Bioavailability of lead to juvenile swine dosed with soil from the Smuggler Mountain NPL Site of Aspen, Colorado.

Bioavailability of lead (Pb) has become an issue in quantifying exposure of sensitive populations and, where necessary, establishing cleanup levels for contaminated soil. Immature swine were used as a model for young children to estimate the degree to which Pb from two fully characterized composite samples from the Smuggler Mountain Superfund Site in Aspen, Colorado may be bioavailable to resident children. The composite soils contained 14,200 and 3870 micrograms Pb/g of soil. Relative and absolute enteric bioavailabilities of Pb in soil (oral dose groups of 75,225, and 675 micrograms Pb/kg body wt/day) were estimated by comparison with an orally administered soluble Pb salt (lead acetate = PbAc2.3H2O) (dose groups of 0, 75, and 225 micrograms Pb/kg body wt/day) and an intravenously administered aqueous solution of Pb (100 micrograms Pb/kg/ day) from the same trihydrate salt administered daily for 15 days to 50 juvenile swine. The biological responses (area under the blood Pb concentration-time curve, and the terminal liver-, kidney-, and bone-lead concentrations) produced by Pb from PbAc2.3H2O and lead-contaminated soils were determined. This study revealed Pb from soil containing 14,200 micrograms Pb/g of soil had a bioavailability relative to Pb from PbAc (RBA), ranging from 56% based on the area under the blood lead concentration-time curve (AUC) versus dose, to 86% based on calculations from liver-Pb loading versus dose. Similarly, Pb from soil containing 3870 micrograms Pb/g of soil had an RBA ranging from 58% based on the AUC versus dose, to 74% based on calculations from liver- and kidney-Pb loading versus dose. Bioavailability of Pb in soils may be more or less than EPA's default RBA of 60%, therefore, measuring site-specific RBAs provides a basis for improved exposure and risk assessment.

Hypersensitivity reactions and specific antibodies in workers exposed to industrial enzymes at a biotechnology plant.

Thirty-six employees who produced industrial enzymes from selected strains of bacteria and fungi were evaluated by epicutaneous threshold testing and enzyme-linked immunosorbent assays (ELISA) for specific IgE and IgG antibodies. The workers complained of 'asthma- and flu-like' symptoms, which generally lessened away from work. The enzymes evaluated were: alpha-amylase (1,4-alpha-d-glucan glucanohydrolase) from Bacillus licheniformis (alpha ABl), B. subtilis formation 1 (alpha A1Bs) and B. subtilis formation 2 (alpha A2Bs); purified alpha-amylase from B. licheniformis (C alpha ABl) and A. oryzae (C alpha AAo); alkaline protease from B. licheniformis (APBl) and purified alkaline protease (CAPBl); amyloglucosidase (1,4-alpha-d-glucan glucohydrolase) from A. niger (AGAn) and purified amyloglucosidase (CAGAn). Statistically significant increases (P > 0.05) in the proportion of workers having positive skin tests to CAPBl, AGAn and CAGAn were found. Significantly elevated (P > 0.05) mean specific IgE results were observed for C alpha AAo CAGAn and AGAn, and elevated (P > 0.05) mean specific IgGs were observed for C alpha AAo, CAGAn, AGAn, alpha A1Bs, alpha AB1 and alpha A2Bs. These results indicate that occupational exposure to some industrial enzymes can cause immediate-onset cutaneous hypersensitivity reactions, pulmonary function deficits and significantly elevated specific antibody levels. Our results are equivocal as to whether work-related respiratory and cutaneous hypersensitivity reactions are antibody mediated, as there was no statistically significant association between these reactions and specific IgE or IgG levels.

Immunologic analyses of peripheral leukocytes from workers at an ethical narcotics manufacturing facility.

