Localization of inhaled trimellitic anhydride to lung with a respiratory lymph node antibody secreting cell response.

Male Sprague-Dawley rats were exposed to trimellitic anhydride (TMA) by inhalation (500 micrograms/m3), 4 hours a day, for 1 to 10 days. TMA was localized to lung cells by immunoelectron microscopy. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis were used in an attempt to localize TMA to lung lavage proteins. The lung-associated lymph node (LALN) B-lymphocyte response was measured by quantitation of immunoglobulin (Ig)G, IgA, and IgM antibody secreting cells specific for TMA rat serum albumin (TM-RSA) by use of the enzyme-linked immunospot assay (ELISPOT) method. The IgG, IgM, and IgA antibody response to TM-RSA in serum and lavage fluid was quantitated by ELISA. Lung injury was assessed by the number of external lung hemorrhagic foci and lung weight. Immunoelectron microscopy localized TMA to alveolar and bronchial cells on all exposure days. ELISA detected trace amounts of TMA haptenized lavage proteins that could not be detected by Western blot analysis. A marked increase occurred in lung injury from day 7 to 10. The LALN IgG, IgA, and IgM antibody secreting cell response to TM-RSA paralleled measures of lung injury. IgG, IgM, and IgA serum and lavage antibody to TM-RSA were correlated with lung injury measures. Lavage and serum IgG antibody levels had the highest correlation with lung injury.

Lung injury induced by short-term intermittent trimellitic anhydride (TMA) inhalation.

We have developed a rat model of lung injury with interstitial pneumonitis, lung hemorrhage, and a systemic and pulmonary immune response to trimellitic anhydride (TMA)-haptenized proteins induced by TMA inhalation for 10 days. The present studies explored the induction of lung injury induced by short-term intermittent TMA inhalation, a model more likely to simulate short-term industrial exposures during inadvertent spills of TMA. Sprague-Dawley rats inhaled TMA powder (500 micrograms/m3) on days 1, 5, and 10, and were necropsied on day 30, 18 hours after a 6-hour TMA-inhalation challenge on day 29. Rats were bled every second day and at necropsy. Serum IgG, IgA, and IgM antibody to trimellityl rat serum albumin was measured by ELISA. There was a rise in IgM and IgA antibody to trimellityl rat serum albumin starting at day 5 that peaked at day 20 with a decline in IgM by day 30. IgG antibody rose at day 7, peaked at day 20, and plateaued. The IgG antibody level was 10 times higher than the IgA or IgM level. In a second experiment, 18 rats were administered TMA-inhalation exposure on days 1, 5, and 10, and a TMA challenge on day 22. The number of hemorrhagic foci, lung weights, and lung-displacement volumes at necropsy on day 23 were highly correlated with IgG, IgA, and IgM serum-antibody levels. In a final experiment, rats developed a mean of 112 hemorrhagic foci per lung on day 30 after receiving only two TMA-inhalation exposures on days 1 and 5 with a rechallenge on day 29. (ABSTRACT TRUNCATED AT 250 WORDS)

Immunologic tolerance in rats during 13 weeks of inhalation exposure to trimellitic anhydride.

