A perspective on the progression of immunotoxicology.

Immunotoxicology originated in the early 1970s, when scientists began investigating chemicals. During the 1970s and early 1980s, a few investigators determined that chemicals were immunotoxic, developed and/or refined immunoassays, and began to characterize immunotoxic responses. In the 1980s, many new investigators entered the field, graduates were being trained as immunotoxicologists, the immune system was identified as a primary target organ, mechanisms of action studies proliferated, a comprehensive immunotoxicological panel was validated, the discipline gained universal credibility, and human studies emerged. The 1990s were ushered in with the concept of biological markers in immunotoxicology, a better understanding of "immune function", inclusion of immunotoxicology in risk assessment analysis, and a focus on molecular immunology. Future investigations will continue to improve and expand this foundation, pursue the relationship of immunotoxic chemicals and adverse health effects in humans, utilize genetically altered rodent models, and use gene expression technology to better understand the pathogenesis of immunotoxicological processes. Immunotoxicology has not only matured since its inception nearly 30 yr ago, but has become a prominent and respected discipline with global recognition; one that has made significant contributions to the advancement of the biomedical sciences.

Pathology of "toxic oils" and selected metals in the MRL/lpr mouse.

The Toxic Oil Syndrome epidemic that occurred in Spain in 1981 and affected nearly 20,000 people was caused by ingestion of oil mixtures that contained analine-denatured rapeseed oil. To date, an animal model in which to identify the actual etiologic agents(s) and to investigate the pathogenesis of the disease has not been discovered. In this study, the MRL/lpr was used to assess the histopathological response of 3 "toxic oils" and 3 metals. The oils tested were a denatured rapeseed oil collected from a family who were affected by the Toxic Oil Syndrome epidemic in Spain (CO756) plus two synthesized oils (RSD and RSA). Female mice, 7 weeks of age, received an undiluted (neat) or a 1:10 diluted dose of each oil; mercury (50 ppm), cadmium (100 ppm), or lead (50 ppm). Half of each group was killed after 5 weeks of exposure and the remaining mice after 10 weeks of exposure. Body and organ weights (liver, kidney, thymus, and spleen) were recorded and selected organs were collected for histopathology. Ten weeks after treatment, body weights (BW) of the cadmium and lead groups were significantly suppressed, and the body weight of the C0756-neat group was significantly increased compared to their respective controls. Kidney/BW were decreased in the RSA-neat and RSA 1:10 groups after 10 weeks of exposure, and the kidney/BW in the mercury and cadmium groups were increased. Spontaneous development (12 weeks of age) and progression (17 weeks of age) of histopathological lesions are described for selected organs examined in the naïve mice as are changes that resulted from exposure to the "toxic oils" and metals. C0756-neat, mercury, and lead suppressed progression of the glomerulonephritis that normally occurs in the MRL/lpr mouse. Also of interest were lesions that included mononuclear cuffing of hepatic bile ducts, progression of the granulomas that formed in the renal glomeruli, vessels in the lymphoid organs that contained tightly packed lymphocytes, and the presence of plasma cells in the thymus. All 3 oils stimulated early development of the lymphoproliferative syndrome characteristic of the MRL/lpr mouse as demonstrated by an increase in the thymus/BW and spleen/BW ratios after 5 weeks of treatment. These data contribute to our knowledge of spontaneous disease progression in the thymus, spleen, lymph nodes, and kidneys in the MRL/lpr mouse and the effects of 3 different "toxic oils" and metals on the development and progression of those lesions.

