The Toxicologist - Society of Toxicology

especially with anti-TNF therapies. Preclinical models of active tuberculosis infection
in the mouse, rabbit and non-human primate provide unique opportunities to
understand disease pathogenesis and assess risk in the development of new immunomodulatory
agents. In some cases, these models recapitulate human tuberculosis
pathogenesis and also can provide insight into the reactivation potential of
LTBI, before larger populations of at risk patients are exposed in clinical trials. Each
model will be presented and discussion of their use during clinical development will
be introduced.
2057 THE USE OF NONCLINICAL ASSESSMENT
STRATEGIES IN THE PREDICTION OF CLINICAL
RISKS OF IMMUNOMODULATORY MOLECULES:
CASE STUDY FOR ABATACEPT, A SELECTIVE CO-
STIMULATION MODULATOR.
H. G. Haggerty. Drug Safety Evaluation, Bristol-Myers Squibb, Syracuse, NY.
While immunosuppressant molecules may protect against the adverse consequences
of their diseases or conditions, they also have the potential to affect how
the body protects itself against infection and oncogenesis by impairing immunosurveillance.
There are a number of assessment strategies that can be undertaken to understand
the effect of such molecules on immunocompetence, including host-resistance
and antigen-response models. Selection of appropriate assessment strategies
must include an understanding of the mechanism of immunomodulation of the
test molecule and the intended patient population for its therapeutic use, allowing
for the prudent use of models that may provide the strongest relevance to identifying
potential risks in patients. This presentation will share some of the approaches
used to evaluate the potential immunotoxicities of one novel immunotherapeutic
protein and how those findings translated to the clinic. Abatacept selectively modulates
T-cell activation by modulating the interaction of CD28 on T-cells with
CD80/86 on antigen presenting cells and is marketed for treatment of rheumatoid
arthritis. The safety of abatacept has been well characterized in a comprehensive
drug-safety evaluation program. Based on an understanding of the pharamacology
of abatacept and how it impacts the various component of the immune system, potential
safety issues were identified and special endpoints that evaluated the immune
system and its potential for adverse consequences included into nonclinical
studies. In addition, the effect of abatacept or murine CTLA4Ig on the host immune
response to different infectious agents including, Pneumocystis carnii;
murine cytomegalovirus; herpes simplex virus; and tuberculosis were evaluated.
Although these types of infections are uncommon, or rare, the clinical data appear
to correlate with the host resistance models. Thus, by understanding the mechanism
of action and intended patient population, the preclinical models evaluated
were predictive of clinical outcomes.
2058 CYTOCHROME P4501A1 (CYP1A1) IS REQUIRED TO
MEDIATE VASCULAR DYSFUNCTION, REACTIVE
OXYGEN SPECIES, AND HYPERTENSION INDUCED
BY 2, 3, 7, 8-TETRACHLORODIBENZO-P-DIOXIN
(TCDD).
P. G. Kopf, L. N. Agbor, J. Scott and M. K. Walker. Pharmaceutical Sciences,
University of New Mexico, Albuquerque, NM.
High dietary fat intake increases the risk of hypertension. Dietary fat intake also
represents the major route of exposure to halogenated aromatic hydrocarbon pollutants,
which include halogenated dibenzo-p-dioxins, dibenzofurans, and biphenyls.
Data from the National Health and Nutrition Examination Survey (NHANES)
link exposure to these pollutants to hypertension in the general U.S. population.
Further, the link with hypertension remains even after controlling for age and body
mass index. The halogenated aromatic hydrocarbons that are associated with hypertension
activate the aryl hydrocarbon receptor (AHR). Nevertheless, the mechanism
linking these environmental pollutants to hypertension is unknown. We
tested the hypothesis that exposure to TCDD induces reactive oxygen species, vascular
dysfunction, and hypertension via a mechanism requiring cytochrome
P4501A1 (CYP1A1). To test this CYP1A1 wildtype (WT) and knockout (KO)
mice were exposed to dietary TCDD for 35 d (150 ng/kg/d). Mean arterial pressure
(MAP) was monitored by radiotelemetry prior to and during exposure.
