Mutagenicity of Diesel Exhaust Soot Dispersed in Phospholipid Surfactants (1ff9a826-3665-4961-9c6b-77d08332944b)

"INTRODUCTIONOrganics extractable from respirable diesel exhaust soot particles by organic solvents have been known for some time to be direct acting frameshift mutagens in the Ames Salmonella typhimurium histidine reversion assay (Huisingh, et al). Upon deposition in a pulmonary alveolus or respiratory bronchiole, respirable diesel soot particles will contact first the hypophase which is coated by and laden with surfactants. To model interactions of soot and pulmonary surfactant, we dispersed soots in vitro in the primary phospholipid pulmonary surfactant dipalmitoyl glycerophosphorylcholine (lecithin) (DPL) in physiological saline. We have shown that diesel soots dispersed in lecithin surfactant can express mutagenic activity, in the Ames assay system using S. typhimurium TA98, comparable to that expressed by equal amounts of soot extracted by dichloromethane/ dimethylsulfoxide (DCM/DMSO)(Wallace, et al, 1987). Here we report additional data on the same system using additional exhaust soots and also using two other phospholipids, dipalmitoyl glycerophosphoryl ethanolamine (DPPE), and dipalmitoyl phosphatidic acid (DPPA), with different ionic character hydrophilic moieties. A preliminary study of the surfactant dispersed soot in an eucaryotic cell test system also is reported.MATERIALS AND METHODSSoots D4 and D6 were collected at two different times as scrapings from the exhaust pipe of a diesel tractor-trailer immediately after the engine was stopped. Surfactant dispersions were made by ultrasonic probe sonicating 25 mg of commercially obtained DPL, DPPE, or DPPA into 1O ml of O.85% physiological sterile saline (PSS) for 10 min at 40 watts power. For surfactant dispersions, 2.5 mg soot was mixed into 2.5 ml of the appropriate phospholipid dispersion. Mixtures were sonicated by 40 watts sonicator probe for one min, then incubated in the sonicator water bath for 30 min at room temperature; the mixture was then incubated for one hour at 37C while being rotated continuously. Dilution series were made with saline. For solvent extraction, 2.5 mg soot was mixed into 2.5 ml of DCM and incubated in a sonicator water bath for 30 min at room temperature. The sample was then mixed with 2.7 ml DMSO, placed in a dry bath at 45C, and the DCM evaporated under a stream of nitrogen gas until 2.5 ml of liquid remained. Ames salmonella/microsome assay utilizing TA98 for frameshift mutation was used for mutagenicity testing, with and without S9 activation, as described by Ames et al (1975). Unfiltered sample extractions or dispersions were preincubated with tester cells for 90 min at 37C : 0.1 ml of sample was mixed with 0.5 ml of PSS, or of S9 mixture, and 0.1 ml of an overnight TA98 culture incubated samples were then added to 2 ml of top agar supplemented with biotin and a trace amount of histidine, and overlayed onto Vogel-Bonner minimal media plates. After 48 hours incubation at 37C, revertant colonies were counted. Solvent controls were DMSO, DPL, DPPE, DPPA, and saline. Positive controls were trinitrofluorenone (1 ug/plate), for samples not treated with S9, and 2-aminoanthracene for S9 treated samples (2.5 ug/plate). All samples were assayed in duplicate. Sister chromatid exchange (SCE) assay was performed, following Lett, et al (1981), using V79 cells, of Chinese hamster pulmonary fibroblast origin. Stock diesel sample suspensions were made as 10 mg soot/ml DPL suspension, or 20 mg soot/ml in DMSO. Aliquots of these soot preparations were added to 15 ml of culture medium per sample flask to result in concentrations of 25, 50, and 75 ug soot/ml medium. After 12 hours incubation, 200 ul of 25 uM 5-bromodeoxyuridine (BrdU) was added to each flask. After another 35 hours incubation, 200 ul of 10 ug/ml colcemid were added; and the cells were harvested three hours later. Cells were swollen by hypotonic saline, and fixed twice with methanol/acetic acid, and burst on microscope slides. Cells on the slide were stained with Hoechst 33258, exp"