Effect of a single or repeated dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on T cell subpopulations in the Long-Evans rat.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure results in adverse effects on the immune system of experimental animals. The purpose of this study was to compare the effects of a single and repeated dosing of TCDD on splenic T-cell subpopulations in Long Evans rats 9 days post-exposure to TCDD. A single dose (25 microg/kg body weight) of TCDD resulted in reduced body weight. The percentage and total number of CD4+ or CD8+ subsets and percentage of CD4+ or CD8+ cell cycling in the S and G2M phases were similar in the single dosed (25 microg/kg body weight) TCDD group compared with the vehicle control. A repeated dose (5 microg/kg/day for 5 days) of TCDD also resulted in a significant reduction in body weight. However, multiple doses of TCDD significantly decreased the percentage of the CD4+ subset and the percentage of CD4+ cells cycling in the S and G2M phases. No significant change occurred in the CD8+ cell subpopulation after single or multiple dosing with TCDD. These results demonstrated that repeated dosing of TCDD decreased the total percentage of CD4+ cells and the percentage of CD4+ cells cycling 9 days post-exposure, while an analogous single dose of TCDD failed to affect the CD4+ cell subpopulation. The difference in biological responses to a single versus 'equivalent' multiple (cumulative) dose of TCDD is discussed.

Superantigen activation and kinetics of cytokines in the Long-Evans rat.

This study was conducted to identify and quantify, over time, selected cytokine responses in Long-Evans rats that were exposed to staphylococcus enterotoxin B (SEB). The kinetics of selected cytokines [interleukin-2 (IL-2), IL-6, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF)] and phenotype and cell cycle analysis of T lymphocytes were determined in Long-Evans rats administered a single intraperitoneal (i.p.) dose of either 50 microg or 500 microg of SEB. Rats injected with 50 microg SEB had significantly elevated levels of IL-2, IL-6 and IFN-gamma in their serum 2 hr post-injection. IL-2 serum levels were significantly elevated at 2 hr and returned to near control values by 12 hr while both IL-6 and IFN-gamma peaked at 6 hr but remained significantly increased at 24 hr post SEB exposure. A 500 microg dose of SEB did not further enhance these cytokine responses. When spleen cells were collected for culture 2 hr after rats were injected i.p. with 50 microg SEB and cocultured with SEB, TNF and IL-6 levels were significantly increased after 2 hr incubation, while IL-2 and IL-6 were significantly elevated at 6 hr. Production of all these cytokines in spleen cell cultures continued to increase over the 24 hr sampled. Peritoneal cells were collected for culture either at 1 hr or 2 hr after injection of either 50 microg or 500 microg of SEB. IL-6 was significantly increased after 1 hr in culture while TNF was significantly increased by 2 hr regardless of whether the cells were harvested 1 or 2 hr after SEB injection. The greatest response for both IL-6 and TNF occurred when cells from animals injected with 50 microg SEB were restimulated in vitro with SEB. The peak levels for IL-6 were at 12 hr post SEB exposure while TNF peaked at 6 hr. The percentage of CD4+ cells was significantly increased at 48 hr and 72 hr post SEB (50 microg) administration while the percentage of CD8+ cells remained similar to control values for the 168-hr test period. A similar pattern was observed in cell cycling where the CD4+ cells proliferated up to 2 days post SEB injection and then were significantly suppressed at day 3. The CD8+ cells were comparable to control values. These studies demonstrate that the cytokine responses in Long-Evans rats exposed to a superantigen are somewhat similar to those that occur in mice and humans, e.g. a rapid short increase in the production of IFN-gamma and TNF that was accompanied by an increase in the production of IL-2. Additional responses noted in this species, however, were a marked increase in IL-6 production, as well as an early increase in the number and cycling of CD4+ cells followed by a down-regulation of these events. These activities occurred in the absence of notable histopathological alteration of lymphoid organs. The results indicate that the Long-Evans rat is an acceptable animal model to investigate the pathogenesis of superantigen-induced disease and that IL-6 may be an active mediator of this process.

Neopterin values in selected groups of normal animals.

To establish baseline information on neopterin concentrations in livestock, companion and laboratory animals and identify the factors that may influence these concentrations, blood samples were taken from normal dairy cattle, horses, llamas, dogs, cats and rats of varying ages and sexes. In addition, neopterin concentrations in normal, adult equines were compared with those found in racing Thoroughbreds. There were no differences due to sex, sexual maturity, pregnancy, castration, or age. For all ages and sexes combined, mean neopterin concentrations were significantly lower in llamas (2.27+/-0.33 nmol litre(-1)) and rats (2.13+/-0.21 nmol litre(-1)) when compared with the other species tested. Racing Thoroughbreds demonstrated higher neopterin values than adult equines not in training (3.54+/-0.64 vs 3.13+/-1.02 nmol litre(-1)).

