Use of the enzyme-linked immunosorbent assay (ELISA) in immunotoxicity testing.

Based on an earlier described macromethod for the routine measurement of IgM and IgG in rat sera, a mechanized micro enzyme-linked immunosorbent assay (ELISA) was developed. The assay was performed in the wells of microtiter plate thus minimizing the quantities of reagents and antisera needed. Data on reproducibility of the assay and calculation of IgM and IgG levels are provided. For the functional assessment of the humoral immunity of the rat, ELISA is a powerful tool. In an earlier report, assays for the titration of thymus-independent IgM antibodies to E. coli LPS and the IgM and IgG response to the thymus-dependent antigen tetanus toxoid were described. More recently it was shown that the antigen ovalbumin elicits a thymus-dependent IgM, IgG and IgE response which could be readily measured with the enzyme immunoassay, as well as a delayed-type hypersensitivity reaction. As the optimum ovalbumin concentration for both types of reactions was the same, it is concluded that the ovalbumin model offers the advantage that both humoral and cellular immunity can be studies simultaneously in the same animal.

Genotoxicity of hormonal steroids.

Hormonal steroids have a widespread use in medicine and their side effects are continuously debated. The possible genotoxic activity of steroids has been the subject of many investigations. The natural estrogens estradiol, estrone and estriol are generally negative in the ICH core battery of tests, but several positive results have been obtained when using additional endpoints of genotoxicity. The genotoxic activity of the 4-hydroxy metabolites of estradiol and estrone is well established. The synthetic steroidal estrogens have a comparable profile of negative and positive test results. Cyproterone acetate and some of its analogues have a special position within the group of progestins. Their genotoxic potential has been established. Other progestins are generally negative in the routine tests. Anti-glucocorticoids, anti-progestins, corticosteroids, androgens, anabolics and anti-androgens appear to be devoid of genotoxic activities. The genotoxic potential of estradiol, estrone and cyproterone acetate with its analogues may play no role under normal physiological and therapeutic conditions. The metabolic conditions that are needed for the formation of DNA-reactive metabolites and oxygen radicals may not be present in humans. Epidemiological cancer data seem to support this view. The importance of thresholds in the dose-effect-relationship of genotoxicity data and their use in risk assessment is discussed.

Acute oral toxicity of inorganic cobalt compounds in rats.

Eight cobalt compounds were administered to rats by gastric intubation, and the following LD50 values (in mg anhydrous compound/kg body weight) were determined: cobalt(II) fluoride 150, cobalt(II) oxide 202, cobalt(II) phosphate 387, cobalt(II) bromide 406, cobalt(II) chloride 418, cobalt(II) sulphate 424, cobalt(II) nitrate 434 and cobalt(II) acetate 503. After administration of the cobalt compounds, body temperatures decreased by 2.5-7.5 degrees C. The liver, heart and kidneys of rats given cobalt(II) fluoride or oxide were examined microscopically. Hyperaemia, haemorrhage and cytoplasmic changes were noted, while the kidney glomeruli were very rich in cells and basal membranes were thickened. Cells of the proximal tubules were swollen and showed vacuolization and degeneration. In the hearts of some rats proliferative and oedematous interstitial tissue and swollen muscle fibres were observed, and focal degeneration, vacuolization and necrosis associated with disappearance of the cross striations were also noted.

The influence of sodium bromide in man: a study in human volunteers with special emphasis on the endocrine and the central nervous system.

Sodium bromide was administered orally in capsules to healthy volunteers in doses of 0, 4 or 9 mg Br-/kg/day using a double-blind design. Each treatment was given to seven males for 12 weeks and to seven non-pregnant females (not using oral contraceptives) over three full cycles. Special attention was paid to possible effects on the endocrine and central nervous systems. At the start and end of the study, a full medical history, the results of a physical examination, haematological studies and standard clinical chemistry and urine analyses were recorded for each subject. These showed no changes for individuals following treatment, except for some incidence of nausea associated with bromide-capsule ingestion. Mean plasma-bromide concentrations at the end of treatment were 0.08, 2.14 and 4.30 mmol/litre for males and 0.07, 3.05 and 4.93 mmol/litre for females of the 0-, 4- and 9-mg Br-/kg/day groups, respectively. Plasma half-life was about 10 days. In the females taking 9 mg Br-/kg/day (but in no other group) there was a significant (P less than 0.01) increase in serum thyroxine and triiodothyronine between the start and end of the study but all concentrations remained within normal limits. No changes were observed in serum concentrations of free thyroxine, thyroxine-binding globulin, cortisol, oestradiol, progesterone or testosterone, or of thyrotropin, prolactin, luteinizing hormone (LH) and follicle-stimulating hormone before or after the administration of thyrotropin-releasing hormone and LH-releasing hormone. Analysis of neurophysiological data (EEG and visual evoked response) showed a decrease in delta 1- and delta 2-activities and increases in beta-activities and in mean frequency (Mobility parameter) in the groups on 9 mg Br-/kg/day, but all the findings were within normal limits.