Little information exists about possible adverse health effects associated with workplace exposure to opiate compounds. We have previously reported opiate-specific IgG antibodies, positive epicutaneous tests, and pulmonary function decrements in workers exposed occupationally to opiates. In the present work, we extended these findings to investigate the effect of occupational opiate exposure on lymphocyte subpopulations and mitogen-induced lymphoblastogenesis. Thirty-three opiate-exposed workers and 8 nonexposed control workers were evaluated for lymphocyte subpopulation absolute numbers and percentages, by evaluating cell surface antigen expression with flow cytometry. A complete blood count with differential, common clinical chemistry parameters, and serum immunoglobulin levels were also evaluated. Opiate-exposed workers showed significantly (p < .05) increased absolute numbers and percentages of HLA-DR+ cells (MHC class II histocompatibility antigen), significantly (p < .01) decreased percentages of T helper-inducer (CD4+) cells, and significantly (p < .05) decreased numbers of basophils, compared with nonexposed opiate workers from the same factory. A trend toward reduction in the T helper-inducer (CD4+)/T cytotoxic-suppressor (CD8+) lymphocyte ratio was also evident. There was also a significant decrease in lymphocyte activity stimulated by pokeweed mitogen (p < .05) in opiate-exposed workers. These data indicate that occupational opiate exposure may change the number and types of circulating peripheral blood leukocytes, or alternatively, alter the expression of receptors on the surface of these cells. In addition, occupational opiate exposure appears to decrease the sensitivity of B-cells to pokeweed mitogen stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Quinolone arthropathy in juvenile New Zealand white rabbits.

Twenty juvenile New Zealand white rabbits were dosed orally once daily with 1,000 mg of nalidixic acid/kg for 3, 7, and 14 consecutive days, then for 14 days followed by a 14-day recovery period. Eighteen age-matched rabbits were allotted to four groups and given corn oil vehicle to serve as controls for the various treatment durations. The articular cartilage from the stifle joints, shoulders, and elbows was studied by gross examination, light microscopy, and toluidine blue histochemistry, and the hips were studied by gross examination and transmission electron microscopy. When examined grossly, raised vesicles could be detected in the joints of some animals after 3 days of treatment. The distal portion of the femur and proximal portion of the ulna were predilection sites for gross lesions. Histologically, the vesicles were fluid-filled clefts in the intermediate layer of the articular cartilage. After 14 days, many of the lacunae in the areas of the defects contained chondrocyte clusters. When treated for 14 days and allowed a 14-day recovery period, territorial matrix had been deposited around individual chondrocytes within the clusters, compressing the matrix between the chondrocyte clusters into thin collagenous bands. For all treatment groups, decreased metachromasia, when stained with toluidine blue, provided histochemical evidence of loss of glycosaminoglycans in the matrix around the clefts.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of cutaneous responses and lung function from exposure to opiate compounds among ethical narcotics-manufacturing workers.

We recently demonstrated morphine-6-hemisuccinate-human serum albumin conjugate (M-6-HS-HSA)-specific IgG in serum from ethic narcotics-manufacturing workers. In this article, we present results of epicutaneous tests to opiate compounds and lung-function studies in these same workers. Thirty-nine workers, exposed to opiates, were evaluated for possible work-related changes in lung function and were administered a questionnaire concerning opiate exposure and health history in February 1988. In December 1988, 33 employees with occupational exposure to opiates, six other workers (New Jersey referent) employed at the same factory with minimal exposure to opiate compounds, and 17 nonexposed individuals from Cincinnati, Ohio, were subjected to epicutaneous threshold testing with a panel of six opiate compounds and nine common aeroallergens. In opiate-exposed workers, significantly lower epicutaneous threshold concentrations were detected (compared to New Jersey referent and Cincinnati control subjects) for dihydrocodeine (p less than 0.01), hydrocodone (p less than 0.05), codeine (p less than 0.01), and morphine (p less than 0.05). Significant associates existed among epicutaneous threshold concentrations between the agents tested; that is, individuals with a positive morphine skin test would generally have a positive codeine skin test, etc. Atopic status (positive cutaneous test results to two or more of nine common aeroallergens) was not significantly associated (p greater than 0.05) with positive opiate skin sensitivity. Although the mean cross-shift decrements in FEV1 for all workers were nonsignificant, five opiate-exposed individuals demonstrated cross-shift decrements in FEV1 of greater than 10%. Daily maximum-minus-minimum changes in workweek PEFR (PEFRmax-min) were significantly reduced for Monday through Thursday (p less than 0.05) compared to PEFRmax-min changes during a nonwork, nonexposure 3-day weekend. Ten exposed workers demonstrated daily PEFRmax-min changes of greater than 20%, suggesting acute airway obstruction. Increased cutaneous reactivity to opiate compounds among opiate-exposed workers may reflect development of pharmacologic hyperresponsiveness to opiate compounds.

