Ronald E. Rasmussen

Arsenic binding proteins from human lymphoblastoid cells.

Arsenic is a ubiquitous contaminant of drinking water and food. The mechanisms of the toxic action of inorganic arsenic are unknown. We report the isolation of proteins having a high affinity for arsenic in the +3 oxidation state that are induced by arsenite (AsIII) in human lymphoblastoid cells. The arsenic-binding proteins were isolated using a p-aminophenylarsine oxide affinity column. At least four proteins of 50, 42, 38.5 and 19.5 kDa were isolated by elution with 10 or 100 mM 2-mercaptoethanol. Two proteins were tentatively identified as tubulin and actin on the basis of their molecular weights and previously reported affinity for the arsenic column. The identities of the remaining proteins are unknown. Heme oxygenase 1 was induced by AsIII but did not bind to the arsenic affinity column. We conclude that AsIII induces multiple proteins that have variable affinities for arsenic in the +3 state as judged by the concentration of 2-mercaptoethanol required for their elution. The arsenic binding motif of these proteins may involve three thiol groups arranged 3-6 A apart by the tertiary structure of the protein as suggested by others. These proteins may serve as high affinity binding sites for AsIII and may be involved in the biological action of AsIII.

An, Acute Light and Electron Microscopic Study of Ultraviolet 193-nm Excimer Laser Corneal Incisions

The 193-nm ultraviolet beam from an argon fluoride excimer laser was focused on the corneas of rabbits to produce incisions of the type necessary for radial keratotomy. The energy densities used were in two ranges, 1.0 to 2.1 J/cm2 per pulse and 200 to 700 mJ/cm2 per pulse. The eyes were enucleated and fixed for histologic and electron microscopic examination immediately after exposure. Structural analysis of the higher energy density exposures showed ridging on the surface of the cornea, micro-pitting on the stromal surface inside the cut, and denudation of the endothelium under the ablation zone. The lower energy density incisions did not exhibit significant surface ridging or endothelial cell loss but did exhibit significant stromal swelling during the laser exposure thus making it difficult to produce incisions of a precisely controlled depth. Beam profile measurements and infrared thermal measurements of the cornea surface during laser exposure were made.

In vitro systems for exposure of lung cells to NO2 and O3

In vitro studies of the effects of NO2 and O3 require development of methods for separation and culture of those lung cells that experience in vivo exposure, and also the design and construction of systems for controlled exposure of the cells to known concentrations of the gases. Separation of lung cell types has been accomplished by enzymatic dispersal of lung tissue and centrifugation of the mixed cells on media of various densities in order to separate the cells on the basis of buoyant density or sedimentation rate. The application of centrifugal elutriation has enabled separation of type II alveolar cells and Clara cells with a high degree of purity. Alveolar macrophages and endothelial cells have also been obtained in good yield. Exposure of cultured cells to test atmospheres requires precise control of pollutant levels, close contact of cells and gas without an intervening layer of medium, capability for prolonged exposure, and maintenance of sterile conditions, so that recovered cells can be cultured further or studied for other biological activity. Systems which meet these criteria include roller bottle cultures, petri dish cultures on rocker platforms, cell cultures on cellulose filters fed by perfusion of medium from the side opposite the cells, and cells grown in dishes with gas-permeable film bottoms. Systems that rely on solution of the gases in the overlaying medium do not resemble exposure conditions in vivo, and may not be suitable for studying effects of the poorly soluble oxidant gases. The cell exposure systems have not been used extensively for studies of the effects of pollutants on freshly isolated specific lung cell types. Such studies should be encouraged.

MUTATION AND SISTER CHROMATID EXCHANGE INDUCTION IN CHINESE HAMSTER OVARY (CHO) CELLS BY PULSED EXCIMER LASER RADIATION AT 93 nm AND 308 nm AND CONTINUOUS UV RADIATION AT 254 nm

Abstract— We compared mutagenesis and sister chromatid exchange (SCE) induction by 193 nm and 308 nm pulsed excimer laser radiation with 254 nm low intensity continuous wave UV light in Chinese hamster ovary (CHO) cells in culture. The 254 nm radiation was most mutagenic of the radiations, in accordance with expectation, and also was most effective in increasing the level of SCEs. The 193 nm radiation was mutagenic at the ouabain resistance locus, but not at the HGPRT locus. However, 193 nm radiation was also strongly cytotoxic at energies producing measurable mutations. This radiation also caused a dose‐related increase in SCEs. Pulsed excimer radiation at 308 nm was mutagenic at both loci, and also increased the incidence of SCEs. Comparison of the ratio of mutants/surviving cells at the D37 after radiation showed similar values for 254 nm and 308 nm at the HGPRT locus, but at the ouabain resistance locus, the ratio for the 308 nm radiation was about 5 times that for 254 nm radiation. These results indicate that some risk for mutagenesis may accompany the use of excimer radiation in the UVA region in therapeutic applications.