Chronic toxicity and carcinogenicity of bis(tri-n-butyltin)oxide (TBTO) in the rat.

In a 106-wk toxicity and carcinogenicity study, groups of 60 male and 60 female weanling Wistar rats were fed 0, 0.5, or 50 mg bis(tri-n-butyltin)oxide (TBTO)/kg diet. In males, feed consumption was increased in all treated groups and increased water consumption occurred at 5 and 50 mg/kg. During the second year, body weight decreased in the 50-mg/kg males, while the females in that group showed no weight gain. Excess mortality was confined to the 50-mg/kg group towards the end of the study. Haematological changes, comprising anaemia, lymphocytopenia and thrombocytosis were noted mainly at the high-dose level. Also, signs of decreased kidney function and increased plasma enzyme activities (alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase) were noted. No effects on serum hormone concentrations (thyrotropin, follicle stimulating hormone, luteinizing hormone or insulin) were observed, except for a decrease in the free thyroxin:thyroxin ratio in both sexes at the high-dose level. Higher serum IgM and IgA levels were present at 50 mg/kg, while, in females, IgG was decreased. At 50 mg/kg, the ovaries, adrenals, spleen (females), heart (males), pituitary, liver and kidneys were increased in weight, but the thyroid weight was decreased in females. The total tin concentrations in liver and kidneys showed a dose relationship and, in general, the concentrations were similar after 1 and 2 yr. Non-neoplastic histological alterations after 1 yr consisted of a decrease in the cell height of the thyroid follicles in all dose groups, with a reduced number of psammoma bodies at 50 mg/kg, a decrease in splenic iron content at 5 (females only) and 50 mg/kg, and a slight bile-duct activation. After 2 yr, only the thyroid changes were still present. In addition, at 2 yr, vacuolation and pigmentation of the proximal tubular epithelium and nephrosis were enhanced at 50 mg/kg. The incidence of benign tumours of the pituitary was significantly elevated and enhanced at 0.5 and 50 mg/kg. At 50 mg/kg increases in pheochromocytomas in the adrenal medulla and in parathyroid adenomas (males) were noted, while adrenal cortical tumours were decreased (males). There was a low, non-dose-related incidence of pancreatic carcinoma. Other tumour rates were in line with control data. It is concluded that lifetime feeding of 50 mg TBTO/kg diet induces toxicity in various organ systems. An increase in some common tumours was found at the high dose, probably due to hormonal or immunological changes.(ABSTRACT TRUNCATED AT 400 WORDS)

Interindividual variability in modeling exposure and toxicokinetics: a case study on cadmium.

This case study on cadmium illustrates a methodological framework for modeling the relation between external and internal dose in which interindividual variability is an integral component. The interindividual variability in intake of cadmium together with the variability in organ weights cannot explain the interindividual variability in internal doses. Therefore, variation in physiology was taken into account as well by assuming that some of the parameters of the toxicokinetic model are stochastic. This "second-order" modeling approach enables the prediction of internal dose distributions relating to the population, as opposed to "first-order" modeling, in which only the internal dose of the average individual is predicted. In the case of cadmium, where the critical internal concentration (in the kidney) is relatively well known, the fraction of the population at risk can be derived immediately. The use of this modeling framework to estimate risks for specific risk groups, defined by (combinations of) specific risk factors, is illustrated.

Immunotoxicity of bis(tri-n-butyltin)oxide in the rat: effects on thymus-dependent immunity and on nonspecific resistance following long-term exposure in young versus aged rats.

