In Vitro exposure of Lung Cells to Diesel Exhaust at the AirCell Interface

1
University of California BerkeleyLawrence
Berkeley National Laboratory

• In Vitro exposure of Lung Cells to Diesel Exhaust
  at the Air-Cell Interface
• Donald Lucas, Amara Holder, Regine
  Goth-Goldstein,
• Catherine Koshland, and Robert Sawyer
• PERF Meeting
• July, 2005

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Introduction
The Health Effects Institute (2005) states that
additional research will be needed to
investigate further the inflammatory mechanisms,
and to identify which components of the diesel
exhaust mixture may contribute to the
inflammatory effects.
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Study Objective

• Design a device for in vitro exposure at the
  air-cell interface
• Characterize diesel exhaust gases and particles
• Investigate effects of diesel particles on human
  lung cells

4
Particle Generation

• Acme Motori diesel generator model ADX-300
• 4 stroke
• direct injection
• 301 cm2 displacement
• 4 kW full load
• Operating Conditions
• California diesel fuel
• 3600 rpm
• 1.5 kW load

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Schematic of Environmental Chamber
Q 1,250 lpm T 19 C RH 35-55
Ventilation
Q 320 lpm Dilution Ratio 1501
Inlet
Recirculation
System
Diesel
Generator
Incubator T 37.5 C
Ventilation
Outlet
Exposed cells
Unexposed cells
Pump
Optical Particle Counter
Scanning Mobility Particle Sizer
2.2 m x 2.4 m x 4.6 m
Filter Pump
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Costartm Transwell Air-Cell Interface

• Human bronchial epithelial cell Line 16HBE14o
  (SV40 immortalized)
• Minimum Essential Medium and 10 Fetal Calf
  Serum
• Cytokine secretion measured with ELISA kit
• Cell Viability measured with MTT assay

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Cell Exposure Device 24 Well Plate

• Growing area 0.33 cm2, 3 µm pore size
• 0.25 ml of medium in the bottom of the well
• 10 ?l aliquot of saline above cell layer
• Exhaust flow rate into the exposure chamber 1300
  cm3/min

Diesel Exhaust Inlet
Outlet to Pump
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Experimental Conditions

• Before experiment - chamber air is HEPA filtered
• N 1.2 x 102 cm-3 dm 42nm
• Stabilize conditions in chamber for 60 minutes
• Characteristic conditions during exposure
• N 3 x 105 cm-3 dm 118 nm (SMPS)
• 1.0 x 105 cm-3 (OPC)
• NOx 7.5 ppm

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Steady State Size Distribution
Total Number Concentration N 3.0 x 105 cm-3
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Experiment 1 One Hour Exposure
Corrected for Cell Viability 59 compared to
controls

• Abe et al.1
• 4 hr exposure/18hr incubation
• 50 increase IL-6
• 420 increase IL-8
• Salvi et al.2
• 1hr exposure on humans
• 50 increase in IL-8

1Abe et al., Am. J. Resp. Cell Mol. 22
(2000) 2Salvi et al., Am. J. Resp. Crit. Care 159
(2000)
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Experiment 2 Time Exposure

• Abe et al.1 observed time dependent increase in
  IL-8 secretion for 2, 4, and 8 hour exposures

1Abe et al., Am. J. Resp. Cell Mol. 22 (2000)
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Experiment 3 Hydrocarbon Denuded Exhaust
No significant difference when volatile and
semi-volatile compounds are removed
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Experiment 4 Filtered Exhaust
Whole exhaust has the largest effect, but the gas
phase species without the particles also produce
significant IL-8
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Summary

• We obtain diesel soot with size distributions
  representative of observed urban atmospheric
  conditions.
• We observe a pro-inflammatory response of the
  cells to the exhaust, with an increasing response
  with increasing dose.
• Removing volatile hydrocarbons has no effect.
• Filtered exhaust has an effect, but it is not as
  large as the exhaust with particles.
• We have a versatile and low cost experiment that
  can be used to investigate the effects of
  particles from varying engine conditions and
  exhaust treatments.

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Future Work

• Separate effects of particulate and gas phase
  species by removal of NOx from the exhaust.
• Perform exposures with size selected particles.
• Measure particle deposition and partitioning
  within the cell versus in the medium with cryo
  X-ray tomagraphy.

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Acknowledgements

• We thank Lara Gundel, Mike Apte, and Doug
  Sullivan at Lawrence Berkeley National
  Laboratory for providing and helping us with the
  environmental chamber.
• We thank Dieter Gruenert at the California
  Pacific Medical Center Research Institute for
  supplying human lung cells.
• This work was supported by the Environmental
  Health Sciences Superfund Basic Research Program
  (Grant Number P42ESO47050-01) from the National
  Institute of Environmental Health Sciences, NIH,
  with funding provided by the EPA, the Wood
  Calvert Chair in Engineering (UCB), and the EETD
  Division of LBNL.