2,3,7,8-Tetrachlorodibenzo-p-dioxin co-stimulates staphylococcal enterotoxin beta (SEB) cytokine production and phenotypic cell cycling in Long-Evans rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that is considered to be a potent immunotoxicant. In the present study, we examined the effect of 25 micrograms/kg TCDD on cytokine production and T lymphocyte phenotype, cell cycling and receptor activity in female Long-Evans rats that had been injected with 50 micrograms of Staphylococcal Enterotoxin B (SEB). In the SEB-injected rats, TCDD increased the serum levels of interleukin-2 (IL-2) but did not affect the serum levels of interleukin-1 (IL-1), interleukin-6 (IL-6) or tumor necrosis factor (TNF). The ability of spleen cells and peritoneal cells to produce cytokines in response to SEB restimulation in vitro was also evaluated. TCDD exposure significantly enhanced IL-2 production by spleen cells from SEB-primed rats after 6 h or 24 h in cultures co-stimulated with SEB in vitro. However, TCDD treatment did not alter the production of IL-6 and TNF in these cultures. Although TCDD did not influence the production of IL-6 and TNF in peritoneal cells from SEB-primed rats with SEB restimultion in vitro, IL-1 production was significantly increased at 2 h. Both the kinetics and extent of SEB-induced IL-2 receptor (IL-2R) and T-cell receptor (TCR) expression on CD4+ cells was unaffected by TCDD. TCDD did not significantly alter the percentage or the total numbers of CD4+ and CD8+ subpopulations at various times after SEB injection. However, flow cytometric analysis showed that TCDD exposure increased the percentage of both CD4+ and CD8+ cells cycling in the S and G2M phase. TCDD, in the absence of SEB priming, did not affect any of the immune parameters tested. Nevertheless, collectively these results showed that TCDD can enhance the production of IL-2 and the percentage of CD4+ and CD8+ cells cycling in SEB-exposed Long-Evans rats. Histopatholgically, there were not observable effects of SEB on lymphoid organs while thymic atrophy and diffuse hepatocellular hypertrophy was observed in the TCDD-treated animals.

The correlation between serum selenium and blood selenium in cattle.

The selenium (Se) concentration of paired blood and serum samples from cattle was determined by 2 methods: 1) atomic absorption spectroscopy using hydride generation (HG-AAS), and 2) inductively coupled argon plasma emission spectroscopy using hydride generation (ICP). Samples from 327 cattle were analyzed by HG-AAS, and samples from 344 cattle were analyzed by ICP. The data were examined by linear regression analysis, and the technique of inverse prediction was utilized to determine prediction intervals for estimating blood Se concentration from known serum Se concentration. The correlation coefficients, by simple linear regression of serum Se on blood Se, were 0.79 (r2 = 0.62) and 0.88 (r2 = 0.77) for the HG-AAS data and the ICP data, respectively. For the HG-AAS data, the inverse prediction formula for estimating blood Se when serum Se is known, at the 95% prediction interval, was [formula; see text]. For the ICP data, the inverse prediction formula for estimating blood Se when serum Se is known, at the 95% prediction interval, was [formula; see text]. The prediction intervals were quite wide, and the accuracy of estimating blood Se from a known serum Se was not useful for diagnostic purposes. The use of serum Se concentration to assess nutritional status of cattle with respect to Se does not appear to be appropriate.

Subchronic 90 day toxicity of dichloroacetic and trichloroacetic acid in rats.