To investigate whether immune function suppression observed in an earlier study after short-term bis(tri-n-butyltin)oxide (TBTO) exposure also occurred after long-term treatment, function studies for specific and nonspecific resistance were performed after exposure of weaned male rats to diets containing 0, 0.5, 5, or 50 mg TBTO/kg for 4-6 and 15-17 months. Treatment for 4.5 months had no effect on body weight but reduced thymus weight at 50 mg/kg. Regarding the thymus-dependent immunity, delayed-type hypersensitivity reactions to ovalbumin and tuberculin were not depressed, in contrast to the results of the short-term study. The resistance to the nematode Trichinella spiralis was dose-relatedly suppressed at the 5 and 50 mg/kg levels, in both experiments (5.5 and 16.5 months exposure), as shown by increased counts of muscle larvae and depressed serum IgE titers. Also the inflammatory reaction around cysts in parasitized musculature was reduced. No significant reduction was found in IgM and IgG titers to T. spiralis, ovalbumin, and sheep red blood cells as determined by enzyme-linked immunosorbent assay. TBTO exposure at 50 mg/kg for 4.5 months significantly reduced thymus weight, but the response of thymocytes to T-cell mitogens was unaltered. TBTO treatment for 4.5 or 16 months did not influence the response of spleen cells to T-and B-cell mitogens and neither influenced spleen weight. A dose-related shift was observed in T- and B- cell numbers in mesenteric lymph nodes as shown by flow cytometry using monoclonal antibodies: treatment for 6 and 18 months reduced the relative count of T-lymphocytes and consequently increased the percentage of B-lymphocytes. As a result, the T:B ratio was reduced in the 5 and 50 mg/kg groups. Concerning the nonspecific resistance, TBTO exposure for 5 and 17 months reduced macrophage function at 50 mg/kg as shown by impaired splenic clearance of Listeria monocytogenes bacteria. Natural cell-mediated cytotoxicity of spleen and peritoneal cells was investigated in a 51Cr-release assay with YAC-lymphoma target cells. TBTO treatment significantly suppressed natural killer (NK) activity in spleen cells. Significant suppression was noted in all treatment groups following 16 months TBTO exposure; in contrast to treatment for 4.5 months. No significant alterations were observed in the spontaneous cytotoxicity of nonadherent and adherent peritoneal cells following 4.5 months treatment. Treatment of aged (i.e., 1-year-old) male rats for 5 months with the 50 mg/kg diet reduced thymus weight but had no effect on body or spleen weight.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunotoxicological screening of morphine and methadone in an extended 28 day study in rats.

Drug addicts are prone to infection with viruses including hepatitis-B and HIV. Besides indirect effects as a consequence of lifestyle, heroin and methadone may also enhance the risk of infections by a direct immunotoxic effect affecting resistance. In addition to general toxicological screening, we therefore performed a screening for potential immunotoxicity of morphine and methadone. Rats treated orally with different dosages of morphine or methadone for 6 weeks showed only a minor effect of overt toxicity on liver and spleen at the high dose, whereas at lower doses an increase in the relative weight of the mesenteric lymph nodes and an increase in cell density in the medullary cords were observed histopathologically, indicating a specific effect on humoral immunity. This specific immunotoxic effect was corroborated by an increased IgG concentration in serum (significant for the methadone-treated group). Further immunotoxicological research is needed aimed at revealing the potential risk of opiate use with respect to immune function. In conclusion, the present paper showed the toxicological profile of morphine and methadone in an extended 28 day subchronic study. Specific immunotoxicological effects were observed at doses where no effects were seen in routine toxicological evaluation, suggesting that the immune system is sensitive to opiates.

Urinary excretion of cadmium, protein, beta-2-microglobulin and glucose in individuals living in a cadmium-polluted area.

Urine was collected from 289 inhabitants of a cadmium-polluted quarter of Stadskanaal. The excretion of cadmium, protein, beta-2-microglobulin and glucose were determined. After being divided according to sex and to smoking habits, the results of the inhabitants were compared with those of 293 controls. In inhabitants as well as controls, cadmium excretion was age-dependent. Cadmium excretion in females increased faster with age than in males. In male-smoker controls, cadmium excretion was significantly higher (p less than 0.001) than in male-non-smoker controls. In male-non-smoker inhabitants, cadmium excretion (p = 0.05), protein excretion (p less than 0.01) and glucose excretion (p less than 0.01) were significantly higher than in corresponding controls. In male-smoker inhabitants, protein excretion (p less than 0.01) and glucose excretion (0.01 less than p less than 0.05) were significantly higher than in corresponding controls. In female-non-smoker inhabitants glucose excretion was significantly higher (0.01 less than p less than 0.05) than in corresponding controls. For some categories, living in the polluted area was associated with an increased cadmium excretion in urine and a slight difference in renal function, possibly related to a difference in cadmium body burden. It was concluded that, considering the actual values of each parameter, the observed differences were not relevant in terms of potential health hazards.