Trimellitic anhydride (TMA) causes several immunologically based pulmonary syndromes in humans. We developed a rat model representative of some of those syndromes whereby rats exposed for 2 weeks to TMA by inhalation developed hemorrhagic lung foci and pneumonitis accompanied by the appearance of TMA-specific serum antibody. The purpose of the study reported here was to examine the long-term, low-dose effects of TMA inhalation. Rats were exposed to target concentrations of 0, 2, 15, or 50 micrograms/m3 TMA 6 hr/day, 5 days/week for 13 weeks. The study included an interim 6.5-week termination and two recovery periods of 3 and 38 weeks, each with and without a final TMA inhalation challenge. Additional rats were bled regularly throughout the study and monitored for the appearance of TMA-specific antibody; other rats were terminated periodically during the 13-week exposure and examined for lung lesions. These serially terminated rats showed that TMA-induced lung lesions reached a maximum after approximately 2 weeks of exposure, but began to diminish thereafter. Rats bled regularly showed increasing TMA-specific antibody titers through the first 6 weeks of exposure, after which antibody titers diminished. Serum antibody levels rose sharply after the 13-week exposure ended and tapered off throughout the recovery period. Rats terminated after 6.5 weeks of exposure showed a dose-dependent increase in lung lesions and serum antibody. However, rats exposed to TMA for 13 weeks showed greatly reduced lung lesions and antibody titers. Rats exposed for 13 weeks and allowed to recover for 3 weeks showed increased antibody titers but few lesions, even after a TMA challenge. Rats exposed for 13 weeks and allowed to recover 38 weeks had reduced but still significant antibody titers; however, no lung lesions were noted even after a TMA inhalation challenge prior to termination. These results indicated that rats became tolerant to TMA and that 13 weeks of exposure to TMA did not produce lesions of any type, even after 38 weeks of recovery.

Antibody response to trimellityl hemoglobin in trimellitic anhydride-induced lung injury.

Sprague-Dawley rats were exposed to trimellitic anhydride by inhalation, and the antibody response to trimellityl (TM)-conjugated hemoglobin (HB) and TM rat serum albumin (RSA) was compared. Groups of rats were exposed to trimellitic anhydride by inhalation 6 hours per day for 2, 6, or 10 days at 100 micrograms/m3 and compared to a control group exposed to filtered air. The IgG antibody response to TM-HB in both serum and bronchoalveolar lavage (BAL) fluid was measured with ELISA. IgG antibody levels to TM-HB rose significantly throughout the exposure. A positive correlation was found between IgG to TM-HB in serum and BAL fluid. In addition, this response in both serum and BAL fluid correlated with the IgG antibody response to TM-RSA. Cross-inhibition studies indicated the existence of shared antigenic determinants on TM-RSA and TM-HB. The IgG antibody to both antigens was specific for new antigenic determinants and not for the TM hapten.

Tumor initiation and promotion effects of petroleum streams in mouse skin.

A furnace oil, a dewaxed heavy paraffinic distillate, and a solvent-extracted lubricant base oil induced skin tumors in 9/43, 26/48, and 1/47 male C3H mice, respectively, during lifetime skin-painting bioassays. An initiation/promotion (I/P) bioassay was conducted to assess the I/P potential of these materials. During a 28-week initiation bioassay, groups of 30 male CD-1 mice were first treated dermally once daily for 5 days with 25 or 50 microliter of test materials or 50 microliter of acetone, rested for 2 weeks, then treated twice per week for 25 weeks with 50 microliter (0.1 mg/ml) phorbol-12-myristate-13-acetate (PMA). Only groups treated with the heavy paraffinic distillate had a significantly higher incidence of papillomas relative to the acetone control group. During a 28-week promotion bioassay, groups of 30 male CD-1 mice were treated once with 50 microliter of either DMBA (1.0 mg/ml) or acetone, rested for 2 weeks, and then treated twice per week with test material for the remaining 25 weeks. The furnace oil and dewaxed heavy paraffinic distillate showed significantly higher incidences of carcinomas and papillomas in DMBA-initiated mice relative to their acetone-initiated controls. Together these bioassay data suggest that only the dewaxed heavy paraffinic distillate is a complete carcinogen, having both initiating and promoting activity; furnace oil is a promoter only, while the solvent-extracted lubricating oil is noncarcinogenic. Overall, the I/P bioassay correlated well with the results of the lifetime skin-painting bioassay.

Fetal alcohol effects: evidence of developmental impairment in the absence of immunotoxicity.

