and T lymphocytes.

There has been continuing interest in the development of synthetic and natural compounds which modify the immune response, particularly for the treatment of AIDS and cancer. Panax ginseng, employed for its putative medicinal properties in South Asia, was examined for its immunomodulatory properties in mice. A systematic evaluation of multiple immune system components revealed that Panax ginseng stimulated basal natural killer (NK) cell activity following subchronic exposure and helped stimulate recovery of NK function in cyclophosphamide-immunosuppressed mice but did not further stimulate NK activity in poly I:C treated mice. Other immunological parameters examined, including T and B cell responses were not affected. Panax ginseng provided a degree of protection against infection with L. monocytes but did not inhibit the growth of transplanted syngeneic tumor cells. Increased resistance to L. monocytogenes was not detected in challenged mice previously given immunosuppressive doses of cyclophosphamide. Taken together, these data suggest that Panax ginseng has some immunomodulatory properties, primarily associated with NK cell activity.

Concern over the potential adverse health effects of chemically contaminated groundwater has existed for many years. In general, these studies have focused on retrospective epidemiological studies for cancer risk. In the present studies, immune function was monitored in female B6C3F1 mice exposed to a chemical mixture in drinking water for either 14 or 90 days. The mixture consisted of 25 common groundwater contaminants frequently found near toxic waste dumps, as determined by EPA surveys. None of the animals developed overt signs of toxicity such as body or liver weight changes. Mice exposed to the highest dose of this mixture for 14 or 90 days showed immune function changes which could be related to rapidly proliferating cells, including suppression of hematopoietic stem cells and of antigen-induced antibody-forming cells. Some of these responses, e.g., granulocyte-macrophage colony formation, were also suppressed at lower concentrations of the chemical mixture. There were no effects on T cell function or T and B cell numbers in any of the treatment groups. Altered resistance to challenge with an infectious agent also occurred in mice given the highest concentration, which correlated with the immune function changes. Paired-water studies indicated that the immune effects were related to chemical exposure and not to decreased water intake. These results suggest that long-term exposure to contaminated groundwater may represent a risk to the immune system in humans.

The effects of methyl isocyanate (MIC) on systemic immunity were evaluated in female B6C3F1 mice exposed via inhalation to 0, 1, or 3 ppm for 6 hr per day on four consecutive days. Humoral immunity, measured as the antibody response to sheep erythrocytes, and natural killer cell activity were not affected by MIC. Furthermore, resistance to the infectious agents Listeria monocytogenes, mouse malaria parasite, and influenza virus, or to B16F10 transplantable tumor cells, was not compromised by MIC exposure. Although lymphoproliferative responses to mitogens were not significantly suppressed, the response of splenic lymphocytes to allogeneic leukocytes in a mixed leukocyte response (MLR) was suppressed in a dose-related fashion and differed significantly from the control response at the 3-ppm level. These studies indicate that MIC exposure in mice does not severely alter systemic immunity. The moderate changes detected in immune function may be a secondary consequence of respiratory toxicity which occurred in these animals.

Methotrexate (MTX) is an antimetabolite commonly used in the treatment of neoplastic disease, while trimetrexate (TMQ) is an investigational antifolate which is currently advocated as a potential alternative to MTX. The cytotoxic properties of antifolates to rapidly proliferating cells suggests that the immune system would be a significant and undesirable target for these drugs. We examined the comparative effects of these two chemotherapeutic agents on the murine immune system using in vivo and in vitro methods. Both drugs were potent suppressors of T-dependent antibody formation in vitro as well as in vivo. While TMQ appeared to be more immunosuppressive than MTX following in vitro addition of the drugs, the converse appeared to be true when dosing was performed in vivo. The drug induced suppression of T-dependent antibody formation was dose dependent for both antifolates. Lymphoproliferative studies demonstrated marked suppressive effects on LPS and PHA induced 3H-uridine and 3H-deoxyuridine incorporation following addition of both drugs in vitro suggesting effects on both RNA and thymidylate biosynthesis. Timed addition studies demonstrated a particularly susceptible time period (hours 24-48 after addition of the mitogen LPS) in stimulated lymphocytes with respect to inhibition of 3H-uridine incorporation. Following in vivo administration of either antifolate, natural killer cell activity was significantly decreased with no substantial differences between the two drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Although the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), via its interaction with the Ah receptor, is an extremely potent carcinogen and immunosuppressive agent in experimental animals, its possible actions on polymorphonuclear (PMN) function have not been determined. In addition to their importance against infectious organisms, PMNs have been implicated in antitumor resistance. The present studies examined the effects of in vivo exposure to TCDD on PMN function in B6C3F1 (TCDD sensitive, presence of high affinity Ah receptor) and DBA/2N (TCDD resistant at low doses, defective Ah receptor) mice. Animals received a single oral exposure of 5 or 10 micrograms/kg of TCDD and PMNs were obtained 5 days later from the peritoneal cavity following elicitation with sodium caseinate. TCDD reduced the cytolytic and cytostatic activity of PMA-activated PMNs in B6C3F1, but not in DBA/2N mice, suggesting that this response segregates with the Ah locus. Furthermore, TCDD was found to bind specifically to PMNs from Ah-responsive mice. Neither the production of superoxide and hydrogen peroxide nor degranulation, the latter measured by beta-glucuronidase release, was impaired. Supernatants recovered from PMN cell cultures of TCDD-sensitive mice, but not from resistant DBA/2N mice, showed reduced killing capacity for actinomycin D-treated L929 tumor cells, while their ability to bind to tumor cells was not altered. These data suggest that TCDD interferes with PMN-mediated tumor cell killing by altering the production or secretion of a cytolytic factor. Examination of bone marrow stem cells revealed that granulocytic but not monocytic colonies were reduced after TCDD exposure in vivo and in vitro. Although mature PMNs had detectable levels of Ah receptor, exposure in vitro of these cells to TCDD had no effect on antitumor activity. Thus, it is possible that TCDD may affect PMNs at the level of hematopoiesis, via a direct interaction with granulocyte precursor cells, or modulate PMNs at different stages of maturation.

