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Summary A major concern in stabled horses is the occurrence of chronic obstructive pulmonary disease (COPD). Chronic obstructive pulmonary disease has been associated with poorly ventilated stables, exposure to dust and mold and as a sequel to bacterial and viral respiratory tract infections. The purpose of this study was to determine the presence of airborne Gram-negative bacteria (GNB), fungi and endotoxin which may be factors in the incidence of COPD in stabled horses. Three bedding materials were used: phone book paper, pine sawdust and wheat straw. Six stalls and six horses were utilized for the study. Two stalls each were bedded with either phone book paper, sawdust or straw. Air samples for GNB and fungi (N=96) were collected inside each stall for eight consecutive days using the time intervals of 15s, 30s, 2 min and 3 min using an Andersen Cascade Impactor. Air samples for endotoxin (N=42) were collected for each bedding type over a seven-day period. Analysis for airborne GNB, fungi and endotoxin were performed on air samples collected from the stalls. No differences were detected in total plate count for either GNB (P>.10) or fungi (P>.10) between the phone book paper, sawdust or straw bedding materials. Gram-negative bacteria and fungi were prevalent in the air of all stalls. The straw stalls had higher levels of airborne endotoxin contamination levels (P

The effects of benzo[ a ]pyrene (BAP) and formaldehyde (HCHO),alone and combined, on cell growth and DNA damage were determined in primary cultures of rat tracheal epithelial cells dissociated from rat tracheas. Cell cultures treated with 25 μM BAP for 24 h or 200 μM HCHO for 90 min did not have a marked reduction in cell growth. However, their combined treatment reduced cell growth by 60% of control when cultures were exposed to BAP followed by HCHO as well as the reverse order. None of these treatments significantly decreased cell viability as judged by dye exclusion, nor did they enhance cell terminal differentiation as measured by confirmed envelope formation. Alkaline elution analysis of DNA damage detected both DNA-protein crosslinks (DPC) and DNA single-strand breaks (SSB) as a result of HCHO treatment, whereas BAP treatment caused only SSB. While HCHO-induced SSB were repaired within 2 h, BAP-induced SSB were detected 3 days after treatment. Combined treatment of cell cultures with BAP followed by HCHO resulted in more SSB than was obtained from either agent alone, but less DPC than was detected from HCHO alone. The increased number of SSB obtained from this combined treatment may be related to the marked enhancement of carcinogenesis observed in earlier in vivo-in vitro studies.

The effects of exposure to benzo[a]pyrene (BAP) and formaldehyde (HCHO), alone and combined, on the induction of carcinogenesis in rat tracheal implants was determined as the number of growth-altered cell populations (tumor-initiation sites, TIS) per trachea. While exposure twice-weekly for 4.5 months to 0.2% HCHO solution gave only a weak response (0.25 TIS/trachea), 2.37 TIS per trachea were detected after exposure to 20 micrograms BAP in the same regimen. The combination of BAP followed by HCHO had a greater response than either agent alone (7.83 TIS/trachea), while the reverse exposure gave 1.49 TIS per trachea, which was less than BAP alone. Thus, the induction of TIS by combined exposure to BAP and HCHO was dependent on the order of exposure, and could not be predicted from their individual exposures.

The DNA damage associated with benzo(a)pyrene (B[a]P) and formaldehyde (HCHO) exposure in rat tracheal implants was determined by alkaline filter elution adapted to measure DNA-protein cross-links (DPC) in vivo. In addition, histopathological responses of the tracheal epithelium were quantitated after multiple exposures to 20 micrograms B[a]P and 0.2% HCHO. Compared to either agent alone, combined exposure for 1-4 weeks caused an increase in cellular atypia and greater thickness of hyperplastic and metaplastic lesions. HCHO exposure resulted in a dose-dependent increase in DPC with a maximal response of 85% DNA filter retention at 0.2% HCHO, which were mostly removed by 72 h. B[a]P did not cause DPC, but when tracheas were pre-exposed to 20 micrograms B[a]P followed by 0.05% HCHO there was a 15% decrease in HCHO-induced DPC. This competition between B[a]P and HCHO for sites presumably on DNA does not offer a clear explanation for their markedly enhanced cocarcinogenicity observed in previous studies, but does demonstrate the interaction between the two agents in tracheal epithelium.

