The Toxicologist - Society of Toxicology

logically & morphologically similar to that of human hair cells, along with our
studies, indicate that megalin is likely located in/near hair cells of the Zfish lateral
line. Zfish exposure to AG & cisplatin resulted in a loss of fluorescently-labeled hair
cells, corresponding to cell death. Furthermore, concomitant application of known
megalin binding agents led to partial protection of drug-induced Zfish hair cell
death. The purpose of this study is to determine whether megalin is located in/near
Zfish hair cells as well as further investigate effectiveness of protective agents in preventing
hair cell death & loss of fluorescence. Zfish at 5 days post fertilization (dpf)
labeled with anti-megalin antibody showed patterns closely resembling neuromast
structures seen with fluorescent labeling. These results show megalin is located in
hair cell neuromast structures along the lateral line of Zfish. Zfish at 4 dpf were
treated with polymyxin B, polymyxin B nonapeptide or gentamicin +/- poly-L-asparagine,
labeled with the fluorescent dye YoPro1 & imaged using fluorescence microscopy
to look for a loss of hair cell fluorescence vs. controls. Only partial protection
was seen with pretreatment of megalin binding agent. Nephrotoxic
cephalosporins that do not go through megalin were also tested & no loss of fluorescence
was seen up to 100 μM, further supporting the conclusion that megalin
plays a key role in the mechanism through which nephrotoxic drugs illicit an ototoxic
effect in Zfish. Drugs that bind to megalin could conceivably be screened in
Zfish using hair cell toxicity as a potential predictor for nephro- and ototoxicity in
mammals.
1747 PERSISTENT LIFE HISTORY EFFECTS OF ATRAZINE
EXPOSURE IN ZEBRAFISH: A MULTI-GENERATIONAL
STUDY ENCOMPASSING GENE EXPRESSION,
FITNESS, AND EPIGENETIC EFFECTS.
S. Lewis1 , G. J. Weber2 , S. M. Peterson2 , J. L. Freeman2 and M. S. Sepulveda1 .
1Forestry and Natural Resources, Purdue University, West Lafayette, IN and 2School of
Health Sciences, Purdue University, West Lafayette, IN.
Atrazine is one of the most widely used herbicides in the United States because it is
highly effective against a wide spectrum of broadleaf and grass weeds and because it
is relatively inexpensive. At present, the maximum contaminant level goals
(MCLG) for atrazine, set by the U.S. EPA, is 3 ppb (parts per billion) for drinking
water. The safety of atrazine exposure is currently being re-evaluated by the EPA
due to recent studies linking exposure with cancer and endocrine disruption.
Therefore, we are studying the lasting effects of a one-time developmental atrazine
exposure in later life stages of the exposed individuals and in their offspring by evaluating
differential gene expression, fitness, survival of offspring and hatching success.
In a previous study using microarray technology, an exposure of embryonic zebrafish
through 72 hours post fertilization (hpf) to 0.3 ppb, 3 ppb, or 30 ppb
atrazine resulted in differentially expressed gene lists enriched with genes associated
with neuroendocrine development, cell proliferation, and cancer development.
From these gene set lists, a subset of candidate genes were chosen as genetic biomarkers
for neuroendocrine functions and gene expression alterations confirmed
with quantitative real-time PCR (qPCR) in the 72 hpf larvae. Lasting effects on
gene expression of these same genes were also measured by qPCR in 33-day old juveniles
and in sexually mature adults. Additionally, the effect of atrazine on sex ratio
in the exposed generation (F0) and on the number of offspring and hatching success
of the F1 was also measured. No alteration in sex ratio was observed, but the
lowest atrazine treatment of 0.3 ppb resulted in decreased survivorship in the F1
generation. No effects on survivorship or hatching rate were observed in the offspring
of the fish exposed to 3 ppb and 30 ppb atrazine.
1748 MOLECULAR EVALUATION OF RABBIT CORNEAL
OCULAR SULFUR MUSTARD INJURY: TOWARDS
MECHANISMS OF PERSISTENT INJURY.
D. Milhorn 2 , P. McNutt 1 , T. Hamilton 1 and R. Lawrence 1 . 1 USAMRICD,
Aberdeen Proving Ground- Edgewood Area, MD and 2 Medical Research and Materiel
Command, Fort Detrick, MD. Sponsor: G. Rockwood.
Sulfur mustard (SM, bis-(2-chloroethyl) sulfide) is a highly reactive, bifunctional
alkylating agent that covalently modifies DNA, proteins, and other macromolecules.
Ocular SM exposure results in a severe acute injury involving inflammation
and corneal erosions that rapidly resolve. Many victims subsequently undergo a delayed
injury that severely compromises eyesight, characterized by corneal erosions,
persistent edema and neovascularization. The etiology of these recurrent keratopathies
is unknown. Diverse therapeutic approaches have failed to mitigate the
delayed injury, suggesting that researchers need an improved understanding of the
mechanisms underlying injury progression. We have developed a novel rabbit
corneal vapor exposure model that exhibits acute and long-term sequelae commensurate
with human clinical reports and characterized consistent and reproducible
metrics of injury progression. Exposure to SM results in sloughing of corneal epithelium
and stromal cell necrosis within 24 hr. The corneal lesion is re-epithelialized
within 96 hr, and there is minimal evidence of injury by 1-2 wk. However,
75% of animals undergo a recurrent injury between 3-5 wk that is progressive and
more severe than the acute injury, characterized by neovascularization, persistent
immune cell infiltration, significant corneal edema and recurrent corneal erosions.
