imaging-approaches-to-assessments-toxicological-oxidative-stress

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency

Oak Ridge Institute for Science and Education

Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm). Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM) for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane.

Darkfield-confocal microscopy detection of nanoscale particle internalization by human lung cells.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the US and its impact continues to increase in women. Oxidant insults during critical periods of early life appear to increase risk of COPD through-out the life course. To better understand susceptibility to early life exposure to oxidant air pollutants we used Fisher (F344), Sprague-Dawley (SD) and Wistar (WIS) male and female neonatal rat pups to assess: (A) if strain (i.e. genetics), sex, or stage of early life development affected baseline lung antioxidant or redox enzyme levels and (B) if these same factors modulated antioxidant responsiveness to acute ozone exposure (1 ppm × 2 h) on post-natal day (PND) 14, 21, or 28. In air-exposed pups from PND14-28, some parameters were unchanged (e.g. uric acid), some decreased (e.g. superoxide dismutase), while others increased (e.g. glutathione recycling enzymes) especially post-weaning. Lung total glutathione levels decreased in F344 and SD pups, but were relatively unchanged in WIS pups. Post-ozone exposure, data suggest that: (1) the youngest (PND14) pups were the most adversely affected; (2) neonatal SD and WIS pups, especially females, were more prone to ozone effects than males of the same age and (3) F344 neonates (females and males) were less susceptible to oxidative lung insult, not unlike F344 adults. Differences in antioxidant levels and responsiveness between sexes and strains and at different periods of development may provide a basis for assessing later life health outcomes - with implications for humans with analogous genetic or dietary-based lung antioxidant deficits.

Eugene A. Gibbs-Flournoy

Environmental Public Health Division

National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

Eugene A. Gibbs-Flournoy

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Environmental Public Health Division

National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

Environmental Public Health Division