Antibodies to morphine in workers exposed to opiates at a narcotics manufacturing facility and evidence for similar antibodies in heroin abusers.

According to the International Narcotics Control Board, over 45,000 kg of morphine and 54,000 kg of codeine were ethically manufactured in 1986 at three facilities in the United States. Little information exists about possible adverse health effects associated with workplace exposure to opiate compounds in this industry. Because there are no specific federal standards for workplace exposure to narcotic dusts, exposure-control defaults to the nuisance dust standard (10 mg/m3, as an 8 hr time-weighted average). Narcotics manufacturing workers were evaluated for anti-morphine IgG before and 10 mo. after the implementation of an improved respiratory protection program (RPP). Significantly elevated IgG levels were measured before the improved RPP (P less than 0.005). After the improved RPP, a significant reduction was observed (P less than 0.001), suggesting that specific antibody levels could be used as biomarkers of exposure. Inhibition studies showed that the antibodies were specifically directed against morphine with some cross reactivity with morphine derivatives. Preliminary results are also shown which indicate that similar anti-morphine antibodies are present in the sera of intravenous heroin abusers. Elevated levels (P less than 0.05) of anti-morphine antibodies were detected in sera from heroin abusers, providing evidence that similar antibodies may be produced from non-occupational exposure to opiates. These finding have potentially far-reaching implications for addiction research and drug testing.

Metabolism of nephrotoxic isopropylcyclohexane in male Fischer 344 rats.

The metabolism of isopropylcyclohexane and associated renal pathology were evaluated in male Fischer 344 rats exposed by oral gavage. The rats experienced moderate proximal tubular damage similar to that produced by acyclic, branched-chain hydrocarbons. The urinary metabolites of isopropylcyclohexane included cis-4-isopropylcyclohexanol, trans-4-isopropylcyclohexanol, 2-cyclohexylpropanoic acid, 2-cyclohexyl-1,3-propanediol, 2t-hydroxy-4t-isopropylcyclohexanol, 2c-hydroxy-4c-isopropyl-cyclohexanol, and 2c-hydroxy-4t-isopropylcyclohexanol. The extent and preferred sites of oxidative metabolism of nephrotoxic hydrocarbons could potentially prove useful in elucidating the pathogenic mechanisms.

The metabolism of t-butylcyclohexane in Fischer-344 male rats with hyaline droplet nephropathy.

The molecule t-butylcyclohexane is one of the first examples of a branched alkyl group attached to a hydrocarbon ring shown to be capable of producing renal damage at the corticomedullary junction of male rats. A metabolic study of t-butylcyclohexane yielded the following urinary metabolites: trans-4-t-butylcyclohexanol, 2c-hydroxy-4t-t-butylcyclohexanol, 2-methyl-2-cyclohexylpropanoic acid, 2c-hydroxy-4c-t-butylcyclohexanol, 2-methyl-2-cyclohexyl-1,3-propanediol, 2t-hydroxy-4t-t-butylcyclohexanol, and cis -4-t-butylcyclohexanol. As with other hydrocarbons of similar molecular weight that induce nephropathy in male rats, preferential sites of oxidative metabolism were observed that could potentially be related to the pathogenesis.