A number of 2'3'-dideoxynucleosides have been reported to markedly inhibit the in vitro growth of HIV, the causative agent of acquired immunodeficiency syndrome (AIDS). Clinical trials have shown that the continued therapeutic use of these nucleoside derivatives can be associated with adverse side effects. Since these side effects include myelotoxicity, as occurs in many patients treated with zidovudine (AZT; 3'-azido'3-deoxythymidine), and AIDS patients already represent an immunologically compromised population, we examined the immunological effects of three nucleoside inhibitors, including zidovudine, 2'3'-dideoxycytidine, and 2'3'-dideoxyadenosine (DDA) in a mouse model. Additional studies were conducted to further determine and characterize the potential toxic effects associated with these drugs on the hematopoietic system. Of the three dideoxynucleosides examined, only DDA altered immune functions following a 30-day subchronic exposure in mice. This was evidenced by a marked suppression of the antibody plaque-forming cell response and a slight alteration in macrophage function. None of the nucleoside derivatives affected bone marrow function following in vivo exposure, although AZT produced a mild macrocytic anemia in vivo and was myelotoxic when added in vitro to bone marrow cell cultures. In vitro studies indicated that AZT was capable of affecting both proliferating stem cells as well as the stromal cell microenvironment, both of which play a role in hematopoiesis. These data indicate that, although the mice may not develop the identical toxicities associated with nucleoside therapy in humans, certain adverse immunological and hematological effects were readily discerned which could have relevance to humans.

The relationship between immune function and chemically induced hepatocarcinogenesis was studied employing an in vivo murine model. Neonatal B6C3F1 mice were given a single carcinogenic dose of diethylnitrosamine (DEN) and the time-response kinetics for the early (foci of alteration) and late (adenomas/carcinomas) phases of hepatocellular carcinogenesis were compared to changes in hematopoiesis and immune functions associated with immune surveillance and natural resistance. Increases in hematopoiesis occurred just prior to or concurrent with the appearance of hepatocellular carcinomas, while increased macrophage and natural killer cell cytotoxicity and suppression of cell-mediated immunity occurred following tumor appearance and progressed with increasing tumor burden. Neither immunological nor hematopoietic changes were associated with early phases of hepatocarcinogenesis, as monitored by the appearance of altered hepatocellular foci. Although changes in hematopoiesis may represent an early indicator for hepatocarcinogenesis in the mouse tumor model, the data suggest that altered immune surveillance and natural resistance are not factors in the development of chemically induced hepatocellular tumors, and the changes in immune function are probably secondary to tumor development.

Ochratoxin A, a naturally occurring mycotoxin, has recently been shown to cause renal and hepatic carcinomas in mice. In the present studies, the effects of ochratoxin A on immune mechanisms associated with tumor resistance were examined in mice using dose levels similar to those that cause neoplasia. Ochratoxin A was shown to specifically inhibit natural killer (NK) cell activity and increase the growth of transplantable tumor cells without altering T-cell- or macrophage-mediated antitumor activity. In contrast, ochratoxin B, a much less toxic ochratoxin, did not influence immune function. Polyinosinic:polycytidylic induced interferon was markedly reduced in mice following exposure to ochratoxin A although total serum protein levels were slightly increased. Injection of polyinosinic:polycytidylic enhanced NK activity in the presence of ochratoxin A, although the level of enhancement was slightly lower than that produced by the agent in the absence of ochratoxin A. Thus, ochratoxin appears to suppress NK cell activity by inhibiting production of basal interferon. Additionally, these findings suggest a possible role for altered NK cell function in the development of mycotoxin-induced carcinogenesis.