In the stratosphere, ozone protects the earth from the sun's harmful UV rays. Image from http://www.unep.org/Ozone/slideshow/images/ozone-cartoons_03_0001_jpg.jpg

Ozone is resonance-stabilized molecule. However, it is very unstable because of its peroxy bonds. Image from http://www.chm.bris.ac.uk/motm/ozone/ozone.jpg

Ozone is an extremely electronegative species, making it a very strong oxidizing agent. Image from http://academic.reed.edu/chemistry/roco/Resonance/images/ozone_pot.jpg

Effect of Ozone on Lung Tissue

• Ozone exposure creates reactive oxidative species, resulting in both lipid and protein peroxidation
  • Ozone may cause the plasma membrane to become more permeable, resulting in cell necrosis
• Lung cells may upregulate Glutathione Reductase to diminish oxidative damage and restore cell to normal conditions
  • The level of glutathione corresponds to the redox state and health of the cell

Ozone and the Inflammatory Response

• Ozone exposure may induce an inflammatory response; the inflammatory response may actually (and ironically) cause more damage than the ozone exposure itself
  • Several molecules can be used to indicate inflammation. Prostaglandin E2 is a particularly useful marker of inflammation
• Chronic inflammation may be an epigenetic marker for cancer

The lung is a complex organ designed to maximize the surface area for gas exchange. Image from http://z.about.com/f/p/440/graphics/images/en/1103.jpg

There are more than 40 unique types of cells in the lung. Above is a picture of type two alveoli cells: these cells are involved in the pulmonary immune response and repair. Image from http://www.lehigh.edu/~influids/images/cagatay/figure3.JPG

Many environmental factors can cause cellular damage, including ozone and the inflammatory response itself. Image from http://www.smokersrx.com/images/why2.jpg

The alveoli are very delicate, as illustrated by the above picture. Image from http://oac.med.jhmi.edu/res_phys/Encyclopedia/Alveoli/AlveoliCellScan.GIF

Pulmonary inflammation can occur throughout lung tissue. Aboe is an image comparing normal bronchiole to an inflammed bronchiole. Image from http://cjcotton.files.wordpress.com/2009/03/asthma.jpg

If the cells in the alveoli become inflammed, as in the picture above, the volume of the lumen, and consequently lung function, is decreased. Image from http://www.ehponline.org/members/2005/7727/fig2.jpg

Estrogen is a sex hormone

• Estrogen is responsible for the development of female sex characteristics
• 17 beta estradiol is the biologically active form
• There are many metabolites of estrogen
  • Estrone is the biologically inactive form of estrogen
  • 2-hydroxy-17-beta estradiol and 4-hydroxy-17-beta estradiol induce oxidative damage and are carcinogenic; their purpose in the cell is not understood
• Estrogen can stimulate cellular changes (i.e. the upregulation of specific proteins) by initiating a signal cascade or by acting as a transcription factor

Estrogen is a Cytoprotectant

• Estrogen is an antioxidant
  • Estrogen can act as a free radical scavenger, like glutathione and other known antioxidants, to protect the cell from reactive oxidative species and to preserve the integrity of cellular membranes and proteins
• Estrogen may be able to preserve mitochondrial membranes, ATP production and induce cell growth in response to oxidative damage by upregulating some unknown proteins
• Estrogen may also be able to upregulate other proteins with antioxidative properties, such as glutathione

Estrogen and the Immune Response

• Estrogen may increase / exaggerate the immune response by ...
  • increasing mitochondrial activity, thus promoting cell growth to replace damaged / dying cells
  • upregulating proteins involved in the inflammatory response by stimulating the NF-kB pathway

The stereochemistry of 17-beta estradiol is very specific and complex. Image from http://medchem.rutgers.edu/courses/graphics/estradiol.png

The metabolism is complex and can be triggered by a multitude of factors. Image from http://www.cellscience.com/Reviews5/Estrogen1.gif

Prostaglandin E2's role in inflammation

• Prostaglandin E2 (PGE2) is a known marker of inflammation
  • It is secreted by many different types of cells (not just those directly involved in the immune system) in response to stress

The Deleterious Side Effects of PGE2

• PGE2 may be involved in carcinogenesis / tumor survival
  • Increased concentrations of PGE2 have been found in tumors

Indicators that Ozone plays a role in lung disease pathogenesis

• Rates of ARDS, asthma and lung cancer have increased as ozone levels have increased

Indicators that Estrogen plays a role in lung disease pathogenesis

• Gender descrepancies in the morbidity and mortality of ARDS, asthma and lung cancers
  • ARDS: women have higher survivorship than do men of simillar health
  • Lung cancers: adenocarcinomas account for 3/4 of all types of lung cancers in women but only 1/3 of all lung cancers in men; women may respond better to surgical removal of tumor
  • Asthma: boys under the age of 15 are more likely to go to the hospital for an asthmatic attack than are girls

So far, we know that ...

• Ozone exposure increases lipid peroxidation
• Increased amounts of lipid peroxidation causes a decrease in the expression of 17-hydroxysteroid dehydrogenases
• Decreased expression of 17-hydroxysteroid dehydrogenases will cause an increase in the concentration of 17-beta estradiol
  • 17-beta estradiol will no longer be converted to estone, the biologically inactive form
• ozone exposure should result in an increase in the amount of estrogen receptors on the plasma, nuclear, and mitochondrial membranes, increaseing estrogen's effect on the cell
• If 17-beta estradiol binds to an estrogen receptor in the presence of ozone, than the concentration of PGE2 will increase
  • These three factors will stimulate the NF-kB pathway
• Increased levels of PGE2 may cause 17-beta estradiol to be metabolized to one of its carcinogenic metabolites
• Also, if 17-beta estradiol binds to an estrogen receptor in the presence of ozone, than the concentration of glutathione proteins will increase
  • Increased glutathione proteins will decrease lipid peroxidation

The Experiment

• Pretreat lung cells with 10 nM 17-beta estradiol for either 48 hours or 30 minutes
• Expose lung cells to 300ppb ozone for 1 hour in either the presence or absence of 10nM 17-beta estradiol
• Allow cells to recover for either 30 minutes or 24 hours in the presence of 10nM 17-beta estradiol
• Perform a typan blue cell count and run MMT, GSH and PGE2 assays to determine cell viability, redox state and inflammation

My Hypothesis

• 17-beta estradiol treatments will increase cell viability, decrease oxidative damage and increase inflammation in lung cells treated with ozone

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