The Toxicologist - Society of Toxicology

corneum thickness, cellular epidermis thickness, number epidermal cell layers, and
dermis thickness). Relative ratios of epidermis and dermis to full thickness measurements
were calculated. The differences in thickness for various Yucatan body
sites is predominantly due to differences in dermis thickness as the dermis makes up
92-98% of full-thickness skin. Rump skin was thinner than back skin for the two
samples analyzed. Female skin was thinner than male skin. Abdomen skin was generally
thinner than the other sites evaluated. The cellular epidermis number of cell
layers varied from 3-7 layers (mean 4.64 layers) and the trichogram from 0-39 hairs
(mean 5.27 hairs) counted per 50.24 sq mm. Data were compared to published
measurements of adult human skin illustrating similarities or differences. These
data support the premise that young adult Yucatan minipig skin thickness is similar
to human skin thickness. These cutaneous measurement data will aid investigators
contemplating a dermal study utilizing the Yucatan miniature swine model.
636 NORMAL PHYSIOLOGICAL RANGES FOR HANFORD
MINIATURE SWINE.
M. Ross, L. Brown, D. Unterreiner, C. Hanks, J. Liu, M. Hodges and G.
Bouchard. Sinclair Research Center, Columbia, MO.
The miniature swine have been increasingly recognized as a non-rodent model in
regulatory toxicity. Members of the FDA have even published on the use of miniature
swine as an alternative to canine and non-human primates in regulatory toxicity.
The similarities between the cardiovascular, renal, and digestive systems make
the miniature swine a suitable animal to model the human counterpart. The miniature
swine are also the most recognized species for dermal toxicology. The Hanford
miniature swine (HMS) has other attractive traits that make them a good substitute
to model humans. They are omnivorous, easy to handle, prone to obesity, and will
develop atherosclerosis and dyslipidemia if fed a high fat diet. With the advent of
new techniques, all routes of compound administration can be used with miniature
swine. The HMS should be considered as one of the non-rodent species in toxicity
testing. In an effort to generate a database on baseline information about the normal
physiological status of the Hanford miniature swine, we report expanded physiological
data from normal intact and naïve juvenile and young adult miniature
swine of both genders. The normal physiological data gathered includes growth parameters,
hematology, serum chemistry, coagulation profile, urinalysis, ECG
rhythm and segment intervals, and organ weights.
637 ROLE OF MAP KINASES IN REGULATING
EXPRESSION OF KERATINOCYTE ANTIOXIDANTS BY
THE ELECTROPHILIC NITRO-FATTY ACIDS
DERIVATIVES 9- AND 10-NITROOLEIC ACID.
R. Zheng 1 , A. T. Black 1 , A. Gow 1 , D. E. Heck 3 , D. L. Laskin 1 and J. D.
Laskin 2 . 1 Rutgers University, Piscataway, NJ, 2 UMDNJ-Robert Wood Johnson
Medical School, Piscataway, NJ and 3 New York Medical College, Valhalla, NY.
ABSTRACT BODY: In many tissues, chemical-induced injury results in the production
of the reactive second messenger nitric oxide. It is well recognized that nitric
oxide and a variety of its byproducts including nitrite can contribute to tissue
damage and form novel signaling mediators via redox mediated nitration reactions.
Nitration of unsaturated fatty acids can generate electrophilic nitro-fatty acid derivatives
such as 9-nitrooleate (9-NO) and 10-nitrooleate (10-NO), which are known
to initiate cell signaling pathways. In the present studies we characterized nitro-fatty
acid-induced signaling in PAM212 mouse keratinocytes. Treatment of the cells
with 5-25 μM 9-NO or 10-NO for 6 hr was found to upregulate mRNA expression
for the primary antioxidants catalase (3-6 fold) and heme oxygenase-1 (HO-1,
50-100 fold), and the secondary antioxidants glutathione-S-transferase (GST)
A1/2 (40-80 fold), GSTA3 (10-70 fold), and GSTA4 (5-15 fold), as determined by
real time PCR. 9-NO also induced phosphorylation of the JNK and p38 MAP kinases
indicating that the nitro-fatty acid activated MAP kinase signaling. Treatment
of keratinocytes with inhibitors of JNK (SP600125, 20 μM) or p38 (SB203580, 10
μM) MAP kinase markedly suppressed 9-NO and 10-NO-induced expression of
heme oxygenase-1 and GSTA4, but not catalase, GSTA1-2 or GSTA3. These data
indicate that the antioxidants are regulated by distinct mechanisms in the cells.
Western blot analysis showed that 9- and 10-NO induced HO-1 protein, a response
markedly reduced by inhibition of JNK, and to a lesser extent by p38 kinase.
Taken together, these data indicate that nitro-fatty acids are important in the
control of antioxidant expression in mouse keratinocytes. Moreover, MAP kinase
signaling plays a key role, at least in part, in regulating responses to the nitro-fatty
acids. Supported by CA132624, ES004738, ES005022, GM034310 and
AR055073.