Toxicity of bis(tri-n-butyltin)oxide in the rat. II. Suppression of thymus-dependent immune responses and of parameters of nonspecific resistance after short-term exposure.

To evaluate the functional significance of bis(tri-n-butyltin)oxide (TBTO)-induced thymus atrophy, lymphocyte depletion in spleen and lymph nodes, lymphopenia, and increased serum IgM and decreased IgG concentrations, in vivo and in vitro function studies were performed for specific and nonspecific resistance. Weaned male rats were fed diets containing 0, 20, or 80 mg TBTO/kg for at least 6 weeks. Regarding the thymus-dependent immunity, delayed-type hypersensitivity reactions to ovalbumin as well as tuberculin were significantly depressed at both dietary concentrations. Resistance to the nematode Trichinella spiralis was significantly suppressed as shown by a retarded expulsion of adult worms from the small intestine, increased counts of muscle larvae, reduced inflammatory reaction in parasitized musculature, and suppressed serum IgE titers. Also the secondary mercaptoethanol-resistant (presumably IgG) hemagglutinating antibody titer to sheep red blood cells was significantly reduced, while no significant alterations were found in IgM and IgG titers to T. spiralis, ovalbumin, and tetanus toxoid. TBTO exposure reduced the response of thymocytes in both treatment groups and of spleen cells in the 80-mg/kg group upon stimulation with T-cell mitogens and increased the response of spleen cells to B-cell mitogens. When calculated per whole spleen, the response to T-cell mitogens was strongly impaired but unaltered by B-cell mitogens. This difference can be explained by a relative increase of splenic B cells as a result of reduced numbers of T cells, as shown by cell surface marker analysis using monoclonal antibodies. Reduced splenic T-cell numbers appeared equally due to a decreased number of T helper and to T suppressor cells. From these data and from results of a time-sequence study in which effects of TBTO on cell count and cell viability of thymus, spleen, and bone marrow were investigated, it is concluded that TBTO-induced immunodeficiency was primarily due to its direct toxic action on thymocytes. When cultured in vitro in the presence of TBTO, viability of thymus and bone marrow cells was equally reduced, while after in vivo treatment viability of bone marrow cells was unaffected. Thus, the in vitro situation does not mimic the in vivo one. Concerning the nonspecific resistance, TBTO reduced macrophage function as shown by impaired splenic clearance of Listeria monocytogenes bacteria. From in vitro studies it is concluded that impaired in vivo splenic clearance was due to a reduction in both the number of adherent cells in the spleen and bacterial digestion on a cell for cell basis.(ABSTRACT TRUNCATED AT 400 WORDS)

Study of sodium bromide in human volunteers, with special emphasis on the endocrine system.

1 Bromide, 1 mg kg-1 daily, was administered to 21 healthy volunteers (11 females not using oral contraceptives and not pregnant and 10 males, during 8 weeks or 2 full cycles to determine whether ingestion of a dose equal to the acceptable daily intake might induce effects. Special attention was paid to the endocrine system because endocrine changes were predominant in rats receiving sodium bromide (NaBr) in their diets. 2 There was no difference between the results of a full medical history and physical examination at the start and at the end of the experiment. 3 The results from the measured haematological, biochemical and urine analyses did not change during the experiment. 4 In females the plasma bromide concentration rose from 0.08 +/- 0.01 mmol 1(-1) to 0.97 +/- 0.18 mmol 1(-1) and in males from 0.08 +/- 0.01 mmol 1(-1) to 0.83 +/- 0.09 mmol 1(-1) (mean +/- s.d.). 5 No changes were observed in the serum concentrations of thyroxine, free thyroxine, thyroxin binding globulin, triiodothyronine, cortisol, testosterone, estradiol and progesterone. Also no changes were observed in the serum concentrations of thyroid stimulating hormone (TSH), prolactin, luteinizing hormone (LH) and follicle stimulating hormone (FSH) before and after the administration of thyroid stimulating hormone releasing hormone (TRH) and luteinizing hormone releasing hormone (LHRH).