Male Sprague-Dawley rats were treated with either dichloroacetic acid (DCA) or trichloroacetic acid (TCA) in the drinking water at levels of 0, 50, 500 and 5000 ppm for a period of 90 days to determine the toxicities associated with subchronic exposure. All animals were sacrificed and examined for gross and histopathologic lesions, serochemical changes, immune dysfunction, hepatic peroxisomal and mixed function oxidase enzyme induction and organ-body weight changes. Animals treated with DCA had decreased body weight gains (500 and 5000 ppm) and decreased total serum protein (all doses). Rats given either TCA (5000 ppm) or DCA (500 or 5000 ppm) had increased liver and kidney organ to body weight ratios. Rats offered DCA had significantly elevated alkaline phosphatase (500 and 5000 ppm) and alanine-amino transferase (5000 ppm). No consistent immunotoxicity was observed in animals exposed to either compound. Rats treated with 5000 ppm TCA or DCA had significantly increased hepatic peroxisomal beta-oxidation activity. These data, along with histopathologic changes, suggest that TCA and DCA produce substantial systemic organ toxicity to the liver and kidney during a 90-day subchronic exposure, although only at doses greater than those expected to occur in the environment.

The effects of methylnitrosourea (MNU) on natural killer (NK) cell cytotoxicity and cytokine production in rats.

Eight-week-old male Sprague-Dawley rats were exposed to the carcinogen methylnitrosourea (MNU) via gastric intubation at doses of either 10 or 20 mg/kg body wt. Rats were treated once a week for 4 weeks, then once every 2 weeks for 1 month, for a total of 6 treatments. MNU was found to exert no consistent significant immunosuppressive effects in vivo as measured by spleen natural killer (NK) cell cytotoxicity, interleukin-2 (IL-2) production by splenic lymphocytes and prostaglandin E2 (PGE2) production by adherent peritoneal macrophages. In contrast, splenic NK cell cytotoxicity and IL-2 production of MNU-treated rats were actually elevated at several of the later sampling periods. PGE2 production was also elevated in MNU-treated rats in the later sampling periods. Body weights of MNU-treated rats were markedly decreased as early as 4 weeks following the initial MNU treatment. This suppression persisted throughout the study. The most dramatic change in organ weights was seen in the thymus. Thymus weights of all MNU-treated rats were significantly decreased 1 day after treatment and persisted for 4 weeks. By the 60 day sampling period, thymus weights were not significantly different from controls. However, by 120 and 180 days, thymus weights again were significantly lowered in those rats receiving MNU. These changes in thymus weights were accompanied histologically by initial cortical thinning and progressive loss of cortical thymocytes followed by the appearance of hyperplastic and neoplastic cells. It thus appears that the carcinogenic effect of MNU is not related to a depression of the immune surveillance system, at least as measured by NK cell activity.

Alterations of rat natural killer (NK) cell cytotoxicity and cytokine production by 3-methylcholanthrene (3-MC).

The carcinogen 3-methylcholanthrene (3-MC) was found to exert immunosuppressive effects both in vitro and in vivo in this study. Spleen cells from 8-week-old male, Sprague-Dawley (S-D) rats exposed to 1, 10 or 100 micrograms/ml 3-MC in vitro for 18 h exhibited a dose-dependent decrease in natural killer (NK) cell cytotoxicity against the YAC-1 tumor target cells in a 4 h 51Cr-release assay. Peritoneal macrophage production of prostaglandin E2 (PGE2) was significantly decreased at all three 3-MC concentrations following a 24 h exposure in vitro. No effect of 3-MC on splenic interleukin-2 (IL-2) production was observed. A separate group of rats was inoculated with a single subcutaneous dose of 5 or 10 mg 3-MC and cytotoxic activity of spleen NK cells was examined at 1, 2, 3, 7, 14, 21, 28, 60, 120 and 180 days after the 3-MC injection. Natural killer cell cytotoxicity was suppressed as early as 24 h after 3-MC injection and persisted up to 21 days. This decrease in NK activity was accompanied by a decreased production of splenic interferon and elevated production of PGE2 by peritoneal macrophages. Natural killer cell cytotoxicity was elevated in the 3-MC-treated rats at 28 and 60 days post-treatment. At 120 and 180 days post-3-MC treatment, when the rats were bearing palpable chemically-induced tumors, NK activity was again significantly depressed. In addition, 3-MC-induced tumors were surgically removed and cultured in vitro. Supernatants from these tumor cell lines were shown to markedly inhibit NK cytotoxicity when tested in vitro. Preliminary results indicate that this inhibition may be mediated by prostaglandins.

