The Toxicologist - Society of Toxicology

global parameter sensitivity analysis (GSA) on various model parameters and studied
their impact on several key metabolites such as ATP, mitochondrial membrane
potential, GSH, NADH, Acetyl CoA, Malonyl-CoA, Cholesterol, DG and TG.
Model parameters are varied by 10%, and the changes in metabolite profiles are
quantified. This analysis allows parameters to be ranked in order of their impact on
liver metabolism.
We mimicked drug-induced perturbations on the rat liver by changing
the kinetic parameters of sensitive enzymatic reactions either singly or in combination
and obtained metabolite profiles. An optimization problem was formulated to
estimate the values of parameters that were potentially responsible for this metabolite
profile. An objective function was defined to minimize the error between predicted
and observed metabolite profiles of key metabolites described above. The estimated
values of the parameters were then compared with the values used in the
simulations and found to match well. This provides a mechanistic basis for relating
metabolite changes to specific pathways being deranged by a drug. This approach
works well for both steady state and time-varying metabolite profiles.
736 DEVELOPMENT OF A PHYSIOLOGICALLY BASED
PHARMACOKINETIC (PBPK) MODEL FOR RDX
(HEXAHYDRO-1, 3, 5-TRINITRO-1, 3, 5-TRIAZINE) IN
FEMALE B6C3F1 MICE.
M. L. Gargas 1 , C. P. Gut 1 , M. R. Okolica 1 , A. Ntamack 1 , L. M. Sweeney 1 , L.
R. Williams 2 , G. Reddy 2 and M. S. Johnson 2 . 1 Naval Medical Research Unit -
Dayton, Wright-Patterson AFB, OH and 2 U.S. Army Public Health Command,
Aberdeen Proving Ground, MD.
RDX is an explosive used in military applications and has been detected in ground
water surrounding US military installations and at manufacturing facilities. The
current cancer slope factor was estimated from the incidence of liver neoplasms in
female mice (no cancers were found in either sex of rats chronically exposed to
RDX) and is based on outdated animal to human scaling techniques and mouse
tumor data that has subsequently been revised using more current pathological criteria.
In addition, dose-response was not evaluated using Benchmark Dose (BMD)
approaches. There is a need to derive an updated cancer potency factor for RDX
using more current data and techniques, including the use of internal dose and
BMD modeling. The purpose of the work reported here was to finalize the development
of a PBPK model for RDX kinetics in the female mouse. An initial PBPK
model structure for female mice was constructed using an existing model for rats
and existing data on tissue partition coefficients. The rate of RDX metabolism for
female mice was determined experimentally in our laboratory using hepatic microsomal
preparations and the resulting rate constant was incorporated as a PBPK
model parameter. Model verification was performed by comparing model predictions
to blood and liver concentration time-course data collected in our laboratory
from female mice orally dosed with RDX at target doses of 1.5, 7, or 35 mg/kg. The
cancer potency of RDX in humans will be determined in future evaluations using
the female mouse PBPK model developed in this current work together with a
PBPK model for RDX kinetics in humans.
737 BIOMARKERS OF EXPOSURE VERSUS PBPK MODEL
ESTIMATES OF INTERNAL DOSE: A CASE STUDY
WITH ACRYLAMIDE.
R. S. DeWoskin. U.S. EPA, Research Triangle Park, NC .
Estimates of internal dose greatly improve the confidence in risk assessments for
systemic toxicants when extrapolating dose-response relationships across species,
routes of exposure, subpopulations, or study conditions (e.g., in vitro to in vivo).
Two leading methods to estimate internal dose are: 1) biomarker of exposure models,
and 2) physiologically based pharmacokinetic (PBPK) models. Hemoglobin
(Hb) adducts are good biomarkers of exposure to acrylamide (AA) and its mutagenic
metabolite, glycidamide (GA). The U.S. EPA posted an IRIS assessment for
acrylamide that estimated internal blood levels in rats and humans with an AA-Hb
and GA-Hb adduct model. Concurrent with the posting of the IRIS assessment, an
updated AA PBPK model was published providing an opportunity to compare results,
and to evaluate the advantages each method has for specific applications. The
Hb-adduct model and the PBPK model gave similar results for the ratio of the rat
external dose (mg/kg/day) to the equivalent human external dose of AA (i.e., the
dose that would produce comparable internal levels of AA and GA in blood).
External dose ratios were in the 4.5-6.5 range for comparable levels of AA in blood,
and in the 0.75-1.0 range for GA in blood. The ratio for AA in blood is similar to
the value from a body weight scaling (BW 2/3 or 3/4), while the lower ratio for
comparable GA internal levels reflects the considerably different production and
clearance of GA in humans compared with rats (i.e., BW scaling would grossly
overestimate this ratio). The Hb-adduct model only estimates internal dose of AA
or GA in blood. Thus, when blood levels suffice as a surrogate dose metric for an
adverse effect, the simpler seven parameter Hb-adduct model is preferred (i.e.,
based on AIC) to the nine compartment, 68 parameter PBPK model. The PBPK
model, however, estimates a variety of dose metrics (e.g., unbound AA or GA in
blood and tissues), and provides more functionality for extrapolating risks and hypothesis
testing. [The views expressed are those of the authors and do not necessarily
reflect the views or policies of the U.S. EPA]
738 IN VITRO 3, 5-DICHLOROANILINE NEPHROTOXICITY
IN FRESHLY ISOLATED RAT RENAL CORTICAL CELLS.