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.

Immune responses in rats supplemented with selenium.

It is generally accepted that Selenium (Se) is necessary for optimum performance of the immune system. Selenium deficiency results in immune suppression but little is known concerning the effect of excess Se on immune function. Recent evidence suggests that oral Se supplementation may impede oncogenesis, but the mechanism of this action is currently unknown. Conversely, under certain conditions, Se is suspected of promoting neoplasia. The studies described herein delineate the effects of excess Se (0.5, 2.0 or 5.0 p.p.m.) on specific immune functions of Se-adequate rats, namely, antibody synthesis, delayed-type hypersensitivity (DTH), natural killer (NK) cell activity, prostaglandin E2 (PGE2) synthesis, and interleukin 1 (IL-1) activity. Selenium administered to female Sprague-Dawley rats for 10 weeks at 0.5 and 2.0 p.p.m. resulted in significant (P less than or equal to 0.01) enhancement of splenic NK activity while the NK response in the 5.0 p.p.m. Se-treated rats was equivalent to the non-Se-treated controls. Conversely, the DTH response was significantly (P less than or equal to 0.01) suppressed at all three dosages while antibody synthesis and prostaglandin E2 activity were significantly (P less than or equal to 0.05) reduced compared to the controls at the highest dosage of Se. IL-1 activity was unaffected by Se exposure. These data could partially explain the contradictory oncogenic characteristics of Se. For instance, tumours that are NK sensitive could be prevented and/or responsive to Se therapy, while NK insensitive neoplasms could be enhanced by Se supplementation due to the impaired function of both humoral and cell-mediated immunity.

The selectivity of isoprinosine, NPT 15392, avridine and cyclophosphamide on multiple immune responses in rats.

Multiple concomitant immune responses were assessed in individual rats following treatment with the immunoenhancing drugs, isoprinosine (5 or 50 mg/kg), NPT 15392 (0.1 or 1.0 mg/kg) and avridine (1 or 25 mg/kg), or the immunosuppressant, cyclophosphamide (75 mg/kg). Immune responses assessed in each rat were specific antibody synthesis, delayed-type hypersensitivity (DTH), natural killer cell (NKC) cytotoxicity and production of three immunoregulatory cytokines, interleukin 1 (IL1), interleukin 2 (IL2) and prostaglandin E2 (PGE2). Spleen and thymus weights and numbers of splenocytes and resident peritoneal cells were also recorded. Rats treated with isoprinosine had dose-related, significant increases in spleen weights and DTH reactions. Rats treated with NPT 15392 had significantly enhanced DTH reactions at the 0.1 mg/kg dose. Rats treated with the 25 mg/kg dose of avridine had significantly increased spleen weights, DTH reactions and NKC cytotoxicity. The effect of avridine treatment on DTH reactions and IL1 and IL2 production was inverse to the dose administered, while the NKC response was directly related to the dose. Thymus weights, antibody production and PGE2 synthesis were not significantly altered in rats treated with isoprinosine, NPT 15392 or avridine. Cyclophosphamide-treated rats had significantly reduced spleen and thymus weights, antibody synthesis, DTH reactions, NKC cytotoxicity and IL2 production, but IL1 and PGE2 synthesis were significantly elevated. It can be concluded that isoprinosine, NPT 15392 and avridine act as general immunostimulants in the rat, with avridine having the greatest effect under these experimental conditions. It also appears that these drugs are differentially immunoselective in the rat and this effect is at least partially related to the dose administered. These results could be of significance in the selective therapeutic manipulation of different arms of the immune system. Also, enhanced production of PGE2 following cyclophosphamide treatment may contribute to the immunosuppressive effects of this drug.

A sensitive delayed-type hypersensitivity model in the rat for assessing in vivo cell-mediated immunity.

