The Toxicologist - Society of Toxicology

nonpregnant and pregnant diabetic mice. Livers from nonpregnant, diabetic mice
exhibited up-regulation of Mrp1, 2, 4, 5 and breast cancer resistance protein (Bcrp)
mRNA that was attenuated in pregnant, diabetic mice. Mrp3 mRNA was similarly
induced in livers of nonpregnant and pregnant diabetic mice. Mrp6 mRNA was decreased
by pregnancy, regardless of glucose level. Collectively, these data suggest
that pregnancy represses expression of efflux transporters in livers of diabetic mice.
Additional studies are necessary to elucidate the transcriptional mechanisms underlying
the opposing regulation of hepatobiliary transporters in response to diabetes
and pregnancy. Supported by NIH ES016042, ES005022, and DK080774.
1080 EFFECT OF TROGLITAZONE (TRO) ON ENDOGENOUS
BILE ACID (BA) DISPOSITION IN RAT AND HUMAN
SANDWICH-CULTURED HEPATOCYTES (SCH).
T. Marion 1 , C. Perry 2 , R. L. St. Claire III 2 and K. Brouwer 1, 3 . 1 Curriculum in
Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 2 Qualyst,
Inc., Research Triangle Park, NC and 3 Eshelman School of Pharmacy, University of
North Carolina at Chapel Hill, Chapel Hill, NC.
Inhibition of hepatic transport may mediate drug-induced liver injury by increasing
intracellular accumulation of potentially toxic compounds such as BAs. Drug-induced
changes in BAs may be indicators of hepatotoxicity. In this study, the endogenous
BA pool was profiled in fresh rat and human SCH on culture day 4 using
B-CLEAR® technology to compare species differences, and to determine effects of
transporter inhibition on cellular BA accumulation and biliary excretion.
Taurocholate (TCA), glycocholate (GCA), taurochenodeoxycholate (TCDCA),
and glycochenodeoxycholate (GCDCA) were measured on day 4 in cells + bile
canaliculi, cells, and medium of SCH by LC-MS/MS 24 h after addition of vehicle
or 10 μM TRO, an inhibitor of the bile salt export pump (BSEP/Bsep). Total BAs
(pmol/mg protein) were ~12-fold greater in human than rat SCH. Consistent with
reports in vivo, ~90% of BAs in rat SCH were taurine-conjugated, while in human
SCH, 99% of BAs were glycine-conjugated. Cellular accumulation of BAs in
human SCH tended to decrease after TRO treatment. In rat SCH, TRO treatment
decreased the biliary excretion index (BEI; [bile]/[cells + bile] calculated for each
BA) of TCA 3%, GCA 12%, TCDCA 15%, and GCDCA 27%; in human SCH,
TRO had no effect on the BEI of GCA, but decreased the BEI of TCA 43%,
TCDCA 67%, and GCDCA 48%, consistent with inhibition of BSEP/Bsep.
Species differences in inhibition of BA BEI by TRO may be due to differences in
intracellular concentrations of TRO and/or metabolites after 24 h in culture. TRO
decreased total BAs (cells + bile + medium) in human SCH by 19% compared to
control, suggesting that TRO may affect BA synthesis pathways in addition to inhibiting
BSEP. Together, these data demonstrate that BA synthesis and secretion
occur in SCH similar to in vivo, and that TRO differentially affects BA disposition
in rat and human SCH. Supported by NIH GM41935
1081 EFFECTS OF SEEDING DENSITY AND DAYS IN
CULTURE ON BILE ACID TRANSPORT AND Mrp4
EXPRESSION IN SANDWICH-CULTURED MOUSE
HEPATOCYTES ARE NOT A RESULT OF OXIDATIVE
STRESS.
B. C. Ferslew 1 , X. Gu 2 , B. Swift 3 , J. E. Manautou 2 and K. L. Brouwer 1 .
1 Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC,
2 Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT and
3 PKPD and Drug Metabolism, Allergan, Inc., Irvine, CA.
Studies were undertaken to determine whether oxidative stress was responsible for
changes in bile acid transport (B-CLEAR ® -MO) and Mrp4 expression in sandwich-cultured
mouse hepatocytes as a function of seeding density and days in culture.
Freshly isolated mouse hepatocytes were seeded at 1.0x10 6 cells/well (low density,
LoD) or 1.5x10 6 cells/well (high density, HiD) in modified DMEM on 6-well
BioCoat plates and overlaid with Matrigel. Gene expression was measured for
Mrp4, Nqo1, Hmox1 and Hes1 using RT-PCR. Mrp4 protein was determined by
Western blot and quantified by densitometry. 3 H-Taurocholate (TC) was used to
assess transport function. Intracellular TC accumulation was reduced on day 4
compared to day 3 for both LoD and HiD. At LoD, TC in vitro biliary clearance
(Cl biliary , mL/min/kg, mean±SEM) and biliary excretion index (BEI, %,
mean±SEM) were 38±5 and 30±2 on day 3, and 14±6 and 24±5 on day 4, respectively.
