GENETIC ANALYSIS OF ELEVATED OZONE RESISTANCE IN VITRO

Project: Research project

Project Details

Description

The objective of this proposal is to understand the nature of the
phenomenon of elevated ozone resistance of conducting airway epithelium.
Previous in vivo animal studies have demonstrated the phenomenon of
"elevated ozone resistance" in conducting airway epithelium after a
repeating ozone exposure. Several years ago, we developed a biphasic
culture system in which conducting airway epithelial cells are able to
grow between air phase and liquid medium. Under the basal feeding,
epithelial cells differentiate and form polarized epithelium resembling
that in vivo. Since ozone is such a highly reactive chemical, this
biphasic culture system allows a direct exposure of airway epithelial
cells to ozone. In the Preliminary Study, the toxicity of ozone on
airway epithelial cells can be demonstrated. However, we observed
different toxicity among different airway epithelial cultures. for most
of human and non-human primate primary airway epithelial cells, the LT50
is more than 30 hrs; in contrast, the immortalized human tracheobronchial
epithelial (TBE) cell lines and established monkey cell lines have LT50
of 4-6 hrs. Numerous explanations can be postulated to address this
difference, such as different levels in antioxidant/oxidant enzymes,
different toxicity response, different membrane structures, and other
mechanisms still undefined. We will test two hypotheses in this
proposal, 1) antioxidant enzymes are involved in the elevation of ozone
resistance; 2) differential gene expressions are involved in the
regulation of different ozone sensitivity in different cells. To address
the former hypothesis, we need to demonstrate the existence of different
levels of antioxidant enzymes in these primary TBE cells and the cell
lines. Most of all, we need to demonstrate that the resistance of cell
lines to ozone can be elevated upon the transfection with antioxidant
enzyme expressional constructs. To address the latter hypothesis, we
will construct the cDNA expressional library from the resistance TBE
cells and use in transfection for selecting cDNA clones that can convert
the sensitive cell lines to be resistant to ozone. This approach will
allow us to identify genes which are previously not recognized in a role
of ozone resistance. Currently, we have obtained expressional cDNA
constructs of manganese-superoxide dismutase (SOD), extracellular form
of SOD, and catalase, and full length cDNA clones of gluthionine
peroxidase and gluthionine-S-transferase. The activity of these enzymes
in transfected cells will be determined biochemically. We will construct
the latter two cDNA constructs in the eukaryotic expressional vectors.
We have considerable experience in cDNA construction. This genetic
approach will help to elucidate the genes underlined the mechanism of the
phenomenon of elevated ozone resistance.
StatusFinished
Effective start/end date8/1/937/31/96

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Environmental Science(all)
  • Medicine(all)

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