The Toxicologist - Society of Toxicology

442 GENE EXPRESSION AND MORPHOLOGICAL
ANALYSIS OF HEART AND LIVER FROM IL-6 KO MICE.
A. Roth, F. Boess, J. Hoflack, C. Landes, M. Bellot, A. Olaharski, L. Suter, S.
Platz, T. Weiser, T. Singer and L. Mueller. F. Hoffmann-La Roche AG, Basel,
Switzerland.
Mice with a deletion of the Interleukin-6 gene (IL-6 KO) were developed in 1994
by Kopf et al. and have been intensively studied, predominantly under conditions
of immunological challenge. IL-6 emerged as an important target for the treatment
of various inflammatory diseases. The fact that IL-6 KO mice do not display an altered
phenotype under normal conditions, supported Il-6 as a suitable drug target
with small inherent risk of undesired adverse effects. A recent publication by
Banerjee et al. (2009), however, suggested that IL-6 KO mice undergo severe morphological
changes in the heart already early in life. To follow-up on these data we
aimed to further examine potential changes in heart and liver induced by deletion
of IL-6 with respect to morphology and gene expression. Method: IL-6 KO
(B6.129S2-Il6tm1Kopf) and C57/B6 wildtype (WT) mice were sacrificed at 6, 16
and 30 weeks of age, and heart and liver tissue was harvested to allow for
histopathological evaluation and microarray gene expression analysis. Results: No
macro- or microscopic morphological differences could be found between KO and
WT mice. Especially the marked morphological alterations in heart tissue published
by Banerjee et al. were not present in this study. No obvious differences in
gene expression were observed between IL-6 KO and age matched WT mice. In
particular, there was no significant effect of IL-6 deletion at any of the time points
analyzed with respect to gluconeogenesis, acute phase response, mitogenesis, apoptosis,
or tissue regeneration – processes well-known to be regulated by cytokines
such as IL-6. Conclusion: In contrast to recently published work, in the present
study absence of IL-6 in IL-6 KO animals did not lead to gross changes in heart or
liver under normal conditions, i.e. in the absence of an inflammatory challenge.
Blockage of IL-6 signaling is thus considered not to lead to overt adverse changes in
these organs, consistent with the view of alternative regulatory mechanisms compensating
for the lack of IL-6.
443 TROVAFLOXACIN ENHANCEMENT OF LPS-
MEDIATED SIGNALING IS ASSOCIATED WITH
REDUCED SOCS-1 EXPRESSION.
R. Singhal, K. L. Poulsen, P. E. Ganey and R. A. Roth. Pharmacology and
Toxicology, Michigan State University, East Lansing, MI.
Trovafloxacin (TVX), a fluoroquinolone antibiotic, has been associated with idiosyncratic
hepatotoxicity in patients. We previously demonstrated that pretreatment
with TVX potentiated lipopolysaccharide (LPS)-induced tumor necrosis factor
(TNF)α production and led to TNFα-dependent hepatotoxicity in mice.
Furthermore, TVX enhanced LPS-stimulated upregulation of TNFα mRNA and
protein in RAW 264.7 transformed macrophage cells. Inhibition of p38 phosphorylation
or NFkB activation abolished TVX-potentiated LPS-induced TNFα
mRNA expression. We hypothesized that TVX pretreatment alters LPS-mediated
signaling by suppressing toll-like receptor 4 (TLR4) negative regulators such as
SOCS-1, TOLLIP, SIGIRR and SARM. TVX (100 μM) treatment reduced basal
and LPS-induced SOCS-1 mRNA expression in RAW cells. It also caused a marginal
reduction in TOLLIP mRNA in RAW cells but did not affect transcript levels
of SIGIRR or SARM. Treatment of mice with TVX (150 mg/kg) reduced the hepatic
levels of SOCS-1 at 3 hr post-treatment without affecting expression in
spleen. These results raise the possibility that TVX enhancement of LPS-induced
hepatotoxicity and TNFα production is mediated, in part, by reducing SOCS-1
and thereby potentiating TLR4 proinflammatory signaling. (Supported by NIH
grant R01DK061315.)
444 ENHANCED ACETAMINOPHEN HEPATOTOXICITY IN
VANIN-1 NULL MICE IS INDEPENDENT OF
DIFFERENCES IN BASAL HEPATIC GLUTATHIONE
LEVELS AND GENE EXPRESSION OF DRUG
METABOLIZING ENZYMES AND TRANSPORTERS.
D. W. Ferreira 1 , P. Naquet 2 , F. Galland 2 and J. E. Manautou 1 . 1 Pharmaceutical
Sciences, University of Connecticut, Storrs, CT and 2 Centre d’Immunologie de
Marseille-Luminy CNRS-INSERM-Université de la Méditerranée, Marseille, France.
The Vanin-1 gene encodes a membrane-bound pantetheinase involved in the production
cysteamine, a potent antioxidant. Previous transcriptome analysis showed a
marked induction of hepatic Vanin-1 gene expression in association with the hepatoprotective
effect of the peroxisome proliferator clofibrate. Our recent studies
show that Vanin-1 knockout mice are more susceptible to acetaminophen (APAP)
hepatotoxicity. Here we investigate whether differential APAP hepatotoxicity in the
absence of Vanin-1 is due to differences in APAP bioactivation, disposition or
detoxification. Livers of naïve Vanin-1 null and wild type male mice were analyzed
for gene expression of APAP metabolizing enzymes and transporters by RT-PCR.
