The Toxicologist - Society of Toxicology

ment of hypertension in companion animals, a sensitive and selective LC-MS/MS
method has been developed and validated for determination of benazepril and benazeprilat
in dog plasma.
In this method, following a solid phase extraction procedure using Oasis WCX
SPE cartridges (3cc, 60 mg) for sample cleanup, the extract was chromatographed
using a Betasil phenyl column (10 X 2.1 mm, 3μ) and methanol-based mobile
phase solutions. A valve switching system is used for eliminating background noise
built up with injections of extracted samples. The analytes were detected by
TurboIonSpray API 4000 LC-MS/MS system under positive MRM mode. The
quantitation ranges were 0.1 to 20 ng/mL and 0.5 to 100 ng/mL for benazepril and
benazeprilat, respectively.
Overall intra-assay accuracy [% bias] and precision [%CV] observed during validation
for four levels of QC samples (six replicates each level, including the LLOQ
level) in three runs were -1.5 to 0.1% and 1.8 to 10.4%, respectively, for benazepril
compared to -0.5 to 0.6% and 1.7 to 10.3% for benazeprilat. Matrix effect was
evaluated for benazepril and benazeprilat in six different lots of blank dog plasma at
concentrations of 0.3 and 1.5 ng/mL, respectively, with variation of response
[CV%] of 0.1% for both analytes. The extraction recovery, potential conversion effect
from benazepril to benazeprilat, stability results, and results for incurred sample
reanalysis will be presented. The validated method can be demonstrated to be reliable
in drug development and GLP studies.
2286 EVALUATION OF ADVERSE EFFECTS ON HUMAN
LUNG FUNCTION CAUSED BY OZONE.
R. L. Prueitt 1 and J. E. Goodman 2 . 1 Gradient, Seattle, WA and 2 Gradient,
Cambridge, MA.
Biological changes in response to an environmental exposure occur along a continuum
that may eventually, depending on exposure dose and duration, lead to adverse
effects. Recently, a framework was described that evaluates biomarkers of exposure
and effect that are found on this continuum to determine whether an
exposure is likely to be causal and an effect is likely to be adverse (Goodman et al.,
2010). We applied this framework to estimate the ozone concentration at which
short-term exposures cause respiratory effects and to characterize the degree of adversity
of these effects. We evaluated human clinical studies and observational epidemiology
studies that examined associations between ambient ozone levels and effects
on lung function. Using the framework, we assessed causality by determining
whether effects observed in these studies are statistically significantly different between
exposed and non-exposed subjects, isolated or independent, secondary, observed
because of study limitations, or exposure related but unrelated to the apical
effect. We assessed the degree of adversity of the observed effects by determining
whether they are adaptive or compensatory, transient, reversible, early precursors of
an apical effect, of low severity, or do not result in functional impairment. Our
analysis indicates that the available evidence supports a short-term exposure threshold
of 70 ppb for effects on lung function, although these effects do not meet the
criteria for adversity as specified in the framework. Below this dose, effects are isolated
or independent and not statistically different in exposed subjects in clinical
studies, and study limitations affect interpretation of results from observational
studies. The available evidence indicates that adverse effects are observed at ozone
exposures of at least 87 ppb, and these effects are reversible and of low severity. We
conclude that the available evidence supports a short-term exposure threshold of 70
ppb ozone for effects on lung function, with adverse effects occurring at ozone concentrations
of at least 87 ppb.
2287 MODELING NF-κB REDOX SENSITIVITY:
APPLICATIONS TO OZONE-INDUCED LUNG
INFLAMMATION.
D. J. Miller and P. D. White. U.S. EPA, Washington, DC. Sponsor: S. Vulimiri.
The NF-κB intracellular signal transduction pathway plays a well-established role
in inflammatory signaling, including transcriptional control of a variety of cytokines
that are critical to inflammatory response to ozone within lung airway epithelial
cells. Less understood are the mechanisms involved in NF-κB pathway activation
in the presence of oxidative stress, despite the absence of canonical pathway
receptor stimuli. We have constructed a redox-sensitive pathway model that is
based upon multiple biological components described in recent literature. This includes
a central role for oxidative inhibition of phosphatases, leading to a buildup
of upstream kinase activity and subsequent downstream signaling of NF-κB.
Computational modeling of this pathway elucidates these testable mechanisms of
activation, and systematic perturbation of the model informs our understanding of
sensitive subpopulations by identifying pathway components that are most critical
to regulation of pathway response. As part of a larger multidisciplinary effort to
apply such next-generation approaches to risk assessment, we describe this model of
the NF-κB signaling pathway, and demonstrate both (1) a potential mechanism for
pathway activation via intracellular reactive oxygen intermediates generated by
ozone and (2) applications of the model to informing human risk.
