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Effect of Sidestream and Mainstream Smoke Exposure on in Vitro Interferon-A/Fl Production by L-929 Cells'

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Effect of Sidestream and Mainstream Smoke Exposure on in Vitro Interferon-A/Fl Production by L-929 Cells' ICANCERRESEARCH46, 2779—2783,June1986J Effect of Sidestream and Mainstream Smoke Exposure on in Vitro Interferon-a/fl Production by L-929 Cells' Gerald Sonnenfeld2 and Russell W. Hudgens Departments ofMicrobiology and immunology and Oral Biology, Schools ofMedicine and Dentistry. University ofLouisvile. Louisville, Kentucky 40292 ABSTRACF tion by L-929 cells. The highest doses of smoke that induced minimal mortality of the cells were used (12). In addition, A peristaltic pump smoking machine that allows simultaneous gener smoke was manipulated by delaying the time of exposure of ation of mainstream (active) and sidestream (passive) smoke from a cells after smoke generation (aging) and by passage of smoke cigarette was used to expose cultures of murine L-929 cells, a potent producer of interferon, to smoke. The cigarette used was the University through activated charcoal. The results of the present study of Kentucky 2R1 reference cigarette. The dosages of smoke used for indicate that smoke exposure can inhibit IFN induction in L exposure were the highest doses possible that generated a minimum toxic 929 cells. The degree of inhibition of IFN induction was much effect, and they were serially diluted to lower doses. Dosages were greater than inhibition that could be accounted for by loss of determined by the number of smoke puffs generated, the volume of smoke viable cells in the smoke-exposed cultures. In addition, manip puffs generated, and the total particulate matter deposited on Cambridge ulations of smoke that decreasedtoxicity to L-929 cells (I 2) filters in the smoke machine. Viability of exposed cells was equivalent to and to yeast (13, 14) also decreased the inhibition of IFN control cell cultures. Interferon-a/fl was induced by addition of polyri induction after smoke exposure. boinosinic-polyribocytidylic acid to the cells. Interferon production was substantially reduced in viable cells exposed to mainstream or sidestream smoke. Aging of smoke by delaying time of exposure of the cells to the MATERIALS AND METHODS smoke, or filtration of smoke through activated charcoal substantially decreased the alteration of interferon production by smoke exposure. CellCultures. L-929 cells were originally obtained from the American These results suggest that actual exposure of cells to mainstream or Type Culture Collection (Rockville, MD) and were maintained in MEM sidestream smoke can inhibit in vitro interferon-a/ft induction, but the (GIBCOLaboratories,GrandIsland,NY) supplementedwith10%fetal cells can be protected from these effects by smoke manipulation. bovineserum.All cultureswereplatedin 25-cm2plastictissueculture flasks (Falcon Plastics, Oxnard, CA). Cultures were utilized for expo INTRODUCTION sure experiments upon reaching confluency (I). Interferon Induction. Mouse IFN-a//3 was induced in cultures of L Several studies have indicated that IFN-a/f33 production was 929 cells as previously described (11). Briefly, 50 @zgofpoly-rI.rC (P inhibited by pretreatment of cell cultures or animals with car L Biochemicals, Milwaukee, WI) were used. One hundred @igofDEAE cinogens (1—5).Treatment of cell cultures or animals with dextran (Pharmacia Fine Chemicals, Piscataway, NJ) were added to each culture to ensure maximum IFN induction (6). After the initial 1- poorly or noncarcinogenic analogues had no effect on IFN h period of incubation, sufficient MEM was added to the cultures to induction (1—5).These studies have been recently extended to bring them to a final volume of 2 ml. The cultures were next incubated include inhibition of murine in vitro and in vivo IFN induction for 23 h at 37T in 5% CO2. Culture supernatant fluids were then by carcinogenic chemical components of cigarette smoke (6, 7); harvestedandassayedforantivirusactivity. however, cigarette smoke is composed of a complex mixture of Interferon Assay. The antivirus activity of putative IFN samples was chemicals in solid and vapor phases, and the effects of individual determined by means of a plaque reduction assay utilizing the Indiana components may not mirror the effect of actual smoke on IFN strain of vesicular stomatitis virus as the test virus (15). L-929 cell production (8, 9). cultures were the target cells for the IFN assay. The IFN titer corre Recently, a new exposure machine utilizing a peristaltic pump spondedto the reciprocalof the greatestdilutionof test samplethat mechanism has been developed (10). This machine allows the reduced virus plaquing by 50%. In this assay system, one antivirus unit simultaneous exposure of animals or tissue cultures to both of IFN was equivalent to 0.88 NIH G-002-904-5l 1 reference standard units. mainstream (active) and sidestream (passive) smoke (10). Uti Smoke Exposures. The peristaltic pump smoking machine, provided lizing this exposure system, we have recently studied the effects by the Kentucky Tobacco and Health Research Institute, was utilized of cigarette smoke