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A Convenient Human Hepatocyte Model for Molecular Mechanisms in Caffeine Preconditioning and Protection from Acetaminophen (APAP)‐Induced Hepatotoxicity
Author(s) -
Gupta Priya,
Sharma Yogeshwar,
Bandi Sriram,
Vishwanathan Preeti,
Gupta Sanjeev
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.1004.12
Subject(s) - caffeine , acetaminophen , cytoprotection , chemistry , hepatocyte , pharmacology , toxicity , population , viability assay , dna damage , cell cycle , flow cytometry , cell , apoptosis , biochemistry , in vitro , biology , medicine , microbiology and biotechnology , dna , endocrinology , environmental health , organic chemistry
Long‐term low‐dose caffeine use may be tissue protective with superior outcomes in liver cirrhosis or other chronic diseases. However, molecular mechanisms underlying these effects of caffeine are unknown and suitable cell‐based models will be helpful for this purpose. Caffeine may affect cells through multiple pathways including interference with DNA damage/repair through ATM, ATR or DNA‐PK activity. Here, we focused on regulation by low‐dose caffeine of ATM‐related mechanisms. To induce hepatotoxicity, we cultured HuH‐7 cells with 10 mM APAP (IC50) for 16‐20h. The effects of no caffeine preconditioning or preconditioning over 5 d with 1‐10 mM caffeine were studied. Cell viability assays with MTT dye reduction indicated low‐dose caffeine preconditioning decreased but short‐term caffeine exposure increased APAP toxicity, p<0.05. Flow cytometry showed low‐dose caffeine preconditioning depleted cells in S and G2/M and enriched cells in G0/G1, indicating replicative stress. This residual cell population contained several‐fold greater numbers of Side Population cells with putative stem cell properties, p<0.05. Use of an ATM promoter construct indicated that caffeine interfered with ATM activity in HuH‐7 cells. Therefore, low‐dose caffeine preconditioning in HuH‐7 cells involved ATM‐related mechanisms, and offers convenient models for studies. Cytoprotection against APAP toxicity in caffeine‐preconditioned cells will allow insights into ATM signaling‐specific pathways, cell cycle controls, and stem‐like cells capable of withstanding injury.