C. Smurthwaite, S. Baksi, T. Ferguson, C. Schuetz, D. Anestis and G. O. Rankin.
Pharmacology, Physiology, and Toxicology, Marshall University, Huntington, WV.
Chloroanilines are widely used in manufacturing dyes, drugs, agricultural chemicals
and industrial intermediates. Previously, we demonstrated that 3,5-dichloroaniline
(3,5-DCA) induced nephrotoxicity in vivo in rats and in vitro in a rat renal cortical
slice model. In this study, freshly isolated renal cortical cells (IRCC) obtained from
male Fischer 344 rats were used to investigate the nephrotoxic potential of 3,5-
DCA and to examine if metabolites and/or oxidative stress contributed to 3,5-
DCA-induced nephrotoxicity in vitro. Nephrotoxicity was determined by incubating
IRCC (~4 million cells/ml; 3 ml total volume) with vehicle (dimethyl sulfoxide,
DMSO; 30 microL) or 3,5-DCA (0 - 1.5 mM) for 60, 90, or 120 minutes and calculating
lactate dehyrogenase (LDH) release as a percent of total LDH. In some experiments,
IRCC were pretreated with antioxidants (2.0 mM ascorbate, 1.0 mM
glutathione, 1.0 mM alpha-tocopherol, or 2.0 mM N-acetyl-L-cysteine) to explore
the role of oxidative stress or pretreated with a cyclooxygenase inhibitor (1.0 mM
indomethacin), CYP inhibitor (1.0 mM metyrapone or 1.0 mM piperonyl butoxide)
or FMO inhibitor (1.0 mM methimazole or 2.0 mM n-octylamine) to explore
the role of metabolites in 3,5-DCA nephrotoxicity. 3,5-DCA nephrotoxicity was
evident at 120 min at 0.5 mM 3,5-DCA and by 60 min with higher 3,5-DCA concentrations.
All pretreatments reduced 3,5-DCA toxicity to varying degrees. These
results suggest that 3,5-DCA is nephrotoxic to IRCC in a time and concentration
dependent manner, metabolites contribute to 3,5-DCA nephrotoxicity, and oxidative
stress may be a mechanism for inducing 3,5-DCA nephrotoxicity. This work
was supported by NIH grant 3P20RR016477-09S1.
739 IN VITRO ANALYSIS OF CYCLOSPORINE A-INDUCED
NEPHROTOXICITY BY GENE EXPRESSION AND 1 H-
NMR-METABONOMICS OF CELL LYSATES.
K. Herrgen 1 , O. Schmal 1 , C. J. Burek 1 , M. Gruene 2 , P. Jennings 3 and W.
Dekant 1 . 1 Department of Toxicology, University of Wuerzburg, Wuerzburg, Germany,
2 Department of Organic Chemistry, University of Wuerzburg, Wuerzburg, Germany
and 3 Department of Physiology and Medical Physics, Medical University of Innsbruck,
Innsbruck, Austria.
Early detection of nephrotoxic side effects of drug candidates combined with reduction
of animal testings is still a challenge for preclinical drug development.
Since some renal biomarker candidates, like KIM-1 and LCN-2 (NGAL) performed
well in vivo, the scope of this study was to test their performance in vitro in
human proximal tubular epithelial cell lines. To gain additional information, the
intracellular metabolite patterns were tracked by 1 H-NMR-Metabonomics and
compared to the gene expression data. In the present study, Cyclosporine A (CsA)
was used as a model compound for drug induced nephrotoxicity. Differentiated
RPTEC/TERT1 and A498 cells were used as cell models and drug treatment was
performed up to 6 days by repeated dosing.
A multivariate data analysis of the hydrophilic cell lysates examined by 1 H-NMR
revealed a clear discrimination between cytotoxic and non-cytotoxic dose groups.
The corresponding metabolites are linked to energy consumption as well as alterations
of renal osmolytes. This can be interpreted as cellular dedifferentiation, a
known effect of Cyclosporine A. Regarding the analyzed biomarker genes, only
LCN-2 and GADD153 showed an upregulation. Other markers, like KIM-1 or
Clusterin, did not show consistent alterations in this experimental set up. The increased
expression of the mechanistic marker GADD153 confirms decompensated
ER stress as an early event in proximal tubular cell damage caused by CsA.
Effective CsA concentrations were monitored by LC-MS and revealed a bound part
of about 20% of the nominal concentration along with varying intracellular concentrations.
This highlights the importance of concentration measurements for in
vitro testings.
SOT 2011 ANNUAL MEETING 159