Pregnant CD-1 mice were intubated from Days 5-17 of gestation with an isocaloric solution of ethanol and/or maltose dextrins in 10% Bio-Serv liquid diet, at doses of 0, 6, and 9 g/kg/day. The control females were food-restricted so that their food consumption matched that of the high-dose females. At delivery, the pups were assigned surrogate mothers and litters were culled to a uniform litter size, when possible. Immediately following birth some pups were examined using a battery of behavioral/developmental tests to monitor postnatal development. At 6-8 weeks of age, other pups were evaluated for changes in immune function. Another group of pups was terminated at 12 weeks of age for hematology and clinical chemistry determinations and histological examination of selected organs. Postnatal developmental delays were seen in pinna detachment, surface righting reflex, eye opening, and incisor eruption. There was no characteristic pattern to immune alterations of the plaque forming cell response to a T-dependent antigen sheep red blood cells, delayed hypersensitivity, and T- and B-lymphocyte blastogenesis. Possible kidney pathology was indicated by a reduction in blood urea nitrogen in the offspring of females treated with 9 g/kg/day. No treatment-related effects were noted in those tissues which were examined histologically.

Evidence of immunologic control of lung injury induced by trimellitic anhydride.

Trimellitic anhydride (TMA) is a chemical intermediate that has been shown to cause immunologically mediated respiratory syndromes in humans. We developed a rat model in which lung lesions accompanied by TMA-specific antibody resembled effects seen in humans. Two sets of experiments were undertaken to determine if TMA lung injury was primarily controlled by the immune system. Experiment 1: Rats were exposed to 95 micrograms/m3 of TMA 6 h/day, 5 days/wk for 2 wk during which time they received daily injections of either the immunosuppressant cyclophosphamide or saline. The TMA-exposed/saline control rats exhibited the usual TMA-induced lung lesions accompanied by TMA-specific antibody. However, the TMA-exposed/cyclophosphamide rats showed no lesions and no antibody. The spleen cells from all rats were subjected to lymphocyte blastogenesis assays using T- and B-cell mitogens. Results confirmed that cyclophosphamide-treated rats showed very little if any blastogenic response, whereas saline-treated rats gave the normal immune response. Thus, cyclophosphamide eliminated T- and B-cell function, which in turn prevented the occurrence of TMA lesions. Experiment 2: An initial passive transfer experiment showed that serum from TMA-sensitized rats could be adoptively transferred into naive recipient rats, which when given a single TMA inhalation challenge exhibited TMA-induced lesions. Similar attempts to transfer spleen cells or spleen cells plus serum did not predispose recipients for lesions. A second modified passive transfer of sensitized serum using a larger number of recipient rats, followed by a TMA challenge, resulted in lesions in 14 of the 16 rats.(ABSTRACT TRUNCATED AT 250 WORDS)

A serial immunologic and histopathologic study of lung injury induced by trimellitic anhydride.

Trimellitic anhydride (TMA) can induce immunologic lung disease in exposed workers. We have developed a rat model of TMA lung injury characterized by lung hemorrhage and an immune response to trimellityl (TM) haptenized lung proteins. The model is similar to the pulmonary disease-anemia syndrome (PDA) seen in workers exposed to TMA fumes. Sprague-Dawley rats, 15 per exposure period, inhaled micronized TMA powder, 100 micrograms/m3, 6 h/day, for 2,6, or 10 days and were sacrificed. At each time period, total, IgG, IgA, and IgM antibody to TM-rat serum albumin (TM-RSA) were measured by radiolabeled antigen binding and enzyme-linked immunosorbent assay (ELISA) in serum and bronchoalveolar lavage fluid (BAL). Hemorrhagic lung foci, weight, and displacement volume were determined, and lungs were examined by light and electron microscopy. There was no lung injury or antibody response at 2 days. There was minimal lung injury at 6 days with low levels of antibody in BAL and serum. At 10 days, there was a marked increase in hemorrhagic foci and in BAL and serum antibody levels. BAL antibody levels at 6 and 10 days had higher correlations with measures of lung injury than corresponding serum levels. There was minimal ultrastructural change at 6 days. By Day 10, there was marked intraalveolar hemorrhage, alveolar septal inflammatory nodules, abundant alveolar macrophages, and evidence of endothelial and epithelial cell injury. These results indicate that the immune response to inhaled TMA occurs parallel with the development of lung lesions, and antibody levels in BAL and serum are highly correlated with lung injury.