CYP1A1 was measured in kidney, heart, aorta, and mesenteric arterioles and lucigenin
luminescence was measured in the heart, kidney, and aorta as an index of superoxide
anion production. We found that TCDD induced CYP1A1 and lucigenin
luminescence in all tissues of WT mice, but not in KOs. In addition, TCDD
steadily increased MAP after 15 d of exposure in CYP1A1 WT mice, reaching hypertension
at +20 mm Hg, but did not alter MAP in KO mice. TCDD also induced
endothelial dysfunction in CYP1A1 WT mice, which was normalized by a
superoxide dismutase mimetic, but had no effect in KO mice. These data demonstrate
that TCDD-induced reactive oxygen species, endothelial dysfunction, and
hypertension require CYP1A1 and suggest that production of reactive oxygen
species may play a role. Supported by R01 HL078914
2059 TCDD TREATMENT INDUCES HBD-3 EXPRESSION IN
HUMAN KERATINOCYTES IN VITRO AND IN ORGAN
CULTURE OF HUMAN SKIN BIOPSIES.
L. Allen-Hoffmann 1, 2 , N. De Abrew 2 and J. Loertscher 2 . 1 Pathology and
Laboratory Medicine, University of Wisconsin - Madison, Madison, WI and
2
Molecular and Environmental Toxicology Center, University of Wisconsin - Madison,
Madison, WI.
TCDD, a ubiquitous and potent environmental contaminant, has been shown to
cause the human skin pathology chloracne. Unlike skin conditions such as acne
vulgaris, chloracne patients exhibit reduced microbial growth in affected areas of
skin. Using an in vitro model, we examined the antimicrobial properties of human
skin following continuous TCDD treatment. TCDD treatment increased the expression
of the β-defensin class of the antimicrobial host defense peptides. Human
β defensin-1 (hBD-1) and human β defensin-3 (hBD-3) mRNA levels were found
to be upregulated by 12 days of treatment. The induction of hBD-3 mRNA and
the concomitant increase in hBD-3 protein was confirmed in adult human skin
biopsies treated with TCDD for 18 days. In monolayer culture, exposure of human
keratinocytes to TCDD resulted in increased hBD-3 mRNA and protein levels.
These results demonstrate that keratinocytes in the absence of mesenchymal cell
types, such as the fibroblasts resident to the dermal compartment, are responsible
for the TCDD-induced HDP effects. In additional studies, treatment of in vitro
skin models with TCDD congeners demonstrated a parallel structure-activity relationship
between the affinity of these chemicals to the aryl hydrocarbon receptor
(AhR) and both increased hBD-3 mRNA levels and hyperkeratinization. These results
provide indirect evidence for the AhR / AhR Nuclear Translocator (ARNT)
pathway in the induction of hBD expression. Cutaneous barrier function of skin
tissues, as measured by electrical impedance-based capacitance, was not reduced by
18 days of TCDD-treatment suggesting that the permeability of skin tissue was not
affected by treatment. Taken together, these results suggest that TCDD enhances
the innate antimicrobial function of human skin through the activation of β-defensins,
providing an explanation for the reduced levels of microbial growth frequently
seen in patients with the skin condition chloracne.
2060 TCDD INDUCES DERMAL ACCUMULATION OF
KERATINOCYTE-DERIVED MATRIX
METALLOPROTEINASE-10 IN A THREE
DIMENSIONAL MODEL OF HUMAN SKIN.
C. Thomas-Virnig 1 , N. De Abrew 2 , C. Rasmussen 1 , E. Bolterstein 2 , S.
Schlosser 1 and L. Allen-Hoffmann 1, 2 . 1 Pathology and Laboratory Medicine,
University of Wisconsin Madison, Madison, WI and 2 Molecular and Environmental
Toxicology Center, University of Wisconsin Madison, Madison, WI.
The epidermis of the skin is the first line of defense against the environment. An
organotypic model of human skin was used to investigate tissue-specific phenotypes
induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD). Continuous treatment of an in vitro model of human skin with TCDD
resulted in intracellular spaces between keratinocytes of the basal and immediately
suprabasal layers as well as thinning of the basement membrane, in addition to the
previously reported hyperkeratinization. These tissue remodeling events were preceded
temporally by changes in expression of the extracellular matrix degrading enzyme,
matrix metalloproteinase-10 (MMP-10). MMP-10 mRNA and protein were
highly induced following TCDD treatment. qPCR and immunoblot results from
TCDD-treated monolayer cultures, as well as indirect immunofluorescence and
immunoblot analysis of TCDD-treated skin tissues, showed MMP-10 was specifically
contributed by the epidermal keratinocytes but not the dermal fibroblasts.
Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment
of the skin model. TCDD-induced epidermal phenotypes were attenuated
by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-
1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and
possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited
in the basement membrane, the basal keratinocyte layer, and the cornified layer
of TCDD-treated skin tissues. Our studies reveal a novel mechanism by which the
epithelial-stromal microenvironment is altered in a tissue-specific manner thereby
inducing structural and functional pathology in the interfollicular epidermis of
human skin.
SOT 2010 ANNUAL MEETING 437