Effect of in vivo ozone exposure on in vitro pulmonary alveolar macrophage mobility

The effect of in vivo O3 exposure on the mobility of pulmonary alveolar macrophages (PAM) in vitro was investigated. Eight randomly selected rats were exposed for 4 h. Four rats were exposed to a clean air (sham) atmosphere, and four to an atmosphere containing 1ppm O3. PAM were obtained by lung lavage and placed on gold-colloid coated coverslips. The area cleared of gold particles by migrating PAM after 24, 48, and 72 h was used as an indicator of cell mobility. The number of PAM recoverable by lavage was similar for both groups (2 x 10(5)), but the percentage of macrophages that made tracks was significantly smaller with 95% certainty in the O3 group. For sham-exposed and O3-exposed groups, the area cleared by PAM increased as the length of incubation increased, with the area cleared by the sham-exposed group being about 50% greater during each time period. When the two groups were compared statistically at each time point, the probability that they differed was, in each case, greater than 95%. It was concluded that the in vitro migrational potential of PAM was most likely decreased by in vivo exposure to O3.

Toxicity of chemical components of ambient fine particulate matter (PM 2.5) inhaled by aged rats

The toxicity of two important chemical components of fine ambient particulate matter (PM 2.5)—ammonium bisulfate (ABS) and elemental carbon (C)—was studied using aged (senescent) rats. The study tested the hypotheses that fine particle exposure can damage lungs and impair host defenses in aged rats and that ozone would potentiate the toxicity of these particles. Ammonium bisulfate aerosols were generated by nebulization of dilute aqueous solutions. Elemental carbon was generated from an aqueous suspension of carbon black. Carbon and ABS mixtures were generated by nebulization of a suspension of carbon black in a dilute aqueous solution of ABS. Rats were exposed, nose‐only, for 4 h a day, three consecutive days a week, for 4 weeks. The rats were exposed to one of six atmospheres: (1) purified air; (2) C, 50 μg m−3, 0.3 μm mass median aerodynamic diameter (MMAD); (3) ABS, 70 μg m−3, 0.3 μm MMAD; (4) O3, 0.2 ppm; (5) ABS + C, 0.46 μm MMAD; and (6) ABS + C + O3, 0.45 μm MMAD. Data were analyzed using ANOVA and Tukey multiple comparison tests; a two‐tailed significance level of 0.05 was used. The nuclei of lung epithelial and interstitial cells were examined to determine the labeling of the DNA of dividing cells by 5‐bromo‐2‐deoxyuridine and to identify the location of injury‐repair‐related cell replication. Increased labeling of both epithelial and interstitial lung cells occurred following all pollutant exposures. Although epithelial cells are most likely impacted by inhaled particles first, the adjacent interstitial cells were the cells that showed the greatest degree of response. Exposure to the ABS + C + O3 mixture resulted in losses of lung collagen and increases in macrophage respiratory burst and phagocytic activities that were statistically significant. Our results demonstrate that ozone can increase the toxicity of inhaled particles (or vice versa), and suggest that detailed study of mixtures could provide a more comprehensive understanding of the mechanisms by which inhaled pollutants adversely affect human health. Copyright

Exposure to environmental tobacco smoke results in an increased production of (+)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide in juvenile ferret lung homogenates

Six-week-old ferrets were exposed head-only to clean air or environmental tobacco smoke (ETS) at an average particulate concentration of 38 +/- 13 mg/m3 for 2 h/d, 5 d/wk for up to 15 wk. Twenty four hours after last exposure, the ferrets were sacrificed and the metabolism of benzo[a]pyrene and (-)-7R-trans-benzo[a]pyrene-7,8-dihydrodiol was studied in lung homogenates. The results show that after ETS exposure total metabolism of benzo[a]pyrene, measured by the accumulation of hexane nonextractable radioactivity, was increased by 35% in the males and 66% in the females (p < .05), respectively, of that observed with air-exposed controls. With (-)-7R-trans-benzo[a]pyrene-7,8-dihydrodiol as substrate, the formation of both benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetraol and (+)-anti-benzo[a]-pyrene-7,8-dihydrodiol-9,10-epoxide-derived tetraols by lung homogenates of ETS-exposed male and female ferrets was significantly increased compared to the air-exposed controls (p < .01). DNA-bound radioactivity was significantly increased in both the males (p < .01) and females (p < .05) compared to the air-exposed ferrets.