At HiD, Cl biliary and BEI were 48±11 and 29±3 on day 3, and 38±15 and
43±5 on day 4, respectively. Mrp4 mRNA and protein expression were inversely related
to seeding density. The influence of seeding density on TC transport and
Mrp4 protein are consistent with previous observations from our laboratory.
mRNA levels for oxidative stress genes Nqo1 and Hmox1 were not significantly affected
by the culture conditions tested. Interestingly, Hes-1, a transcription factor
involved in regulation of cell differentiation and proliferation, increased as a function
of days in culture, but was not influenced by seeding density. Sandwich-cultured
mouse hepatocytes seeded at LoD and HiD do not exhibit increased oxidative
stress under the conditions investigated. Therefore, changes in bile acid transport
and increased Mrp4 expression at LoD compared to HiD do not appear to be a
function of oxidative stress.
Supported by NIH GM41935; DK069557
1082 THE CELL SURFACE MARKER CD90 IDENTIFIES A
SUBSET OF HEPATOCYTES PRIMED FOR INCREASED
MATRIX PRODUCTION: IMPLICATIONS FOR
ETHANOL-INDUCED LIVER FIBROSIS.
B. A. Hocevar, Z. Wang and L. M. Kamendulis. Department of Environmental
Health, Indiana University, Bloomington, IN.
Hepatic fibrosis, initiated by excessive alcohol consumption, is a predisposing factor
in the development of cirrhosis. Excessive production of extracellular matrix
(ECM) leads to cirrhosis, loss of liver function, and eventually death. Deposition of
ECM is stimulated by TGFβ, which is produced and activated during the fibrotic
process. We hypothesize that TGFβ leads to epithelial to mesenchymal transition
(EMT) in hepatocytes, which contributes significantly to ECM deposition seen in
alcohol-induced liver fibrosis. Mice treated with 5% ethanol (w/v) for 2 weeks displayed
~2-fold increase in collagen deposition in the liver providing validation for
our experimental model. To characterize the transdifferentiation of hepatocytes, cell
surface marker expression was profiled in control and ethanol treated livers. We
found that ethanol produced a 2.1-fold increase (6.9% to 14.3%) in CD90 cell surface
expression and a 1.3-fold increase in CD44 expression while expression of
CD133, CD29, CD326, and CD49f did not change. Differential gene expression
in CD90 + versus CD90- hepatocytes was assessed by microarray analysis. Reporters
were selected as differentially regulated in a non-statistical approach yielding 2248
upregulated and 1413 downregulated reporters. Initial examination of genes differentially
expressed in CD90 + hepatocytes identified increased expression of genes involved
in inflammatory processes (IL1α, IL1β, IL10, TNFα), leukocyte recruitment
(CCL2, CCL3, CCL19) and genes regulating ECM (COL1A1, COL1A2,
COL4A1, TIMP2, TIMP3). In addition, the CD90 + population expressed markers
identifying hepatic progenitor cells (CD44, Procr) and cells that have undergone
EMT (ZEB2, Vimentin, PDGFRβ, TGFβ1). These results show that ethanol treatment
leads to the expansion of the CD90 + population. Characterization of the
CD90 + hepatocytes indicates that this population may be responsible for the excess
matrix production that occurs during ethanol-induced fibrosis.
1083 INVESTIGATING ER STRESS PATHWAY ACTIVATION IN
LIVER CELLS AND TISSUE AS A TOXICOLOGICAL TOOL.
M. Lafleur, R. Hernandez and J. W. Lawrence. Investigative Toxicology, Amgen,
Thousand Oaks, CA. Sponsor: S. Sawant.
One of the many functions of the endoplasmic reticulum (ER) is the proper folding
and assembly of newly synthesized secretory and membrane proteins. ER stress occurs
when there is an accumulation of unfolded proteins in the ER and can be
elicited by normal physiological processes such as the differentiation of B cells into
plasma cells, by pathological conditions such as oxidative injury or viral infections,
or induced by a number of drugs or compounds that interfere with the protein
folding machinery. An evolutionary conserved adaptive or protective response
known as the unfolded protein response (UPR) is activated in response to ER stress.
The UPR consists of 3 principal pathways that collectively, help to ameliorate the
accumulation of unfolded proteins in the ER by increasing the protein folding machinery
or eliminating unfolded proteins. If the UPR is unable to dampen the unfolded
protein load in the ER, a set of cellular responses lead to apoptosis. We have
characterized ER stress responses to various chemicals. ER stress inducers tested included
a number of marketed pharmaceutical compounds as well as agents that interfere
with the cellular protein folding machinery such as Tunicamycin and
Thapsigargin. ER stress was assessed in liver cell lines as well as primary liver cells
and tissue. We observed that pharmaceutical agents and classical ER stress inducers
trigger the UPR pathways in a general manner; however, Atazanavir and Nelfinavir
pathway responses were not similar. These data suggest that agents associated with
ER stress may not all behave the same in vitro.
1084 PROFILING IMPAIRED HEPATIC ENDOPLASMIC
RETICULUM GLYCOSYLATION AS A CONSEQUENCE
OF ETHANOL INGESTION.
J. Galligan 1 , K. S. Fritz 2 , H. Tipney 1 , R. L. Smathers 2 , C. T. Shearn 2 , L. J.
Hunter 1 and D. R. Petersen 2 . 1 Pharmacology, University of Colorado Denver,
Aurora, CO and 2 Toxicology, University of Colorado Denver, Aurora, CO.
Chronic ethanol consumption remains a predominant cause of liver injury in the
United States. The precise mechanisms underlying the progression of alcoholic liver
disease (ALD) are poorly understood; however, alterations in post-translational
SOT 2011 ANNUAL MEETING 231