638 A STUDY OF PHOTOTOXICITY FOLLOWING
INTRAVENOUS ADMINISTRATION OF
CIPROFLOXACIN HYDROCHLORIDE IN THE
MICROMINIPIG.
H. Kawaguchi 1 , N. Miyoshi 1 , A. Tanimoto 1 , Y. Takahashi 2 , S. Utsunomiya 2 , T.
Motokado 2 , Y. Ooshima 2 , H. Izumi 2 , T. Sukamoto 2 and R. Nagata 2 . 1 Kagoshima
University, Kagoshima, Japan and 2 Shin Nippon Biomedical Laboratories (SNBL),
Ltd., Kagoshima, Japan.
Purpose: We previously reported (2008 and 2009 SOT) on in vivo phototoxicity in
Balb/c mice. Recently, the Microminipig (MMP; Fuji Micra Inc.) has emerged as a
possible experimental animal model for non-clinical pharmacological/toxicological
use. The body weight of the young mature MMP is less than 7 kg, enabling ease of
handling. In this study, we evaluated phototoxicity in the MMP following intravenous
administration of ciprofloxacin hydrochloride (CPFX) because little information
is available on in vivo phototoxicity. Method: Five female MMPs aged 5-7
months were used. The backs of the animals were shaved, and divided into four
areas on both the right and left sides before administration. The areas on the right
side were not irradiated. CPFX (a phototoxic substance) was continuously administered
intravenously once at 100 mg/kg. Approximately fifteen minutes after the
start of administration, the four areas on the left side of each animal were irradiated
with long-wave ultraviolet rays (UVA) at approximately 5, 10, 15, or 20 J/cm2 with
an Ultraviolet Irradiation Apparatus (Dermaray, M-DMR-50, Eisai Co., Ltd.).
Skin reactions (erythema and edema) were evaluated in accordance with the Draize
method criteria before administration and at approximately 1, 4, 8, 12, 24, 48, and
72 hours after irradiation. After gross examination at each observation point, the irradiated
sites were prepared for histopathology. Result: At approximately 1 hour
after irradiation, no skin reaction (erythema and edema) was grossly observed at
any irradiation site in any animal. Erythema scored as 1-3 was observed at all irradiated
sites from 12 to 72 hours after irradiation. Edema scored as 1 was observed
at the 15 and 20 J/cm2 UVA irradiation sites from 48 to 72 hours after irradiation.
Positive reactions were clearly observed, from which it was considered possible to
evaluate the in vivo phototoxicity test using the MMP.
639 INFLUENCE OF THE ARYL HYDROCARBON
RECEPTOR ON PROLIFERATION OF HUMAN
KERATINOCYTES (HACAT).
M. Kalmes, J. Hennen, J. Clemens and B. Blömeke. Environmental Toxicology,
University of Trier, Trier, Germany.
While activation of the aryl hydrocarbon receptor (AhR) by exogenous ligands is
well investigated, its physiological function is less understood. By extending research
in AhR biology, evidence appeared that the receptor generally plays an important
role in cell physiology. In keratinocytes, little is known about endogenous
functions of the AhR. In order to expand this knowledge, we analyzed the impact of
AhR knockdown on cell cycle progression in HaCaT cells and showed that proliferation
of siAhR HaCaT cells was significantly decreased. In line with that result,
western blot analysis revealed that protein level of the cyclin dependent kinase inhibitor
p27KIP1 was increased, whereas protein level of the cyclin dependent kinase
(CDK) 2 was reduced. CDK4 and CDK6 protein levels remained unchanged,
whereas protein level of the retinoblastoma protein (pRB) was reduced. By measuring
ethoxyresorufin-O-deethylase (EROD) activity we showed that endogenous cytochrome
P450 is required for normal cell cycle in HaCaT cells, as well. To the best
of our knowledge we provide evidence for the first time in human skin cells that in
the absence of exogenous ligands the AhR promotes cell cycle progression in
HaCaT cells and one can speculate that this is the physiological function of this receptor
in keratinocytes.
640 WHAT IS THE BEST SKIN PREPARATION FOR
DERMAL PENETRATION STUDIES IN VITRO ? A
COMPARISON OF FULL-THICKNESS SKIN AND
DERMATOMED SKIN.
K. Guth 1 , M. Schaefer-Korting 2 , E. Fabian 1 , B. van Ravenzwaay 1 and R.
Landsiedel 1 . 1 BASF SE, Experimental Toxicology and Ecology, Ludwigshafen,
Germany and 2 Freie Universitaet, Berlin, Germany.
Skin Absorption in vitro is an alternative method accepted by the OECD. The respective
guidelines (TG 428 and GD 28) give technical guidance how to perform
valid experiments. Within these guidelines many experimental parameters can be
varied, with potential influence on the study results. We investigated the influence
of skin preparations on dermal penetration and measured dermal penetration with
full-thickness skin (FTS) and dermatomed skin (DMS) from the same human
SOT 2011 ANNUAL MEETING 137