Immunotoxicity assessment of gentamycin and liquamycin.

Rats were injected with gentamycin (GEN) or the oxytetracycline, liquamycin 200 (LA200), and multiple immune responses were measured to assess the immunotoxicity of these antibiotics. Animals were treated by sc or im injection for 12 days with either GEN or LA200 given at a therapeutic dose or at 10-, 50-, or 100-fold greater concentrations. The following immune parameters were assessed in each animal: delayed-type hypersensitivity (DTH), natural killer (NK) cell cytotoxicity, antibody production, and synthesis of interleukin 2 (IL2) and gamma interferon (IFN). The DTH response and IFN production in GEN- and LA200-injected animals were suppressed in a dose-dependent manner. Production of IFN was significantly suppressed in all groups treated with LA200. Suppression of DTH was evident when GEN or LA200 were given at the recommended therapeutic dose. Both LA200- and GEN-treated rats had suppressed NK cell cytotoxicity, but only at the highest dosages. Antibody production was suppressed in a dose-dependent manner in LA200-treated rats. Synthesis of IL2 was suppressed in rats treated with the high dose of GEN. Body weight loss occurred, and hepatic and renal toxicity were apparent at the 2 highest dosages of GEN or LA200. It was apparent that relatively high doses of GEN and LA200 can suppress specific and non-specific cell-mediated immune responses. Also, the data suggest the certain immune responses (eg, DTH reaction and possibly IFN production) may be suppressed at the recommended therapeutic doses in the absence of other signs of toxicity. These results indicate that these antibiotics should not be administered at doses or exposure periods in excess of those recommended.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of CI-949 and CI-959 on immune function and lymphoid organs in rats.

The immunotoxic properties of two experimental antiallergic drugs, CI-949 and CI-959, were investigated. Wistar rats were gavaged once (CI-949) or twice (CI-959) daily for 21 days with the drugs. Immunotoxicity was assessed using the enzyme-linked immunoabsorbant assay (ELISA) for humoral immunity, a delayed-type hypersensitivity (DTH) procedure for cell-mediated immunity, and natural killer cell (NKC) activity to evaluate spontaneous cytotoxicity. Ratios of body weight to spleen, thymus, liver and kidney weights were determined. Routine histopathology was performed on lymphoid tissue and other body organs. Although 100 mg/kg/day of CI-949 had some stimulating effect on antibody production and NKC cytotoxicity, no consistent immunomodulation was apparent. Except for a significant increase in liver weight at the 100 mg/kg dose of CI-949, no other toxic effects were observed. In contrast to CI-949, CI-959 significantly (P less than 0.05) suppressed antibody production at the 100 mg/kg dose and impaired the DTH reaction, although not significantly. Natural killer cell cytotoxicity was unaffected by 100 mg/kg CI-959. Decreased body weight and histopathological lesions were observed in the thymus and spleen of rats administered 100 mg/kg CI-959. These lesions ranged from mild to severe lymphoid depletion which was also reflected in significantly (P less than 0.05) reduced spleen and thymus organ weight to body weight ratios. Since 100 mg/kg of CI-959 produced toxicological and pathological alterations in the exposed rats, these data suggest that CI-959 is not highly or specifically immunotoxic at dosages lower than those that alter conventional toxicological parameters used in new drug testing programs.(ABSTRACT TRUNCATED AT 250 WORDS)

Severe or marginal copper deficiency results in a graded reduction in immune status in mice.

