Investigating the Inflammatory Effects of Traditional Petrodiesel and Alternative Biodiesel Exhaust Using an Improved In Vitro Exposure System

EPA Grant Number: FP917247
Title: Investigating the Inflammatory Effects of Traditional Petrodiesel and Alternative Biodiesel Exhaust Using an Improved In Vitro Exposure System
Investigators: Hawley, Brie Michelle
Institution: Colorado State University
EPA Project Officer: Lee, Sonja
Project Period: August 24, 2010 through August 23, 2013
Project Amount: $111,000
RFA: STAR Graduate Fellowships (2010) RFA Text |  Recipients Lists
Research Category: Academic Fellowships , Fellowship - Human Health: Public Health Sciences


Humans have been rapidly altering the air around us ever since the discovery of fire. Today, there is widespread combustion of fossil fuels to meet our transportation, energy, and industrial needs. In a country that relies heavily upon the combustion of diesel fuel for construction, agricultural, and transportation needs, exposure to diesel exhaust (DE) is widespread. DE is a large contributor to soot and fine particles in the air and inhalational exposures have been implicated in increasing one’s risk for chronic lung inflammation and fibrosis, allergic immune responses in the lungs, asthma, lung cancer, and cardiovascular events. However, much of this understanding stems from population, animal, or limited in vitro studies. There exists a need for an improved understanding of what occurs at the cellular level following diesel exposure. Further, the nature of diesel exhaust is changing as environmental and public health concerns dictate stricter EPA regulation of emissions from diesel engines. Environmental and energy concerns have called for the use of alternative fuels like biodiesel, which also changes the physical and chemical nature of diesel exhaust. Given the changing dynamics of the particulate matter (PM) found in DE, there exists a need to more fully understand the human health implications of switching to more efficient and “cleaner” (Tier 4) diesel engines, and engines that run on alternative fuels like soy-based or algae-based diesel. This project seeks to utilize an improved lung cell culture and exposure system to (1) compare the health effects of diesel exhaust from a Tier 4 engine vs. a Tier 2 engine and (2) compare the health effects of DE from engines run on traditional petrodiesel vs. alternative biodiesel fuels.


In a country that relies heavily on diesel fuel combustion, exposure to DE is widespread. DE is a large contributor to soot and fine particles in the air and inhalational exposure is associated with cardiopulmonary compromise. Tighter emissions regulations and increased biodiesel use are changing the physicochemical nature of DE. This project will compare the health effects of DE from an engine run with Tier 4 vs. Tier 2 control technologies and petro vs. biodiesel fuels.


An improved aerosol in vitro exposure system will be used to compare the inflammatory effects of exhaust from an off road diesel engine meeting Tier 2 emissions guidelines and a diesel engine meeting Tier 4 emissions guidelines. The inflammatory effects will be assessed by measuring the mRNA transcript profiles for interleukin-8, heme oxygenase-1, cyclooxygenase-2, heat-shock protein 70, and the release of lactate dehydrogenase from cultured normal human bronchial epithelial cells at 1 and 24 hours post-exposure.

Expected Results:

This project will investigate if emissions from engines meeting the EPA Tier 4 guidelines are truly less damaging to the human lung cell than their predecessors (a Tier 2 engine) by investigating changes in the expression of selected genes associated with inflammation, and oxidative stress. Cell death by necrosis and apoptosis will also be assessed. The same cellular endpoints will be utilized to assess differences in the health impacts from exposures to traditional petrodiesel exhaust vs. exhaust from soy-based and algae-based diesel fuel. Three hypotheses regarding the inflammatory potential of diesel exhaust will be tested: (1) An increase in diesel exhaust particle number (and not necessarily mass) increases cellular inflammation, oxidative stress, and necrosis; (2) The redox potential of the diesel particulate matter produced is associated with cellular stress and inflammatory response; and (3) Biodiesel emissions contain less sulfur, NOx, and PAHS than petrodiesel emissions, and therefore there is less of a marked response in cells exposed to biodiesel emissions than in cells exposed to petrodiesel emissions.

Potential to Further Environmental/Human Health Protection:

As emissions standards become stricter, attempts to understand the health impacts of DE are complicated by the changing nature of DE. The EPA has sought to improve air quality and public welfare by reducing the allowable mass of PM produced by diesel engines. However, the question remains as to whether reducing the mass output from diesel engines will lessen the burden upon human health. It is not clearly understood if mass is a good determinant in the dose-response to diesel exhaust exposure, as it may not be the mass of diesel particles, but rather the number concentration, or surface area, or composition of the particles that produces toxicological effects. Previous studies have shown that particle surface area is a better dose metric than particle mass or number. By reducing the allowable mass of PM output from diesel engines, the particle mode may be shifted to the ultrafine and nano range (dp < 0.1 µm) of particle sizes. This shift would greatly increase the particle surface area to mass ratio. Further, the small size of nanoparticles has also been speculated to increase their uptake into cells. This investigation seeks to identify the physical characteristics of the PM in DE that make DE most inflammatory to human lung cells. The results from this investigation may be used to rethink DE PM standards (perhaps shifting from a standard based on PM mass to one based on PM size and surface area) such that the PM standards more fully protect human health. The same research methodology will be used to investigate the human health implications of using bio-based diesel fuel versus traditional petrodiesel fuel. The global dependence upon the combustion of fossil fuels to meet energy demands has created a need to look to alternative fuels as the petroleum supply remains fixed while global energy demands increase. From an environmental and public health perspective, bio-based diesel fuel has an appeal as it is naturally lower in sulfur, NOx, and PAHs emissions.

Supplemental Keywords:

air pollution effects, air, in vitro toxicology, diesel exhaust, petrodiesel, biodiesel, particulate matter, inflammatory biomarkers,

Progress and Final Reports:

  • 2011
  • 2012
  • Final