The Toxicologist - Society of Toxicology

(ABC) transporters actively transport xenobiotic compounds in vivo, but are also
involved in multidrug resistance of tumors. The study of these transporters is relevant
to the development of chemotherapeutic agents. Here we report the successful
creation of the suite of ABC transporters knockout models Mdr1a, Mrp1, Mrp2,
and Bcrp using ZFN technology. Homozygous knockout animals were confirmed
by the lack of respective proteins in Western Blots. Mrp2 homozygous animals are
obviously jaundice after birth, a phenotype seen in TR- rats and patients with the
Dubin-Johnson syndrome, all of which have mutations that disrupt the Mrp2 gene.
All models are being tested in pharmacokinetics studies.
536 PATHOLOGY OF A BROWN-NORWAY RAT MODEL OF
AMODIAQUINE-INDUCED LIVER INJURY.
H. C. Workman 1 , J. Li 2 , P. Cai 3 , M. Odin 1 , M. David 1 and J. Uetrecht 3, 4 .
1 Non-Clinical Safety, Hoffmann-La Roche, Nutley, NJ, 2 Immunotoxicology, Bristol-
Myers Squibb, New Brunswick, NJ, 3 Pharmacy, University of Toronto, Toronto, ON,
Canada and 4 Medicine, University of Toronto, Toronto, ON, Canada.
Rationale Amodiaquine (AQ), an anti-malarial drug still available in some countries,
is reported to cause idiosyncratic drug induced liver injury (I-DILI) in people.
Lesions in people are poorly described; findings usually include mononuclear cell
infiltration, Kupffer cell activation (occasionally containing cytoplasmic pigment),
hepatocellular necrosis, bile duct hyperplasia and fibrosis. The underlying mechanism
causing the pathology of DILI is not well understood nor is an animal model
for this type of DILI currently available. We used the BN rat, prone to develop immune-type
response, to help further characterize the pathology of this disease.
Experimental procedures Male Brown-Norway (BN) rats were treated with AQ at
62.5 mg/kg/day PO for 42 days and a second study for 49 days was repeated.
Histopathology included hematoxylin and eosin (H&E), immunohistochemistry
and special histochemical stained sections of a complete tissue set. Results Liver
findings included scattered individual Kupffer cell increase and activation as well as
small microgranulomas of activated macrophages admixed with few lymphocytes.
Large aggregates of macrophages with iron positive intracytoplasmic pigment
showed a bridging pattern, primarily around portal tracts and occasionally random.
Other findings included (scattered) single hepatocellular necrosis/apoptosis and
bile duct hyperplasia. Spleen findings included macrophage activation in the red
pulp, follicular hyperplasia and variable depletion of the marginal zone in the white
pulp. Other findings were those reported with the BN rat strain. Conclusions The
pathology reported in this AQ BN rat model exhibited similarities with that reported
in AQ-induced liver injury in patients. Additional work is needed to further
characterize this model and understand the immunological mechanisms therein.
537 ALTERED BEHAVIOR, BODY WEIGHT, AND BRAIN
CHEMISTRY IN PAK5-/-, PAK6 -/-AND PAK5-/-/PAK6-/-
MICE.
M. A. Furnari 1 , M. L. Jobes 1 , T. Nekrasova 2 , A. Minden 2 and G. C. Wagner 1, 3 .
1 Joint Graduate Program in Toxicology, Rutgers University, Piscataway, NJ, 2 Susan
Lehman Cullman Laboratory for Cancer Research, Rutgers University, Piscataway, NJ
and 3 Psychology, Rutgers University, Piscataway, NJ.
PAK5 and PAK6 are protein kinases highly expressed in brain. We observed that
PAK5 and PAK6 single knockout mice and PAK5/PAK6 double-knockout
mice(DKO) displayed altered behavior and that PAK6 single knockout mice gained
significantly more weight than other genotypes, including wildtypes(WT). Based
on this work, further investigation of these genotypes was done. Mice of each genotype
were given free access to run wheel exercise or placed in cages without run
wheels for a total of 74 days. Body weight, fluid and food consumption were measured
weekly. PAK5 and PAK6 single knockouts, DKOs, and WT mice were treated
with a single dose of amphetamine (50mg/kg) and sacrificed 72 hours later. The
striatum was dissected and assayed for dopamine(DA), serotonin(5-HT) and their
metabolites dihydryoxyphenylacetic acid(DOPAC), homovanillic acid(HVA), and
5-hydroxyindoleacetic acid(5-HIAA) by high pressure liquid
chromatography(HPLC). While run wheel mice consumed significantly more
food, they weighed less than non-run wheel mice. In addition, although PAK6
knockout mice consumed the same amount of food as wildtype mice, they were significantly
heavier regardless of run wheel condition. These data suggest that PAK6
may be involved in the regulation of body fat deposition. Dopamine significantly
lower in all genotypes that received amphetamine while DOPAC, 5-HT, HVA, and
5-HIAA were not different from saline treated controls. There was no significant
difference in any neurotransmitter or metabolite between saline- and amphetamine-treated
groups in WT, PAK5 knockouts and DKs. The PAK6 knockout mice
treated with amphetamine did show a significant decrease in dopamine compared
to controls. These data suggest that PAK6 may be involved in the regulation of
body fat deposition and that the PAK6 mice have an increased sensitivity to the
neurotoxic effects of amphetamine. Supported in part by ES005022
&T32ES007148.