No differences in hepatic gene expression of phase I and II enzymes (Cyp1a2, 2e1,
3a11, Ugt1a6) or transporters (Abcc2, Abcc3, Abcc4) were detected between genotypes.
Markers of oxidative stress (Hmox1) and inflammation (Tnfα, Cxcl1) were
also unchanged. Gene expression of the catalytic subunit of gamma glutamylcysteine
synthetase was slightly increased in Vanin-1 null mice. However, no differences
in hepatic total glutathione (GSH) concentrations were detected. In mice
treated with APAP (400mg/kg, i.p.), induction of Hmox1 was more dramatic in
Vanin-1 null mice (~8 fold) compared to wild types (~4 fold) at 24 hours, indicating
higher hepatic oxidative stress response in these mice. Collectively, these data
suggest that the higher susceptibility of Vanin-1 mice to APAP hepatotoxicity is unlikely
due to differences in APAP bioactivation, disposition or basal GSH concentration.
Future studies will investigate the role of the Vanin-1 catalytic product cysteamine
in APAP hepatotoxicity.
445 DIFFERENTIAL HEPATIC EXPRESSION OF ID GENES
BY ACETAMINOPHEN TREATMENT IN MICE.
X. Gu and J. E. Manautou. University of Connecticut, Storrs, CT.
Hepatotoxicity by acetaminophen (APAP) is one of the leading causes of acute liver
failure in the United States. Hepatocellular damage by APAP is followed by a recovery
phase in which normally quiescent hepatocytes replicate to replace necrotic
cells. During this regeneration phase, rodents exhibit resistance to toxic APAP reexposure,
a phenomenon known as autoprotection. Inhibitor of DNA binding
(ID) genes encode a family of helix-loop-helix (HLH) proteins that lack a basic
DNA-binding domain and inhibit transcription of basic HLH (bHLH) transcription
factors through formation of heterodimers incapable of binding to DNA. ID
proteins could play a role in cell growth, senescence and differentiation. To examine
potential changes in expression of Id genes during APAP hepatotoxicity and autoprotection,
mice were treated with either 400 mg/kg APAP only or APAP (400
mg/kg) followed by a second dose of 600 mg/kg 48 hr later (APAP autoprotection
dosing regimen). Id gene expression was examined over a 72 hrs time course. ID
gene expression was also examined in vitro in human HC04 liver cells exposed to
several proxidant chemicals. The results show that mouse liver Id1 and Id3 gene expression
decreased by 2-3 folds at 2 hrs, followed by a 6-10 fold increase between 4
to 12 hrs after a single dose of APAP, returning to normal values by 24 hrs.
Similarly, increases in Id2 and Id4 gene expression were detected at several time
points after single APAP treatment. With the APAP autoprotection dosing regimen,
Id1 gene expression increased 3.8 and 5.6 fold at 4 and 24 hrs, respectively,
while induction of Id2 and Id3 gene expression (3-2 fold) was only detected at 4
hrs. No changes in Id4 were detected. Treatment of HCO4 cells with APAP, tBHP
or tBHQ also resulted in induction of ID gene expression over time. Collectively,
expression of the ID/Id family of genes increases in response to both a single dose of
APAP and the autoprotection dosing regimen in mice and in vitro in response to
oxidative stress. Supported by NIH DK069557.
446 PROFILING THE HEPATIC PROTEOME TO EXPLORE
THE MOLECULAR BASIS OF THE CHRONOTOXICITY
OF ACETAMINOPHEN.
P. J. Starkey Lewis 1 , C. E. Goldring 1 , V. Platt 1 , A. M. Obeng 1 , L. Randle 1 , C.
Rowe 1 , J. Moggs 2 and K. Park 1 . 1 Molecular & Clinical Pharmacology, University of
Liverpool, Liverpool, Merseyside, United Kingdom and 2 Investigative Toxicology,
Novartis, Basel, CH-4132, Switzerland. Sponsor: D. Mendrick.
The mammalian biological clock has been found to exert a powerful influence on
the physiology of mammalian systems. This regulation hinges on the complex interplay
between the clock genes and their products that oscillate over a twenty-four
hour period and promote a diurnal variation in numerous output pathways.
Emerging evidence suggests that the efficacy and toxicity of many therapeutic compounds
follow a diurnal rhythm and that this may be at least partly attributable to
the clock-mediated regulation of drug targets and pathways of drug metabolism. In
this study, we have confirmed a significant circadian variation in acetaminopheninduced
liver injury in the mouse (CD-1). We have utilized conventional bioanalytical
techniques to explore differences in hepatic glutathione status and several
CYP450s at the protein and activity levels to begin to address the basis for this variation.
Moreover, we have now carried out a more global profile of the circadian hepatic
proteome using mass spectrometry, which enabled the determination of the
relative levels of approximately four hundred hepatic proteins across a 12h daylight
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