The Toxicologist - Society of Toxicology

period. We have identified proteins implicated in bioactivation and detoxification
pathways of acetaminophen and other relevant pharmaceutical agents that follow a
diurnal variation in expression (e.g. cytochrome b5, cytokeratin-18,
peroxiredoxin-6, aromatic-L-amino-acid decarboxylase and regucalcin). This
dataset reveals new areas of investigation and testable hypotheses with which to explore
the temporal relationship between circadian physiology and DILI.
447 ACUTE LIVER TOXICITY OF ACETAMINOPHEN IS
NOT POTENTIATED IN HCV TRANSGENIC MICE.
T. Uehara 1 , O. Kosyk 1 , E. Jeannot 1 , B. Bradford 1 , J. Grimes 2 , T. O’Connell 2 ,
G. Boorman 3 , S. Melnyk 4 , S. Weinman 5 and I. Rusyn 1 . 1 Department of
Environmental Sciences and Engineering, UNC, Chapel Hill, NC, 2 The Hamner
Institutes, Research Triangle Park, NC, 3 Covance, Princeton, NJ, 4 University of
Arkansas for Medical Sciences, Little Rock, AR and 5 University of Kansas Medical
center, Kansas City, KS.
The global burden of hepatitis C virus (HCV) infection is increasing and chronic
liver disease in subjects positive for HCV is becoming a significant public health
concern in the developed and developing countries alike. While HCV infection
may result in steatohepatitis, cirrhosis and hepatocellular carcinoma, it has been
shown recently that it also potentiates the hepatotoxicity of acetaminophen
(APAP), a drug which is a leading cause of acute liver toxicity. Since the mechanisms
of such potentiation are not known, we hypothesized that HCV-Tg mice
may be more susceptible to APAP hepatotoxicity and that an animal model may be
established to elucidate the mechanisms for co-morbidity. Mice expressing core, E1,
E2 and p7 proteins (HCV-Tg) and wild-type C57BL/6J mice were treated with a
single dose (300 mg/kg) of APAP and liver toxicity was evaluated at 4 and 24 hrs
after dosing. Acute liver injury due to APAP was not exacerbated in HCV-Tg mice
and there were few differences between wild type and HCV-Tg in markers of oxidative
stress, inflammation, or ER-stress in the liver. Interestingly, the amount of
mitochondrial total and reduced glutathione was elevated in HCV-Tg mice, and
was decreased upon APAP treatment, an effect not observed in wild-type mice. This
study shows that the extent of liver disease in HCV-Tg mice over-expressing core,
E1, E2 and p7 alone are not sufficient to produce the sensitivity to APAP that has
been observed clinically. However, our findings move the field forward by narrowing
down the possibilities of how HCV affects drug toxicity. Our data suggest that
either the nonstructural viral proteins are critical for the hyper-sensitivity to druginduced
liver injury in humans, or that it may be due to an immune component
that is difficult to reproduce in HCV-Tg mice.
448 INTRAVITAL IMAGING OF ACETAMINOPHEN (APAP)
HEPATOTOXICITY.
J. Hu 1 , V. K. Ramshesh 1 , H. Jaeschke 2 and J. J. Lemasters 1 . 1 Medical University
of South Carolina, Charleston, SC and 2 University of Kansas Medical Center, Kansas
City, KS.
APAP overdose causes liver injury involving mitochondrial dysfunction and onset
of the mitochondrial permeability transition (MPT). NIM811 (N-methyl-4isoleucine
cyclosporin) is a nonimmunosuppressive derivative of cyclosporin A that
inhibits the MPT in vitro and in vivo. Monitoring mitochondrial function in living
animals has been difficult. Recent developments in intravital confocal/multiphoton
microscopy provide a novel approach to visualize mitochondrial dysfunction and
cell death in vivo. Here we have two aims. The first is to investigate whether
NIM811 decreases APAP-induced liver toxicity. Our second aim is to understand
the mechanisms of APAP hepatotoxicity and the effect of APAP on mitochondrial
polarization and cell death in vivo. Male C57BL/6 mice were fasted overnight and
then administered APAP (300 mg/kg, i.p.) or vehicle. NIM811 was gavaged (10
mg/kg) 1 h before APAP. For imaging, the abdomen of each mouse was opened,
and the exposed liver was positioned on a #1.5 glass coverslip mounted on the inverted
stage of an Olympus FV1000 multiphoton microscope. Mitochondrial polarization
and cell death were assessed from the respective green and red fluorescence
of rhodamine 123 (Rh123) and propidium iodide(PI) using 820-nm
multiphoton excitation and a 25X, 1.1 NA water-immersion objective lens. After
vehicle, green Rh123 fluorescence was punctate in most hepatocytes, indicating mitochondrial
polarization, and nuclear PI staining signifying cell death was absent.
At 6 h after APAP, loss of mitochondrial Rh123 fluorescence occurred in pericentral
hepatocyes often accompanied by PI labeling. NIM811 decreased both mitochondrial
depolarization and cell death. In conclusion, NIM811 attenuates APAP-induced
mitochondrial depolarization and necrotic cell death in mouse liver in vivo.