Reducing the number of rabbits in the Draize eye irritancy test: a statistical analysis of 155 studies conducted over 6 years.

The Draize eye irritancy test in rabbits has been the focus of recent efforts to reduce the use of live animals in toxicity testing. A suitable alternative is not yet available; therefore, we studied the adequacy of reducing the number of rabbits used per test. Data generated from 6-rabbit eye irritation tests of 155 various materials were used to determine the ability of irritation scores from all possible combinations of 5-, 4-, 3-, or 2-rabbit subsets to predict the Draize score derived from six rabbits. There are 930, 2325, 3100, and 2325 possible combinations of 155 studies for the 5-, 4-, 3-, and 2-rabbit subsets, respectively. We classify materials using a four-level adjectival rating system based on (among other factors) the Draize score. Comparisons indicated that 5-, 4-, 3-, and 2-rabbit scores were in 98, 96, 94, and 91% agreement, respectively, with the classification assigned on the basis of the 6-rabbit score. The correlation coefficients for randomly selected subsets of 5-, 4-, 3-, and 2-rabbit scores versus the Draize score for six rabbits were 0.998, 0.996, 0.992, and 0.984, respectively. This study confirms the findings of an earlier report by De Sousa et al. (1984), and indicates that a high level of accuracy can be obtained with reduced numbers of rabbits per test.

The pathologic and immunologic effects of inhaled acrolein in rats.

Four groups of 40 male Sprague-Dawley rats each were exposed by inhalation to target concentrations of 0, 0.1, 1.0, and 3.0 ppm of acrolein 6 h/day, 5 days/week for 3 weeks. Subsequent changes in local pulmonary immunity were determined by examining the number of antibody plaque-forming cells in the lung-associated lymph nodes following intratracheal immunization with sheep red blood cells. Separate groups of rats were evaluated for blastogenic responsiveness to phytohemagglutinin-P and Salmonella typhimurium antigen using spleen- and lung-associated lymph node cells. In vivo resistance was evaluated utilizing acrolein-exposed rats subsequently challenged with intravenous Listeria monocytogenes. Local pulmonary antibody responsiveness was not affected by acrolein exposure. Lymphocyte blastogenesis and resistance to Listeria challenge were not altered. Body weights and spleen weights were decreased in the 3 ppm-exposed group only. Microscopic examination of the nasal turbinates revealed acrolein-induced exfoliation, erosion, and necrosis of the respiratory epithelium as well as squamous metaplasia, however, lung histology was not affected. Thus at environmental concentrations, acrolein toxicity appeared to be confined to local nasal pathologic changes with no alterations in lung histology or immune function.

Levels and specificity of antibody in bronchoalveolar lavage (BAL) and serum in an animal model of trimellitic anhydride-induced lung injury.

A study was undertaken to characterize the antibody response in rats exposed to trimellitic anhydride (TMA) by inhalation. Total antibody levels directed to trimellitic rat serum albumin (TM-RSA) from TMA-exposed rats were assayed by an ammonium sulfate technique. Total antibody levels in bronchoalveolar lavage (BAL) and the matched serum were compared by correction for the albumin content of each. An ELISA was developed to detect IgG, IgA, and IgM directed toward TM-RSA in BAL and serum and to compare class-specific antibody levels in BAL and serum by normalizing for albumin content. The specificity of the rat IgG response was determined by ELISA inhibition with TM-RSA and TM-human serum albumin (TM-HSA) and compared with reciprocal inhibition studies with serum from TMA-exposed workers. The levels of total antibody in BAL were three to 15 times greater than the levels found in the matched serum pair. IgG, IgA, and IgM antibodies were detected in the BAL and the serum of TMA-exposed rats but not in control rats. In each of the four rats tested, all antibody classes were present in equal or greater amounts in the BAL than in the serum. Complete inhibition of the rat IgG binding in ELISA was observed when TM-RSA or TM-HSA were added as inhibitors. Human IgG was inhibited in ELISA only by TM-HSA. In an animal model of human lung disease, the levels of total antibody as well as class-specific antibodies directed against TM-RSA were greater in BAL than in serum.(ABSTRACT TRUNCATED AT 250 WORDS)

A model of immunologic lung injury induced by trimellitic anhydride inhalation: antibody response.