From birth mice received diets containing copper at 0.5, 1, 2 or 6 mg/kg diet. At 8 wk of age they were killed and copper status and immune responsiveness were determined. Only the groups that received copper at 0.5 or 1 mg/kg showed signs of copper deficiency, such as reduced serum ceruloplasmin, hemoglobin, hematocrit and red blood cell counts and characteristic changes in organ pathology. Body and lymphoid organ weights were altered in the groups that received copper at 0.5 or 1 mg/kg. Males were more severely affected than females. A dose-related reduction in splenic T-cell subpopulations was noted in the 0.5 and 1 mg/kg groups. Responses to lipopolysaccharide challenge were reduced, and an increase in spontaneous cycling cells was noted in the groups receiving copper at 0.5 or 1 mg/kg. Only the group receiving copper at 0.5 mg/kg had increased stem cell activity; this increase was probably due to increased erythropoiesis to meet increased demands for red blood cells in this group. These data indicate that only groups receiving copper at 0.5 or 1 mg/kg in the diet were depleted and marginally depleted in copper, respectively, and that immune hyporesponsiveness differs between the depleted and marginally depleted groups.

Immunotoxicologic evaluation of chlorine-based drinking water disinfectants, sodium hypochlorite and monochloramine.

Male Sprague-Dawley rats were exposed to chlorine-based disinfectants in the drinking water from weaning to 12 weeks of age, at which time they were terminated and assessed for immune competence. Chlorine-based drinking water disinfectants used were sodium hypochlorite (5, 15 and 30 ppm) and monochloramine (9, 19 and 38 ppm). Parameters of immunity measured were spleen and thymus weights, antibody production, delayed-type hypersensitivity (DTH) reactions, natural killer cell (NKC) cytotoxicity, oxidative metabolism response (i.e chemiluminescence-CL) and phagocytosis by macrophages, and production of 2 immunoregulatory cytokines, interleukin 2 (IL2) and prostaglandin E2 (PGE2). Significant (P less than or equal to 0.05) reductions of spleen weight, DTH reactions, and oxidative metabolism by macrophages were observed only in groups of rats exposed to high levels (30 ppm) of sodium hypochlorite, while PGE2 production was elevated. Rats exposed to the higher doses of monochloramine had reduced spleen weights (38 ppm), decreased antibody synthesis (9 and 19 ppm) and augmented PGE2 production (19 and 38 ppm). These results extend the earlier observations of others that macrophage function of laboratory rodents may be impaired by exposure to high concentrations of chlorinated drinking water. Furthermore, the function of other major populations of immunocytes and types of immune responses may also be altered following subchronic exposure to high concentrations of chlorinated drinking water. These types of effects on the immune system are a previously unrecognized potential side-effect of the ubiquitous practice of disinfection of water with chlorine compounds. Alteration of immune function of chlorine-based disinfectant-exposed rats in this study was only evident at relatively high doses, and only selected immune responses were altered. It appears, therefore, that these chlorine-based disinfectants are not particularly strong immunodepressants. However, further studies in different species may be warranted in order to better extrapolate to implications to human health following chronic low-level exposure.

Immune dysfunction in rats fed a diet deficient in copper.

Rat pups maintained on copper (Cu)-adequate (6ppm), Cu-deficient (2 ppm) or Cu-depleted (0 ppm) diets from parturition were killed at 8-wk. Liver Cu and serum-ceruloplasmin levels confirmed that on the 0- and 2-ppm diets, a Cu-deficient state was induced. Although body weight was unaffected by the deficiency, the liver, heart, and thymus weights (% body weight) were altered. Hepatomegaly occurred in females fed 0-ppm Cu and males fed 2-ppm Cu. Heart weights increased in both sexes fed 0-ppm Cu. Thymus weights decreased in male rats fed 0-ppm Cu. Antibody titers and natural killer-cell cytotoxicity were markedly suppressed in the animals fed 0-ppm Cu. Male rats given 2-ppm Cu showed reduced antibody titer. Delayed-type hypersensitivity and prostaglandin E2 levels were not significantly affected. These studies suggest that certain components of the immune system are Cu dependent.

Immunotoxicology today.