Effects of ozone, hexachlorobenzene, and bis(tri-n-butyltin)oxide on natural killer activity in the rat lung.

The respiratory tract is a major route of exposure to noxious agents as well as pathogens such as viruses. Natural killer (NK) activity is an important first line of defense to virally infected cells as well as certain neoplasms; therefore, testing the effects of exposure to toxic compounds on this activity is important in understanding the immunotoxic potential of the compound. Lymphoid cell suspensions, obtained after enzymatic dispersion of rat lungs and purification over nylon wool columns, showed in vitro natural killer activity toward YAC lymphoma cells. Validation of the test with well-known NK activity stimulators such as Bacillus Calmette-Guérin (BCG), interleukin-2 (IL-2), interferon (IFN), and inhibitors like anti-asialo-GM1 (ganglio-n-tetrasylceramide) antibody confirmed the reliability of the test as an assay for detecting NK activity in rat lungs. Using this assay, we studied the effects of exposure to ozone (O3), hexachlorobenzene (HCB), and bis(tri-n-butyltin)oxide (TBTO) on NK activity in rat lung. Inhalation exposure to O3 for 7 days at 0.4 and 0.8 mg/m3 resulted in stimulation, and exposure at 1.6 mg O3/m3 resulted in suppression of NK activity. Oral exposure to HCB in concentrations of 150 and 450 mg/kg food for 6 weeks suppressed NK activity in rat lungs in a dose-related manner. This was also true for 6 weeks of oral exposure of rats to 20 and 80 mg TBTO/kg food, but to a lesser extent. In summary, we have developed and validated a method to measure the effects of (toxic) substances on NK activity in rat lung.

Toxicity of bis(tri-n-butyltin)oxide in the rat. I. Short-term effects on general parameters and on the endocrine and lymphoid systems.

Male and female Wistar rats were fed bis(tri-n-butyltin)oxide (TBTO) at 0, 5, 20, 80, or 320 mg/kg diet for 4 weeks. Clinical signs and decreases in feed and water consumption were observed in the 80 and 320 mg/kg groups. The serum transferase activities (alanine amino transferase and aspartate amino transferase were increased at 20 (males only), 80, and 320 mg/kg. The serum glucose and liver glycogen concentrations were lowered in the 320 mg/kg group. At 80 and 320 mg/kg the serum IgG level was reduced and IgM level was increased. Compared to controls the mean relative weight of the thymus was decreased at 20 (males), 80, and 320 mg/kg. In the groups receiving 80 or 320 mg/kg microcytic anemia was found. The white blood cell counts were decreased, due to the reduction in the number of lymphocytes in the 80 (males) and 320 mg/kg groups. The concentration of neutrophilic granulocytes was increased in the highest dose group. Histopathologic effects included a dose-related lymphocyte depletion of thymic cortex and of T lymphocytes in spleen and mesenteric lymph nodes. In the spleen also depletion of iron stores was found, and in the medullary sinuses of mesenteric lymph nodes, rosettes of erythrocytes were found around mononuclear cells; the occurrence of rosettes increased with dose from 5 to 80 mg/kg, and appeared to be the most sensitive parameter. A low incidence of areas of liver necrosis with inflammatory reaction and bile duct hyperplasia was found in the 320 mg/kg group. A viral or bacterial etiology could be demonstrated for these liver lesions, but they appeared associated with TBTO-induced ulcerative inflammation of the common bile duct as shown in an additional study. In 6-week studies exposure of male weanlings to the 0, 20, and 80 mg/kg diets, the serum insulin concentration in the treated groups was decreased, although the response to glucose challenge was unaffected. The serum thyroxin and thyrotropin (TSH) concentrations were reduced, whereas the luteinizing hormone (LH) concentration was increased in the 80 mg/kg group. The concentrations of follicle-stimulating hormone (FSH) and corticosterone were not changed. The release of LH and FSH was enhanced in the 80 mg/kg group and a tendency toward reduced release was found for TSH. Using immunocytochemistry a dose-related reduction was found in the number and staining intensity of TSH-producing cells in the pituitary, correlating with histopathologically decreased activity of the thyroid.(ABSTRACT TRUNCATED AT 400 WORDS)