We studied lung injury induced in Sprague-Dawley rats by trimellitic anhydride (TMA) inhalation. Groups of 40 male and 20 female rats were exposed to TMA by inhalation at target concentrations of 0, 10, 30, 100, and 300 micrograms/m3, 6 hours per day, 5 days per week, for 2 weeks. Rats in each exposure group were sacrificed after 10 exposures or rested for 12 days and either sacrificed or received a 6-hour TMA challenge at their initial exposure levels and sacrificed at 24 hours. At each sacrifice, serum antibody to radiolabeled trimellityl rat serum albumin (RSA-TM) was measured by an ammonium sulfate technique, and lung pathology was determined. After 10 days of exposure, external hemorrhagic lung foci were directly related to the exposure concentration of TMA. Serum antibody binding of RSA-TM correlated with exposure concentration, hemorrhagic lung foci, and lung weight. There was healing of lung lesions 12 days after exposure with a return of lung lesions only 18 hours after the 6-hour inhalation challenge. A correlation between serum antibody to RSA-TM, hemorrhagic foci, and lung weight existed after challenge. This model clarifies two clinical entities observed in exposed workers, the late respiratory systemic and the pulmonary disease-anemia syndromes.

The pathologic and immunologic response to inhaled trimellitic anhydride in rats.

Trimellitic anhydride (TMA) is a chemical intermediate used in the paint and plastics industry. Inhalation of TMA can induce four types of syndromes in TMA workers; three are immunologically based whereas the fourth, an irritant syndrome, is nonimmunologic. To evaluate the potential inhalation hazard of TMA under controlled conditions, Sprague-Dawley rats were exposed 6 hr/day via inhalation to target concentrations of 0, 10, 30, 100, and 300 micrograms/m3 for varying durations. Two sets of rats received either 5 or 10 exposures and were terminated. A third set received 10 exposures, and was held 12 days and terminated. A fourth set received 10 exposures, and was held 12 days, challenged with a single 6-hr exposure, and terminated. A fifth set received 10 exposures, and was held 12 weeks and terminated. There were no effects after 5 exposures; however, after 10 exposures the following parameters were increased in a concentration-related manner: absolute and relative lung weights, external hemorrhagic lung foci, alveolar macrophage accumulation, alveolar hemorrhage, pneumonitis, and lung and mediastinal lymph node nonspecific IgG and complement (C3). The rats exposed and rested 12 days were nearly recovered from these effects; however, rats rested 12 days and subsequently challenged exhibited lesions similar to those seen immediately following exposure. Exposed rats rested 12 weeks were completely normal in all of the above parameters. The timing and nature of the lung lesions, along with the presence of lung IgG and complement, are consistent with some of the known aspects of TMA-induced lesions in humans, and are reflective of results obtained from other hypersensitivity pneumonitis models.

Effects of acrolein on macrophage functions in rats.

Male Sprague-Dawley rats were exposed to 0.1, 1.0 or 3.0 ppm acrolein or filtered air 6 h/day, 5 days/week for 3 weeks. Rats were tested one day following the last exposure and exhibited no change in pulmonary clearance of inhaled 35S-labeled Klebsiella pneumoniae at any acrolein concentration. Decreased numbers of peritoneal cells were obtained from exposed rats while the number of cells lavaged from the lungs was unchanged. Macrophages of acrolein-exposed rats had altered phagocytic and enzymatic patterns as compared to macrophages from animals breathing filtered air. However, these changes had no apparent effect on macrophage killing of inhaled bacteria and were therefore probably not indicative of extreme chemical toxicity.