Immunotoxicology is a science which deals with the effects of physical and chemical agents and other toxic substances on the immune system. The discipline includes study of the detection, occurrence, adverse effects, and mechanisms of chemically-induced immune dysfunction. Many drugs and chemicals are known to compromise the immune response of a host which is extremely vulnerable and sensitive to perturbation by these agents. The consequences of immune dysfunction may be expressed in reduced resistance to infectious and neoplastic diseases, or enhanced hypersensitivity and autoimmunity. The immunomodulating profiles of xenobiotics may be diverse, involving several components of the immune system, or they may selectively compromise an individual compartment of the immune response. Many drugs and chemicals are known to result in immune disregulation in animals, but a paucity of information is available to indicate the effects of xenobiotics on systemic immunity in humans. Epidemiological studies and research designed to assess chemical immunomodulation in man are necessary to confirm animal data. Further, a definitive diagnosis for chemically-induced immune dysfunction must include several etiologies and body organ systems, since a triad of reciprocal interactions exists between the immune, endocrine, and central nervous systems.

Trichloroacetic acid effects on rat liver peroxisomes and enzyme-altered foci.

The initiating and promoting effects of trichloroacetic acid (TCA) were investigated using a rat hepatic enzyme-altered foci bioassay. The experimental protocol used has been shown to induce gamma-glutamyltranspeptidase (GGT)-positive foci in hepatic tissue following an initiating dose with a genotoxic carcinogen. Twenty-four hours following 2/3 partial hepatectomy, rats received either a single oral dose (1500 mg/kg) or 5000 ppm TCA in drinking water for 10, 20, or 30 days. Two weeks after the end of TCA exposure, the rats were promoted for 3 or 6 months with 500 ppm phenobarbital in drinking water. TCA failed to induce GGT-positive foci using this initiation protocol. In addition, groups of 2/3 partially hepatectomized rats were initiated with a single oral dose of diethylnitrosamine (10 mg/kg) and then administered 50, 500, or 5000 ppm TCA drinking water. In this promotion protocol, TCA exposure resulted in a significant increase in the number of GGT-positive foci. The ability of TCA to stimulate peroxisomal-dependent palmitoyl-coenzyme A oxidation was also investigated. Only the 5000 ppm TCA treatment within the promotion protocol resulted in a significant, although minor, stimulation of peroxisomal enzyme activity. The findings support the hypothesis that TCA may possess weak promoting activity in the rat liver.

Immunotoxicity testing: an economical multiple-assay approach.

A model for assessing immunotoxicologic effects of chemicals and drugs was developed in the Sprague-Dawley rat whereby multiple concomitant immunoassays were performed in a single animal. The multiple parameters of immunity assessed in each rat included T cell-dependent IgG antibody production, delayed hypersensitivity, natural killer cell cytotoxicity, and production of three potent immune regulating immunocytokines: macrophage-derived interleukin 1 and prostaglandin E2, and lymphocyte-derived interleukin 2. Splenocyte and resident peritoneal macrophage numbers were also quantitated and spleen and thymus weights recorded. The sensitivity of this animal model was tested by treating rats with the immune-potentiating drugs, NPT 15392 (erythro-9-[2-hydroxy,3-nonyl]hypoxanthine) and avridine (N,N-dioctadecyl-N',N'-bis-[2-hydroxyethyl]propanediamine, or the immune-suppressive drugs, cyclophosphamide (N,N-bis[2-chloroethyl]tetrahydro-2H-1,3,2-oxazaphosphorin-2-amine -2-oxide) and dexamethasone. Rats treated with NPT 15392 or avridine generally had enhanced immune responses, while those treated with cyclophosphamide or dexamethasone had decreased immune responses. Differential responsiveness of various immunocyte populations within individual rats to different drugs, or to doses of the same drug, indicates the efficacy of measuring multiple responses within the same animal. The multiassay-single animal approach represents an economical, versatile, sensitive, and relatively comprehensive paradigm for assessing immunotoxicologic/pharmacologic properties of chemicals and drugs. The approach is extremely economical since multiple immune responses are evaluated in each animal. The approach is versatile because it is amenable to incorporation of a variety of in vitro and in vivo assays and could be applied to almost any species. The model is relatively comprehensive because major types of immune responses/immunocyte populations and immunoregulatory pathways are tested. Finally, the model is sensitive for detecting immunosuppression as well as immunoenhancement, as validated by the use of known immune response modifiers in this study.