These results also illustrate the utility of intravital confocal/multiphoton microscopy
as a powerful technology to study disruption of liver function in living animals
due to APAP.
96 SOT 2011 ANNUAL MEETING
449 THE ALPHA MUPA MICE, A MODEL OF CALORIC
RESTRICTION, ARE MORE SUSCEPTIBLE TO
ACETAMINOPHEN HEPATOTOXICITY.
Z. Fu 1 , Y. Zhang 1 , R. Miskin 2 and C. D. Klaassen 1 . 1 Department of
Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center,
Kansas City, KS and 2 Department of Biological Chemistry, The Weizmann Institute of
Science, Rehovot, Israel.
Murine urokinase plasminogen activator (αMUPA) transgenic mice spontaneously
eat less and live longer (approximately 20%), therefore, they have been considered
a caloric restriction (CR) model. Previous studies indicate that CR protects against
the hepatotoxic effects of bleomycin and thioacetamide, whereas two other longlived
models, GHR-KO and Snell dwarf mice, are more susceptible to acetaminophen
hepatotoxicity. The goal of the present study was to examine the resistance of
αMUPA mice to acetaminophen (APAP). Male αMUPA mice and wild-type (WT)
mice (14-months of age) were given a single dose of APAP (375 mg/kg, ip).
Whereas 92% of WT mice were alive at 48h, the survival of αMUPA mice was only
58% at 24h, and 29% at 48h. In αMUPA mice, APAP caused more severe liver injury,
as indicated by higher serum levels of alanine aminotransferase (4.3-fold) and
lactate dehydrogenase (5.7-fold), as well as more severe liver necrosis. In unchallenged
αMUPA and WT mice, the mRNAs of Cyp2e1 and Cyp3a11, two enzymes
important for converting APAP to its toxic intermediate, NAPQI, were similar.
However, αMUPA mice exhibited higher mRNAs of some other phase-I drug metabolizing
enzymes, such as Cyp1a1 (2-fold), Cyp1a2 (1.6-fold), and Cyp4a14 (2fold).
In addition, αMUPA mice exhibited higher mRNAs of phase-II enzymes
that detoxify APAP, Ugt1a1 (1.6-fold) and Ugt1a6 (1.6-fold); that detoxifies
NAPQI, Gstp (2.9-fold); and hepatic transporters that transport xenobiotics and
bile acids into liver, such as Oatp1a1 (1.6-fold) and Ntcp (1.7-fold). In summary,
the present study shows that αMUPA mice exhibit similar expression of key enzymes
for APAP bioactivation, and higher expression of enzymes for detoxifying
APAP and NAPQI. Therefore, the higher susceptibility of αMUPA mice to APAP
hepatotoxicity is likely due to differences in toxicodynamics, rather than toxicokinetics.
(Supported by NIH grants ES-09649, ES-09716, ES-07079, DK-081461,
and RR-021940)
450 ACTIVATION OF CASPASES DURING
ACETAMINOPHEN TOXICITY IS A STRAIN
DEPENDENT PHENOMENON.
H. Jaeschke, M. Koerner and C. D. Williams. University of Kansas Medical Center,
Kansas City, KS.
Acetaminophen (APAP)-induced liver injury resulting from overdose is the most
frequent cause of acute hepatic failure in the US. The mechanisms of APAP-mediated
cell death have been extensively characterized in both laboratory animals and
man and it is generally accepted that liver cells die by oncotic necrosis (Gujral et al.,
Toxicol Sci. 2002;67:322-8). Recently it was demonstrated in fed but not in fasted
CD-1 mice (outbred strain) that there is transient caspase activation after APAP
overdose. It was speculated that apoptosis is limited in APAP hepatotoxicity due to
the use of fasted animals (Antoine et al., Toxicol Sci. 2009;112:521-31). To evaluate
these findings in more detail, APAP-induced liver injury in an outbred strain
(Swiss Webster, SW) was compared with the more commonly used inbred mouse
strain (C57BL/6). In these studies fed and fasted mice were treated with 530 mg/kg
APAP for 3, 5 and 24h with or without pan-caspase inhibitor (10 mg/kg Z-VDfmk).
As a positive control for caspase-dependent apoptosis, mice were treated with
700 mg/kg galactosamine (GalN) and 100 μg/kg endotoxin (ET). Fasted or fed
APAP-treated C57BL/6 mice showed no evidence of caspase-3 processing by western
blot or caspase-3 activity by Ac-DEVD-AFC fluorogenic assay at any timepoint.
Interestingly, a minor, temporary increase in caspase-3 processing and enzyme
activity (250% above baseline) was observed after APAP treatment in SW
mice, but this could be observed in both fed and fasted animals. Z-VD-fmk in vivo
partially attenuated this caspase activation but did not alter injury (plasma ALT,
area of necrosis). The degree of caspase-3 processing and activation in SW mice
after APAP was much less than that observed in the GalN/ET-treated mice (1700%
above baseline). Conclusion: Caspase-3 processing and activation after APAP-overdose
can be observed only in some outbred mouse strains independent of the fed or
fasted state. In contrast to the GalN/ET-induced apoptosis, this transient, minor
caspase activation does not seem to impact the overall liver injury and toxicity during
APAP overdose.