For the first time we present correlations between ultrastructural and clinical aspects
of the acute and recurrent injuries from 0-5 wk, correlating architectural
changes in the corneal epithelium, basement membrane and stroma with the onset
of recurrent sequelae. We evaluate these results in terms of injury mechanism(s) and
novel, evidence-based therapeutic approaches as well as propose future research directions
to identify the contribution of specific corneal tissues to SM injury.
1749 INDUCTION OF AUTOPHAGY IN A MOUSE
KERATINOCYTE CONSTRUCT MODEL BY THE
VESICANT 2-CHLOROETHYL ETHYL SULFIDE;
REGULATION BY MAP KINASES AND CAVEOLAE.
A. T. Black 1 , R. P. Casillas 2 , D. E. Heck 3 , D. R. Gerecke 1 , D. L. Laskin 1 and J.
D. Laskin 4 . 1 Rutgers University, Piscataway, NJ, 2 Battelle Biomedical Research
Center, Columbus, OH, 3 New York Medical College, Valhalla, NY and 4 UMDNJ-
RWJ Medical School, Piscataway, NJ.
Sulfur mustard is a potent vesicant that causes inflammation, edema and blistering
in the skin. These effects are associated with basal keratinocyte damage and cytotoxicity.
Autophagy is a membrane-trafficking mechanism for protein and cytoplasmic
organelle degradation and is enhanced in stressed cells. In the present studies,
we analyzed the effects of the half mustard 2-chloroethyl ethyl sulfide (CEES) on
the autophagic process in keratinocytes using a skin construct model in which cells
are grown at an air-surface interface. CEES (100-1000 μM) caused a concentration-dependent
increase in autophagy in keratinocytes as detected by flow cytometry
in conjunction with acridine orange staining. CEES treatment also upregulated
gene and protein expression of LC3B, beclin-1, and p62, proteins known to participate
in autophagy, as well as caveolin-1, the primary structural protein of caveolae.
Isolation of cytosolic and caveolar fractions from keratinocytes showed that LC3B
and p62 were specifically localized within caveolae, while beclin-1 was found primarily
in the cytosol of control cells, and in both cytosolic and caveolar fractions of
CEES-treated cells. Disruption of caveolae using filipin (20 μM) or methyl-β-cyclodextrin
(5 mM) markedly suppressed expression of LC3B, beclin-1 and p62 in
CEES-treated keratinocytes. CEES also activated the JNK and p38 MAP kinase
signaling pathways in keratinocytes. Treatment of the cells with inhibitors of JNK
(SP600125, 20 μM) or p38 (SB203580, 10 μM) markedly suppressed expression
of LC3B, beclin-1 and p62, as well as caveolin-1. Taken together, these data indicate
that caveolae play a key role in CEES-induced autophagy in keratinocytes and
that this process is regulated JNK and p38 MAP kinases. Supported by CA093798,
ES004738, ES005022, GM034310 and AR055073.
1750 ROLE OF CAVEOLAE IN REGULATING EXPRESSION
OF HSP27 AND HSP70 FOLLOWING EXPOSURE OF
HUMAN AND MOUSE SKIN MODELS TO THE
VESICANT 2-CHLOROETHYL ETHYL SULFIDE.
J. D. Laskin 1 , A. T. Black 2 , P. J. Hayden 3 , R. P. Casillas 4 , D. E. Heck 5 , D. R.
Gerecke 2 and D. L. Laskin 2 . 1 UMDNJ-Robert Wood Johnson Medical School,
Piscataway, NJ, 2 Rutgers University, Piscataway, NJ, 3 MatTek Corporation, Ashland,
MA, 4 Batelle Memorial Institute, Columbus, OH and 5 New York Medical College,
Valhalla, NY.
Dermal exposure to the vesicant sulfur mustard causes marked inflammation and
tissue damage. Although sulfur mustard is a non-specific alkylating agent, basal keratinocytes
appear to be a major target. In the present studies, mechanisms mediating
sulfur mustard-induced skin toxicity were examined using a full-thickness
human skin equivalent (EpiDerm-FT TM ) and a mouse keratinocyte skin construct
model. In both models, treatment of keratinocytes at the air surface with 2chloroethyl
ethyl sulfide (CEES, 100-1000 μM) induced gene and protein expression
of caveolin-1, a major component of caveolae which are membrane structural
elements important in regulating intracellular signaling. CEES treatment also increased
gene and protein expression for heat shock proteins 27 and 70 (Hsp27 and
Hsp70). In the full-thickness human skin equivalent, immunohistochemistry revealed
that Hsp27, Hsp70 and caveolin-1 were localized in basal and suprabasal
layers of the epidermis. Caveolin-1 was also detected in fibroblasts in the dermal
component. Isolation of caveolar fractions from control and CEES-treated mouse
keratinocytes from the skin constructs using sucrose density gradient ultracentrifugation
showed that Hsp27 and Hsp70 were localized in caveolae. Treatment of keratinocytes
with filipin (20 μM) or methyl-β-cyclodextrin (5 mM), two agents
which disrupt caveolar structure, markedly suppressed CEES-induced Hsp27 and
Hsp70 gene and protein expression. These data indicate that caveolae-mediated
regulation of heat shock protein expression is likely to play an important role in the
pathophysiology of vesicant-induced skin toxicity. Supported by CA093798,
CA132624, ES004738, ES005022, GM034310 and AR055073.
SOT 2011 ANNUAL MEETING 375