* Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269; and
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269
Received July 21, 1999; accepted February 4, 2000
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ABSTRACT |
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Key Words: acetaminophen; NF-B; I
B
; Hepa 1-6; hepatotoxicity; reactive oxygen; antioxidants.
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INTRODUCTION |
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In addition to cellular damage inflicted by NAPQI, APAP itself may directly contribute to toxicity. APAP has been found to interfere with growth factor signal transduction (Boulares et al., 1999), mitochondrial respiration (Meyers et al., 1988
), and ribonucleotide reductase (required for deoxyribose nucleotide synthesis) (Holme et al., 1988
; Hongslo et al., 1989
; Hongslo et al., 1990
; Richard et al., 1991
), even in the absence of cytochrome P450 conversion to NAPQI. Although the contribution of these direct effects of APAP on toxicity are not yet known, it has been proposed that APAP may interfere with liver regeneration after injury sustained from NAPQI or other forms of chemical or physical damage (Boulares et al., 1999
; Chanda et al., 1995
).
It has recently been reported that APAP influences the transcription factor NF-B. Immediately following a toxic dose of APAP, the NF-
B DNA binding activity isolated from the liver is dramatically reduced, with activity returning only after several hours (Blazka et al., 1996
). This transient APAP inhibition of NF-
B could potentially play an important role in APAP toxicity. Studies utilizing knockout mice deficient in the RelA subunit of NF-
B reveal that NF-
B is required for maintaining hepatic viability; embryonic livers in RelA knockout animals suffer extensive apoptosis (Beg et al., 1995
). NF-
B has also been found to protect numerous cell types from cytokine and drug-induced cell death (Baichwal and Baeuerle, 1997
; Beg and Baltimore, 1996
; Bellas et al., 1997
; Iimuro et al., 1998
; Van Antwerp et al., 1996
; Wang et al., 1996
; Wu et al., 1996
). The influence of NF-
B on cell death appears to be due to its ability to activate protective genes, such as that for the superoxide scavenging enzyme MnSOD (Li and Oberley, 1997
; Wong et al., 1989
). In addition, the APAP inhibition of NF-
B may suppress hepatocyte proliferation and subsequent liver regeneration (Baldwin et al., 1991
; Boulares et al., 1999
).
We recently demonstrated that the APAP inhibition of NF-B can be reproduced in Hepa 1-6 cells (Boulares et al., 1999
). Interestingly, NF-
B inhibition is observed in Hepa 1-6 cells in the absence of cytochrome P450 activation, glutathione oxidation, cytosolic enzyme leakage, and cell death. However, even in the absence of overt cellular toxicity, APAP still inhibits proliferation of Hepa 1-6 cells. The APAP inhibition of cell proliferation appears to involve the inhibition of a number of G1 signaling events including Raf-1 kinase activation and NF-
B activation (Boulares et al., 1999
). Here we present evidence that APAP inhibits NF-
B activation in part by interfering with the oxidant signal required for activation of this transcription factor.
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MATERIALS AND METHODS |
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Cytoplasmic and nuclear extract preparations and electrophoretic mobility shift assay (EMSA).
Cytoplasmic and nuclear extracts were prepared, and EMSA analysis of DNA binding activity was performed as described previously (Boulares et al., 1999). The NF-
B binding oligonucleotide used for these studies was a double-stranded DNA probe synthesized by the University of Connecticut Biotechnology Center with the sequence TCGACAGAGGGGACTTTCCGAGAGGCTCGA (Boulares et al., 1999
). This oligonucleotide was end labeled with T4 polynucleotide kinase using
[32P]ATP (Boulares et al., 1999
). In the DNA binding reaction, the radioactive DNA probe was in excess to ensure that it was not the limiting component of the binding reaction. Typically, only a few percent of the DNA in the reaction was bound by protein.
Western immunoblotting.