Gasoline vapor exposures. Part I. Characterization of workplace exposures.

Monitoring surveys of gasoline vapor exposures were conducted on truck drivers and terminal operators from five terminal loading facilities, on dockmen and seamen at two tanker/barge loading facilities, and on attendants at a single expressway service plaza. Results revealed wide variations in total C6+ hydrocarbon exposures for each location, with overall 8-hr time-weighted averaged (TWA) geometric means of 5.7 mg/m3 (1.4 ppm) for the terminals, and 4.0 mg/m3 (1.0 ppm) for the service plaza, respectively. The exposures ranged from 0.8 to 120.8 mg/m3 (0.2-30.1 ppm) for the terminals, and from 1.1 to 130.3 mg/m3 (0.3-32.5 ppm) for the service plaza. For the terminals, exposures were not significantly different regardless of loading method or the presence or absence of vapor recovery systems. Comprehensive chemical analyses of terminal employee exposure samples revealed that the C4 and C5 hydrocarbon components constituted 74.8 +/- 9.2% of the total exposure sample on a microgram/sample basis. The C6, C7, and C8+ components constituted 13.0 +/- 1.9, 6.2 +/- 3.0, and 5.9 +/- 7.2% of the total samples, respectively. Comprehensive analyses of the marine employee exposure samples resulted in a similar distribution of components; that is, 66.6 +/- 7.9, 17.5 +/- 4.7, 9.2 +/- 3.1, and 6.4 +/- 1.9% for the C4/C5, C6, C7, and C8+ components, respectively. The composition of the exposures, however, was weighted more toward the C5, C6 and C7 components when compared to the bulk terminal employee exposures.(ABSTRACT TRUNCATED AT 250 WORDS)

Gasoline vapor exposures. Part II. Evaluation of the nephrotoxicity of the major C4/C5 hydrocarbon components.

Analysis of workplace exposures to gasoline vapors revealed that C4 and C5 hydrocarbons constitute anywhere from 67 to 74% by weight of a typical vapor. Furthermore, it was found that n-butane, isobutane, n-pentane, and isopentane together comprise greater than 90% of all the C4/C5 vapor components and approximately 61 to 67% by weight of the total vapor. Accordingly, a 21-day inhalation toxicity study of a blend consisting of 25% (w/w) each of these four hydrocarbons was conducted using rats to assess the potential for these major gasoline vapor components to induce kidney damage. No evidence of the kidney lesions typically associated with hydrocarbon-induced nephropathy was observed in rats exposed at up to 11 800 mg/m3 (4437 ppm) of the blend.

Neurobehavioral actions of cannabichromene and interactions with delta 9-tetrahydrocannabinol.

1. Neither cannabichromene (CBC) nor delta 9-tetrahydrocannabinol (THC) protected mice from electroshock-induced seizures, although THC inhibited postictal mortality. Minor effects were produced on seizure latency and duration. 2. CBC had a weak analgetic action in mice; THC had a moderate and lengthy effect, which was potentiated at 2 hr by concurrent CBC. 3. Both CBC (10-75 mg/kg, i.p.) and THC (20 mg/kg) reduced motility of mice, the THC equalling the highest dose of CBC. 4. Performance of a conditioned avoidance response was strongly impaired by THC, but not by CBC, nor did CBC combined with THC have influence on the effects of THC.

Perinatal exposure to cannabichromene and delta 9-tetrahydrocannabinol: separate and combined effects on viability of pups and on male reproductive system at maturity.

The effects of cannabichromene (CBC), delta 9-tetrahydrocannabinol (delta 9-THC) and their combination (all doses 50 mg/kg orally) were determined after being administered to female mice for 7 days beginning on the 20th day of gestation. The THC treatment reduced postnatal viability, impaired male reproductive behavior at maturity and significantly reduced seminal vesicle weights. No changes from control values occurred after CBC or CBC + THC. Thus, CBC alone at this dosage did not act like THC; moreover, it antagonized the effects of THC when the two were given in combination.