Many drugs and other chemicals can alter cell-mediated immunity (CMI), a response that often correlates with delayed-type hypersensitivity (DTH). Several DTH assays were evaluated to determine a method best suited for assessing chemically induced modulation of CMI in rats. The effects of various antigens, adjuvants, doses, routes, and immunosuppressants were investigated. The DTH method which produced optimum results in rats uses a footpad swelling reaction elicited by specific preparations of bovine serum albumin (BSA) and Freund's complete adjuvant (FCA). The rats were sensitized with 100 micrograms BSA in FCA injected subcutaneously at the base of the tail, and were challenged 7 days later with 75 microliter of 2% heat-aggregated BSA suspension injected into the left rear footpad. Footpad swelling was measured with pressure calipers 24 h later and compared to the contralateral footpad which was sham-injected with 75 microliters of physiological saline. Antigen-injected footpads were nearly double the thickness (7-8 mm) of the control footpads (3-4 mm), and variation between animals was small (CV = 5%). Dexamethasone and cyclophosphamide significantly suppressed the DTH reaction. Histopathological examination of the DTH reaction sites revealed a mononuclear cell infiltrate which is characteristic of type IV hypersensitivity. In addition to being highly quantitative and sensitive, this method provides a simple and reproducible assessment of CMI responses in the rat.

Multiple immunoassay in a single animal: a practical approach to immunotoxicologic testing.

A battery of immunoassays were developed for the rat which measured the major arms of the immune system, namely, humoral immunity, cell-mediated immunity (CMI), and macrophage function. Humoral immunity was assessed by the enzyme-linked immunosorbent assay (using keyhole limpet hemocyanin as antigen). CMI was measured by delayed-type hypersensitivity to heat-aggregated bovine serum albumin using a footpad swelling technique. Macrophage function was assessed by measuring the ability of macrophages to phagocytize sheep red blood cells in vitro. The feasibility of performing all three immunoassays in the same animal was investigated by treating groups of rats with all combinations of antigenic exposure used to induce each type of response. Multiple antigenic treatment regimens did not significantly alter responses to individual antigens used in this study. Animals exposed to the concurrent antigen treatment regimens remained sensitive to immunosuppressants. These data suggest that multiple immunoassays can be successfully performed utilizing a single animal which has been concurrently treated with different antigens. The inclusion of additional assays into this protocol and the efficacy to immunotoxicologic testing is discussed.

Toxicologic, pathologic, and immunotoxic effects of 2,4-dichlorophenol in rats.

2,4-Dichlorophenol (DCP) is a drinking and waste-water contaminant formed by the spontaneous reaction of chlorine with phenols following chlorination of water for disinfection and deodorization. Rats were exposed to 0, 3, 30, or 300 ppm DCP in drinking water either in utero or for 12 wk postnatally following in utero exposure. Toxicity to DCP was assessed by organ and body weight changes, histopathology, and effects on reproduction and immunocompetence. Reproductive parameters measured included conception, litter size, pup birth weight, number stillborn, survival to weaning, and weaning weight. Immune parameters assessed were humoral immunity (antibody production) by an indirect enzyme-linked immunosorbent assay (ELISA), cell-mediated immunity by a delayed-type hypersensitivity response, and macrophage function by phagocytosis of radiolabeled blood cells. Rats that received the combined in utero and postnatal treatment with 300 ppm DCP had significantly increased liver and spleen weights, enhanced humoral immune responsiveness, and depressed cell-mediated immunity. Histopathologic changes were unremarkable in DCP-exposed rats, even in the presence of increased liver and spleen weights. The 6-wk-old progeny of DCP-treated dams had normal immune functions and showed no signs of DCP toxicity, other than increased spleen weights in the 300-ppm exposure group. The results indicate that (1) the immune system may be a sensitive target for chlorinated phenolic compounds, (2) DCP may exert different effects on separate major immune responses, and (3) unlike some other chlorinated phenols, DCP does not appear to alter reproductive performance in rats.