Cytoplasmic extracts (50 µg of protein per lane) were run on a 10% SDS-polyacrylamide gel, and transferred to Immobilon-P membranes (Millipore). Transfers were performed using a Trans-Blot Electrophoretic Transfer Cell (Bio-Rad), following the manufacturer instructions. IB
protein was detected with an affinity purified polyclonal anti-I
B
/MAD3 antibody (Santa Cruz Biotechnology) and visualized by enhanced chemiluminescent staining using ECL reagents (Amersham). Antibody probings were performed in phosphate buffered saline plus 0.1% Tween-20, with antibody dilutions of 1:1000.
Dihydrodichlorofluorescein (H2DCF) oxidation assay for peroxide.
The assay for reactive oxygen was performed as described (Giardina and Inan, 1998). In brief, cells were grown on 96-well tissue culture plates to near confluency, and loaded with H2DCF by adding the diacetate form of this compound (Molecular Probes) to the medium at a final concentration of 50 µM. After 30 min, the medium was completely removed and replaced with 100 µl of fresh medium containing APAP or antioxidants as indicated. H2O2 was then added to a concentration of 500 µM. In the presence of reactive oxygen species such as H2O2, the nonfluorescent H2DCF is oxidized to fluorescent DCF, which can then be quantified. DCF production was detected after a 30-min exposure with a Cytofluor microplate-reading fluorometer (PerSeptive), with excitation and emission wavelengths of 475 nm and 525 nm, respectively.
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RESULTS |
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DISCUSSION |
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The role of NF-B inhibition in APAP toxicity is not clear. A wealth of evidence has implicated this transcription factor in regulating cell death in a number of situations. For example, knockout mice lacking the RelA subunit of NF-
B suffer extensive hepatocyte apoptosis during embryogenesis (Beg et al., 1995
). A direct role for NF-
B in maintaining hepatocyte viability has also been demonstrated in cell culture models (Bellas et al., 1997
; Xu et al., 1998
). Whether the transient inhibition of NF-
B observed after APAP administration sensitizes cells to a NAPQI-induced cell death is not yet known. NF-
B activation has, however, been shown to protect cells from toxicity induced by a number of chemotherapeutic agents (Wang et al., 1996
), suggesting that it can modulate cellular sensitivity to toxins.
Since the initial discovery that APAP has antioxidant properties (DuBois et al., 1983; Garrido et al., 1991
), evidence has been obtained that reactive oxygen species can play a role in cell signaling. For example, growth factor signal transduction appears to require a reactive oxygen signal (Sundaresan et al., 1995
). An emerging paradigm envisions a modest level of reactive oxygen generation as being essential for cell viability and proliferation (Khan and Wilson, 1995
). Here we show that APAP's interference with growth factor signal transduction to NF-
B can be accounted for (at least in part) by its antioxidant properties. NF-
B can influence cell cycle progression through its ability to modulate c-myc expression (Baldwin et al., 1991
; La Rosa et al., 1994
), and we have previously shown that APAP also blocks c-myc expression in Hepa 1-6 cells (Boulares et al., 1999
). However, other cellular processes required for maintaining cell viability and proliferative capacity may also be suppressed by APAP's antioxidant properties. Although evidence has been obtained indicating that the stimulation of hepatocyte proliferation may enhance recovery from APAP poisoning (Chanda et al., 1995
), we have proposed that APAP's inhibition of hepatocyte proliferation may interfere with liver regeneration following injury (Boulares et al., 1999
). The present findings suggest that APAP's interference with cell proliferation is mediated through its antioxidant action. Additional studies will be required to determine if APAP's antioxidant action can block cell proliferation in vivo and whether this activity contributes to hepatic injury.
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ACKNOWLEDGMENTS |
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NOTES |
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3 To whom correspondence should be addressed at University of Connecticut, Department of Pharmaceutical Sciences, 372 Fairfield Rd., Storrs, CT 06269. Fax: (860) 486-4998. E-mail: cohens{at}uconnvm.uconn.edu.
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