538 THE TOXICITY OF METHIMAZOLE IN THE MOUSE
OLFACTORY MUCOSA IS AT LEAST PARTLY
MEDIATED THROUGH TARGET-TISSUE METABOLIC
ACTIVATION BY CYP2A5.
J. Gu 1 , F. Xie 1 , X. Zhou 1 , M. Genter 2 and X. Ding 1 . 1 Wadsworth Center, New
York State Department of Health, Albany, NY and 2 Department of Environmental
Health, University of Cincinnati, Cincinnati, OH.
The anti-thyroid drug methimazole (MMZ) can cause severe, tissue-specific toxicity
in mouse olfactory mucosa (OM). The toxicity of MMZ is mediated through its
reactive intermediates (sulfenic and sulfinic acids), formed presumably by a sequential
activation of MMZ, by P450 and flavin monooxygenases (FMO). The specific
P450 enzymes responsible for MMZ metabolic activation have not been identified,
and it is unclear whether target-tissue metabolic activation is essential for toxicity.
The aims of the present study were to determine whether CYP2A5, one of the most
abundant P450 enzymes in the mouse OM, is involved in MMZ metabolic activation,
by comparing Cyp2a5-null with wild-type (WT) mice, and whether hepatic
microsomal P450 enzymes, including CYP2A5, are essential for MMZ-induced
OM toxicity, by comparing liver-Cpr-null (LCN) mice, which have little P450 activity
in hepatocytes, with WT mice. Our results showed that the loss of CYP2A5
expression did not alter systemic clearance of MMZ (at 50 mg/kg, i.p.); but it did
significantly decrease the rates of MMZ metabolism in the OM, while olfactory
FMO expression was not decreased. MMZ induced depletion of non-protein thiols,
as well as pathological changes, in the OM of WT mice; the extent of these
changes was much reduced in the Cyp2a5-null mice. Thus, CYP2A5 plays an important
role in mediating MMZ toxicity in the OM. In contrast, the rate of systemic
clearance of MMZ was significantly reduced in the LCN mice, compared to
WT mice, whereas the MMZ-induced OM toxicity was not prevented. Therefore,
hepatic P450 enzymes are essential for systemic MMZ clearance, but they are not
required for MMZ-induced OM toxicity. We conclude that the tissue-specific toxicity
of MMZ is mediated by target tissue metabolic activation, and the reaction is
at least partly catalyzed by CYP2A5 in the OM. (Supported in part by NIH grant
ES007462)
539 MODELS OF ACUTE LUNG INFLAMMATION IN NON
HUMAN PRIMATES: LPS, MECHANICAL
VENTILATION, PSEUDOMONAS AERUGINOSA, AND
CIGARETTE SMOKE.
R. W. Spindle, M. Doyle-Eisele, O. Denise, J. Tipper, K. Harrod, R. Jaramillo
and J. McDonald. LRRI, Albuquerque, NM.
Non-human primates (NHP) may serve as an important translational research tool
for the evaluation of mechanisms of disease pathogenesis and efficacy of new therapeutics.
We have defined the pathogenesis of several acute lung injury models in
NHPs, including inhaled lipopolysaccharides (LPS), instilled Pseudomonas
Aeruginosa (P. Aeruginosa), inhaled cigarette smoke (CS) and mechanical ventilation.
For each animal model, inflammation is assessed in bronchoalveolar lavage
(BAL) collected prior to the initial challenge and prior to necropsy. For LPS, animals
received a single inhalation exposure (5 mg/m3) by face mask 24 hrs prior to
necropsy. Significant increases in neutrophils, IL5, IL8 and TNFα were observed in
BAL compared to control animals. This inflammation was reduced by treatment
with inhaled budesonide (0.7 mg/kg). To evaluate the ability to potentiate the LPS
injury to represent an acute lung injury model, the same LPS dose was administered
to animals approximately 24 hours prior to being placed on an Intensive care
unit (ICU) ventilator. Animals were held at high-peak pressure ventilation for 3-6
hours under systemic anesthesia. Inflammation and protein measured in BAL was
elevated immediately post ventilation compared to the control. These results were
greater than ventilation or LPS alone. For P. Aeruginosa, animals were instilled with
10^9 colony forming units or saline on Day 0 and were observed out through Day
4. Neutrophil infiltration and pneumonitis was observed compared to control animals.
Inflammation in NHPs exposed to CS was confirmed after exposure for up to
4 weeks of exposure. Each of these models identifies new tools for the assessment of
acute inflammation in NHPs.
540 EVALUATING THE CELLULAR CHANGES IN
BLEOMYCIN-INDUCED PULMONARY FIBROSIS
MODELS: IT VS INHALATION.
M. Doyle-Eisele, R. W. Spindle, A. Gigliotti and J. D. McDonald. LRRI,
Albuquerque, NM.
Intratracheal (IT) bleomycin sulfate (BS) is one of the most commonly used animal
models for pulmonary fibrosis (PF). This model regularly causes patchy fibrosis in
the lung. While IT models do not elucidate the human condition, they are representative
of many of the clinical and subclinical PF characteristics. Inhalation
SOT 2011 ANNUAL MEETING 115