on viability of L-929 cells in culture (1 1). for all exposurestudies(10). A completedescriptionof the system, We have shown that either mainstream or sidestream smoke including diagrams and photographs, has been previously published in exposure resulted in dose-dependent mortality to the cells. Ref. 10. This machine generates smoke through a puffing mechanism, Dilution, aging, and filtration of the smoke through activated and mainstream smoke is pumped directly to an exposure system able charcoal each decreasedthe toxic effects of the mainstream or to expose four tissue culture flasks at the same time. Sidestream smoke sidestream smoke exposure on L-929 cells (12). was trapped off the end of a burning cigarette in a plastic chamber and The current study was designed to determine the effects of pumped to an additional exposure system capable of exposing four mainstream or sidestream smoke exposure on IFN-a/fl produc tissue culture flasks at the same time. In this fashion, tissue cultures were exposed to mainstream and sidestream smoke generated from the Received 11/20/85; revised 2/27/86; accepted 3/5/86. same cigarette. The cigarette used was the University of Kentucky 2Rl The costs of publication of this article were defrayed in part by the payment reference cigarette (16). The cigarettes were not selected for weight or of page charges. This article must therefore be hereby marked advertisement in pressure drop. TPM for each cigarette exposure was determined by accordance with 18 U.S.C. Section 1734 solely to indicate this fact. @ Supported by a grant from the Kentucky Tobacco and Health Research extracting Cambridge filters placed in the path of smoke with acetone Institute. measuring the absorbance of an extract at 390 nm, and multiplying the 2To whom requests for reprints should be addressed, at Department of results by the dilution factor (10). Mainstream smoke was generated at Microbiology and Immunology, School of Medicine, University of Louisville, a rate of one puff per mm with a puff duration of 2.4 s. Puff volume Louisville, KY 40292. 3 The abbreviations used are: IFN, interferon; MEM, minimal essential me averaged17ml and wasobtainedby useof a calibrateddilution tip dium; poly-rIrC, polyriboinosinic-polyribocytidylic acid; TPM, total particulate placed at the top of the puff chamber (10). The smoke exposure matter. apparatus was washed with 70% ethanol to decrease the possibility of 2779 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1986 American Association for Cancer Research. TOBACCO SMOKE AND IFN contamination. All medium was removed from the tissue culture flasks ulations were carried out using four puffs of sidestream smoke. andreplacedwith 4 ml of MEM. The tissuecultureflaskswerefitted Passageof smoke through charcoal filters or aging of smoke onto the apparatus, and a tight seal was obtained (10). The cultures prior to exposure ofcells to sidestream smoke yielded low levels were next exposed to smoke as will be described for each individual of toxicity to the cells (Table 1). experiment. “Sham―exposure was obtained in some instances by pass Cultures ofconfluent L-929 cells were exposed to mainstream ing air through the system to the tissue cultures. After exposure, the cells were returned to a 37'C incubator with 5% CO2for 1 h. Then, the smoke. Exposure of cells to greater than eight puffs of smoke medium was removed from each flask and was replaced with 2 ml of resulted in appreciable mortality to the cells (Table 2). Exposure MEM with 2% fetal bovine serum. Twenty-four h later, IFN was of cells to up to six puffs of mainstream smoke resulted in a induced, and the cells were allowed to incubate for another 24 h. Then, lesser degree of mortality to the cells, so all additional main culture supernatant fluids were harvested and assayed for IFN antivirus stream smoke manipulations were carried out using an exposure activity, and viability was determined by means of a trypan blue dye of six puffs. Manipulation of smoke through charcoal filters or exclusion assay. In this study, the percentage of mortality is defmed as aging of mainstream smoke resulted in decreased mortality of L-929 cells as measured by trypan blue viability staining. — . (control viability sample viability) % ofmortality = ... x 100 In addition, cells that were exposed to dosages of smoke that (control viability) produced minimal mortality were harvested from the cell cul ture plates. These cells were then diluted, replated, and repli (10). Smoke Manipulation. Aging ofsmoke was obtained by placing plastic cated at the same rate as control unexposed cells (data not chambers of different size between the smoke source and the tissue shown). cultureflask.This servedto delaythe timeof exposureof the cellsto Effect of Sidestream Smoke Exposure on IFN Induction. Cell the smoke after the generation of smoke. Filter studies were carried cultures were exposed to four puffs of sidestream smoke, and out by placing filters between the puffchamber and the peristaltic pump then IFN-a//3 was induced with poly-rI .rC. The number of for mainstream smoke exposure, and between a dilution chamber and exposures for each type of treatment is indicated in Table 1.
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