Separate cultures of beating myocytes (M cells), endothelial cells (E cells) and fibroblasts (F cells) from neonatal rat hearts were compared for their susceptibilities to the toxic effects of doxorubicin, 5-fluorouracil and cyclophosphamide. Toxic injury was measured as inhibition of metabolism and changes in morphology for all cell types, and changes in beating of myocytes. Cells were exposed to the anti-tumor agents for 3 days with myocytes and 7 days for endothelial cells and fibroblasts. All measures of injury were made several times the first day and daily thereafter, with the same cultures used throughout. Toxic effects of DOX and 5-FU were greater for E and F cells than for M cells, and all measures of toxicity were generally parallel. Cyclophosphamide, whether activated by liver microsomes or unactivated, was less toxic in general than the other agents, but it was more toxic for M cells than for E cells or F cells.

Neonatal rat hearts were separated into separate cultures of beating myocytes (M cells), endothelial cells (E cells) and fibroblasts (F cells). Their susceptibilities to the toxic effects of emetine, chloroquine and metronidazole were then compared using a quantitative metabolic inhibition test (QMIT) and morphologic and beating changes as indices of injury. Measurements on the same cultures were made at 6 h and 12 h daily for 7 days with E and F cells; with M cells for 3 days. Metronidazole was non-toxic for all cell types at 810 micrograms/ml, whether as the parent compound or after attempted rat liver microsomal activation. QMIT data, integrated as time-concentration effects, showed all cell responses to either emetine or chloroquine to be parallel (P less than 0.05), and their order of susceptibility to be: E greater than M greater than F cells. Although morphologic signs of injury and changes in beating are not readily evaluated statistically, there was a general parallelism between these indices of injury and those of QMIT. As judged by QMIT emetine was more toxic than chloroquine. However, at equivalent QMIT grades chloroquine produced greater effects on morphology and beating. Toxic concentration-50 values for chloroquine with the QMIT were similar to reported human toxic and lethal blood concentrations.

The single and combined effects of 2 environmental carcinogens, benzo[ a ]pyrene (BAP) and formaldehyde (HCHO), on cell growth and DNA damage were assessed in a rat tracheal epithelial cell line, C18. Treatment of C18 cells with HCHO for 90 min reduced the calculated growth index at the highest concentration tested, 400 μM, while no growth effects were observed with BAP treatments. Combination treatments reduced the growth index to 75% of control values. Alkaline elution analysis of C18 cell DNA detected both DNA-protein crosslinks (DPC) and single-strand breaks (SSB) as a result of HCHO treatment, while BAP caused only SSB. HCHO-induced SSB were repaired within 2 h, whereas BAP-induced SSB persisted for at least 48 h. Combination treatment of cells with BAP followed by HCHO enhanced the number of SSB, but reduced DPC. The results are discussed in reference to earlier work which demonstrated the interaction in vivo between BAP and HCHO with respect to their carcinogenicity in rat tracheal implants.

We have developed an immunological assay to detect DNA-protein complexes (DPCs) in cell cultures treated with environmental toxicants. The assay uses an antiserum developed against K(2)CrO(4)-induced DPCs, which recognizes an acidic protein with a molecular weight of 95 kDaltons. The method uses a filter-binding assay to trap the DPCs from SDS-lysed cell cultures on polycarbonate filters, after which they are immunologically probed with the DPC antiserum. Cultures of Chinese hamster ovary cells were treated with K(2)CrO(4), formaldehyde or UV irradiation. DPCs were detected immunologically in cells treated with K(2)CrO(4) or UV irradiation, but not in those treated with formaldehyde. These results were similar to those obtained when DPCs were detected by filter-binding assay using radiolabelled cell cultures to measure the adherence of protein and DNA to the filters. In addition, both methods gave analogous dose responses of DPC formation in K(2)CrO(4)-treated cells. This novel immunological assay for detecting DNA lesions allows the rapid analysis of samples, which do not need to be radiolabelled, and thus it may have an application as a non-invasive test to screen for DNA-protein complexes.

The toxicity of substances for monolayer cultures of rat lung fibroblasts with phenol red as a pH indicator in the medium was measured by comparing the changes produced in medium color against 10 stable color standards. The toxicities of 6 concentrations each of actinomycin D, amphotericin B, chloroquine, 2-deoxyglucose, oligomycin, puromycin, and silicon dioxide were measured at 12 h and daily for 7 days on the same cultures throughout. Morphologic changes were monitored at the same times. The method, which simultaneously measures both concentration and time effects, was rapid, simple-to-perform, reproducible, low-in-cost, and quite sensitive. Its reproducibility depended on details of the cell culture methodology. The method is unsuitable for acidic or basic substances, or substances poorly-soluble in culture medium. Unless combined with morphologic evaluation, substances producing delayed toxicity may give misleading results.