Inhalation of resuspended road dust, but not ammonium nitrate, decreases the expression of the pulmonary macrophage Fc receptor

Pulmonary macrophages (PM) play a key role in the immune defenses of the lung. When stimulated, PM express Fc receptors (FcR) that regulate the immune response. PM were assayed for FcR expression following subchronic inhalation exposure of adult Fischer 344 rats to either 90 micrograms/m3 nitrate (NH4NO3), 300 micrograms/m3 road dust, or clean air, for 4 h/day, 4 days/week, for 8 weeks. PM were lavaged from the lungs and attached to glass coverslips for 18 h. PM FcR were labelled with rat IgG conjugated with cyanine-3. For each exposure, FcR were determined with a Meridian ACAS 570 confocal cytometer by imaging the fluorescence of 50 cells. We found that the IgG binding to FcR (in arbitrary fluorescence units, FU, per cell) for PM from road dust exposed rats was less (835 +/- 39.3 FU/cell) than that for PM from both ammonium nitrate or clean air-exposed rats (1115 +/- 58.0 FU/cell and 1123 +/- 46.6 FU/cell, respectively). While acid incubation conditions in vitro (pH 5.5 for 30 min to simulate the acid environment of ammonium nitrate inhalation) resulted in a 16% decrease in IgG binding (P < 0.05), IgG binding to PM from acid aerosol exposed rats was no different than the IgG bound to PM from clean air-exposed rats. PM exposed to road dust in vivo expressed 25% fewer FcR (P < 0.05). Three-dimensional images of PM failed to show any major alterations in FcR distribution. These preliminary results indicate cellular recognition of antibody-immune complexes may be impaired by subchronic exposure to road dust, which could decrease the immune response of road dust exposed animals.

Metabolism and macromolecular binding of 1-nitronaphthalene in the mouse

The metabolism and binding of 1-nitronaphthalene (1-NN) to tissue macromolecules was studied in the mouse using tissue microsomes, lung slices, and isolated lung cells. With microsomes, binding was NADPH-dependent. CO inhibited binding in lung microsomes by more than 90% and in liver microsomes by 60-85%. Nitrogen inhibited binding by 70-80% in lung microsomes and 50-75% in liver microsomes. Incubation of the microsomes in pure O2 did not affect the binding of 1-NN. SKF525A inhibited binding in a dose-dependent manner with approximately 50% inhibition of binding obtained at a molar ratio of 1:4, SKF525A/1-NN. The rate of 1-NN binding by liver microsomes was increased by pretreatment of the mice with phenobarbital (1.2 vs. 0.8 nmol/min/mg protein). beta-Naphthoflavone (BNF) pretreatment increased slightly the rate of 1-NN binding by lung microsomes (1.2 vs 0.9 nmol/min/mg protein), but had no effect on binding by liver microsomes. Studies with isolated lung cells indicated that cell cultures enriched in Clara cells were 6-15-fold more active in metabolism and binding of 1-NN than cultures not containing Clara cells. Autoradiography of lung slices incubated in vitro with [14C]1-NN showed the label to be concentrated in the cells of the bronchiolar epithelium. The results indicate that 1-NN is metabolized in vitro by cytochrome P-450 enzymes via an oxidative pathway to bind to tissue macromolecules. Further, this pathway exists in lung cells, and can lead to binding of the compound without the need for extrapulmonary metabolism.

Inhibition of DNA replication by ozone in chinese hamster V79 cells

DNA replication in Chinese hamster lung fibroblasts, line V79, was depressed in a dose-dependent manner over an ozone concentration range of 1-10 ppm. When the cells were exposed for 1 h at concentrations up to 6 ppm, the rate of DNA replication, as measured by [3H]thymidine incorporation, declined further during a 3-h period immediately following exposure. At higher ozone concentrations, at which more than 99.9% of the cells were killed, no further decline in DNA replication was seen beyond that immediately following exposure. Cultures exposed for 1 h to 10 mM ethyl methanesulfonate or to 10 J/m2 of ultraviolet (UV) light showed a similar progressive decline in the rate of DNA replication. The inhibition of DNA replication by ozone resembled that seen after exposure of cells to chemical mutagens or radiation and did not resemble the inhibition produced by metabolic poisons. The results may indicate that ozone or its reaction products interact directly with DNA in a way that inhibits replication.