2288 COMBINED CFD/PBPK MODELS FOR DETERMINING
SITE-SPECIFIC UPTAKE AND TISSUE
CONCENTRATIONS OF REACTIVE GASES IN THE
RESPIRATORY TRACT OF RATS AND HUMANS.
R. A. Corley 1 , S. Kabilan 1 , J. E. Carson 1 , R. Jacob 1 , K. R. Minard 1 , R. W.
Glenny 2 , S. Pipavath 2 , M. Fanucchi 3 and D. R. Einstein 1 . 1 Pacific Northwest
National Laboratory, Richland, WA, 2 University of Washington, Seattle, WA and
3 University of Alabama at Birmingham, Birmingham, AL.
3D computational fluid dynamic (CFD) airflow models have been developed for
the full respiratory system of rats and humans. The CFD models include the external
nares and mouth to provide more realistic airway inlets for nasal vs. oral breathing.
Site-specific airflow patterns are heavily influenced by species-specific 3D
anatomy and breathing patterns. Since potentially significant human exposures to
reactive gases occurs via cigarette smoking, we incorporated a two-compartment
PBPK model for acetaldehyde as airway boundary conditions for the CFD model
to demonstrate the impact of using full respiratory airflow models in comparative
target tissue dosimetry. The airway PBPK models were compartmentalized according
to species-specific cell type (nose) or anatomic region (rest of respiratory tract).
Physiological constants for the two compartments representing mucus and surface
epithelium (compartment 1) and submucosa with blood perfusion (compartment
2) were taken from the literature or measured directly. Chemical-specific constants
were taken from the PBPK model of Teeguarden et al. Inhal. Toxicol. 20 (2008)
375-390. Site-specific flux rates, Cmax’s, AUC’s and Lifetime average Daily Dose
(LADD) levels for each compartment and region were determined at the NOAEL
for subchronic nasal olfactory degeneration in the rat (50 ppm) and at an average
yield of acetaldehyde in cigarette smoke for humans. Although the concentrations
of acetaldehyde can achieve very high initial concentrations (>1000 ppm) in the
mouth, the transient nature of a bolus, puff inhalation results in 13- to 50-fold
lower LADD’s in human tissues than the rat olfactory region depending upon the
number of cigarettes smoked/day, with the larynx in humans being the region of
greatest tissue concentration following oral inhalation. Funded by NHLBI R01
HL073598 and RJR Project 56296.
2289 MODELING VAPOR UPTAKE AND TISSUE
DISPOSITION IN HUMAN LUNGS.
M. Singal 1 , B. Asgharian 2 , O. T. Price 2 , J. S. Schroeter 3 and J. S. Kimbell 4 .
1 Research Institute for Fragrance Materials, Inc., Woodcliff Lake, NJ, 2 Health Effects
& Medical Response, Applied Research Associates, Raleigh, NC, 3 Division of
Computational Biology, The Hamner Institutes for Health Sciences, Research Triangle
Park, NC and 4 Department of Otolaryngology/Head and Neck Surgery, University of
North Carolina, Chapel Hill, NC.
Estimates of the uptake of inhaled vapors in the lung and transport to other tissues
and organs of the body are needed to assess body burden and biological response.
Vapor transport through the lung is usually modeled as a steady flow process
through a representative cylindrical tube or simply an input parameter to a pharmacokinetic
(PK) model. When the target site in toxicology studies is the lung and
improved PK predictions are desired, a mechanistic model is needed that can predict
regional lung vapor uptake and tissue concentration using a realistic lung
geometry and ventilation pattern. A vapor uptake model in the airways and adjacent
tissues was developed based on the momentum and mass conservation of air
and inhaled material through the airways. Mass transfer coefficients for unsteady
breathing during inhalation, pause, and exhalation were developed to uncouple
transport equations in the air and tissue phases. Vapor transport in the tissue occurred
by diffusion while the absorbed vapor was eliminated by linear and saturable
pathways. The model was used to study the fate of vapors of different solubilities
and reactivities in the tissue. Highly soluble vapors such as formaldehyde were
found to reach only the first few airway generations of the lung while less soluble
vapors such as acetaldehyde penetrated deep into the lung with a significant accumulation
in the lung tissue at the end of a breathing cycle. A lung-tissue vapor uptake
model coupled with a PK model alleviates, or reduces the need for parameter
optimization and enables capturing physical and biological mechanisms of action
to study potential health effects of exposure to vapors.
SOT 2011 ANNUAL MEETING 491