3-Aminobenzamide Prevents Concanavalin A-Induced Acute Hepatitis by an Anti-inflammatory and Anti-oxidative Mechanism

Abstract

Background and Aims

Concanavalin A is known to activate T cells and to cause liver injury and hepatitis, mediated in part by secretion of TNFα from macrophages. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have been shown to prevent tissue damage in various animal models of inflammation. The objectives of this study were to evaluate the efficacy and mechanism of the PARP-1 inhibitor 3-aminobenzamide (3-AB) in preventing concanavalin A-induced liver damage.

Methods

We tested the in vivo effects of 3-AB on concanavalin A-treated mice, its effects on lipopolysaccharide (LPS)-stimulated macrophages in culture, and its ability to act as a scavenger in in vitro assays.

Results

3-AB markedly reduced inflammation, oxidative stress, and liver tissue damage in concanavalin A-treated mice. In LPS-stimulated RAW264.7 macrophages, 3-AB inhibited NFκB transcriptional activity and subsequent expression of TNFα and iNOS and blocked NO production. In vitro, 3-AB acted as a hydrogen peroxide scavenger. The ROS scavenger N-acetylcysteine (NAC) and the ROS formation inhibitor diphenyleneiodonium (DPI) also inhibited TNFα expression in stimulated macrophages, but unlike 3-AB, NAC and DPI were unable to abolish NFκB activity. PARP-1 knockout failed to affect NFκB and TNFα suppression by 3-AB in stimulated macrophages.

Conclusions

Our results suggest that 3-AB has a therapeutic effect on concanavalin A-induced liver injury by inhibiting expression of the key pro-inflammatory cytokine TNFα, via PARP-1-independent NFκB suppression and via an NFκB-independent anti-oxidative mechanism.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Abbreviations

3-AB:

3-Aminobenzamide

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

ConA:

Concanavalin A

DPI:

Diphenyleneiodonium

HRP:

Horseradish peroxidase

IFNγ:

Interferon γ

LPS:

Lipopolysaccharide

MDH:

Malate dehydrogenase

NAC:

N-acetylcysteine

NFκB:

Nuclear factor kappa B

NO:

Nitric oxide

Nox:

NADPH oxidase

PARP-1:

Poly(ADP-ribose) polymerase-1

PF2:

Peroxyfluor-2

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

TNFα:

Tumor necrosis factor α

References

  1. 1.

    Tiegs G, Hentschel J, Wendel A. A T cell-dependent experimental liver injury in mice inducible by concanavalin A. J Clin Invest. 1992;90:196–203.

    CAS  Article  Google Scholar 

  2. 2.

    Takeda K, Hayakawa Y, Van Kaer L, Matsuda H, Yagita H, Okumura K. Critical contribution of liver natural killer T cells to a murine model of hepatitis. Proc Natl Acad Sci USA. 2000;97:5498–5503.

    CAS  Article  Google Scholar 

  3. 3.

    Schumann J, Wolf D, Pahl A, et al. Importance of Kupffer cells for T-cell-dependent liver injury in mice. Am J Pathol. 2000;157:1671–1683.

    CAS  Article  Google Scholar 

  4. 4.

    Gantner F, Leist M, Kusters S, Vogt K, Volk HD, Tiegs G. T cell stimulus-induced crosstalk between lymphocytes and liver macrophages results in augmented cytokine release. Exp Cell Res. 1996;229:137–146.

    CAS  Article  Google Scholar 

  5. 5.

    Kusters S, Gantner F, Kunstle G, Tiegs G. Interferon gamma plays a critical role in T cell-dependent liver injury in mice initiated by concanavalin A. Gastroenterology. 1996;111:462–471.

    CAS  Article  Google Scholar 

  6. 6.

    Essani NA, Fisher MA, Jaeschke H. Inhibition of NF-kappa B activation by dimethyl sulfoxide correlates with suppression of TNF-alpha formation, reduced ICAM-1 gene transcription, and protection against endotoxin-induced liver injury. Shock. 1997;7:90–96.

    CAS  Article  Google Scholar 

  7. 7.

    Gill R, Tsung A, Billiar T. Linking oxidative stress to inflammation: toll-like receptors. Free Radic Biol Med. 2010;48:1121–1132.

    CAS  Article  Google Scholar 

  8. 8.

    Roh YS, Seki E. Toll-like receptors in alcoholic liver disease, nonalcoholic steatohepatitis and carcinogenesis. J Gastroenterol Hepatol. 2013;28:38–42.

    CAS  Article  Google Scholar 

  9. 9.

    Xu J, Zhang X, Monestier M, Esmon NL, Esmon CT. Extracellular histones are mediators of death through TLR2 and TLR4 in mouse fatal liver injury. J Immunol. 2011;187:2626–2631.

    CAS  Article  Google Scholar 

  10. 10.

    Gong Q, Zhang H, Li JH, et al. High-mobility group box 1 exacerbates concanavalin A-induced hepatic injury in mice. J Mol Med (Berl). 2010;88:1289–1298.

    CAS  Article  Google Scholar 

  11. 11.

    Woodhouse BC, Dianov GL. Poly(ADP-ribose) polymerase-1: an international molecule of mystery. DNA Repair (Amst). 2008;7:1077–1086.

    CAS  Article  Google Scholar 

  12. 12.

    Liu L, Ke Y, Jiang X, et al. Lipopolysaccharide activates ERK-PARP-1-RelA pathway and promotes nuclear factor-kappaB transcription in murine macrophages. Hum Immunol. 2012;73:439–447.

    CAS  Article  Google Scholar 

  13. 13.

    Huang D, Yang CZ, Yao L, Wang Y, Liao YH, Huang K. Activation and overexpression of PARP-1 in circulating mononuclear cells promote TNF-alpha and IL-6 expression in patients with unstable angina. Arch Med Res. 2008;39:775–784.

    CAS  Article  Google Scholar 

  14. 14.

    Peralta-Leal A, Rodriguez-Vargas JM, Aguilar-Quesada R, et al. PARP inhibitors: new partners in the therapy of cancer and inflammatory diseases. Free Radic Biol Med. 2009;47:13–26.

    CAS  Article  Google Scholar 

  15. 15.

    Mukhopadhyay P, Rajesh M, Cao Z, et al. Poly(ADP-ribose) polymerase-1 is a key mediator of liver inflammation and fibrosis. Hepatology. 2014;59:1998–2009.

    CAS  Article  Google Scholar 

  16. 16.

    Donmez M, Uysal B, Poyrazoglu Y, et al. PARP inhibition prevents acetaminophen-induced liver injury and increases survival rate in rats. Turk J Med Sci. 2015;45:18–26.

    CAS  Article  Google Scholar 

  17. 17.

    Zhang Y, Wang C, Tian Y, et al. Inhibition of poly(ADP-Ribose) polymerase-1 protects chronic alcoholic liver injury. Am J Pathol. 2016;186:3117–3130.

    CAS  Article  Google Scholar 

  18. 18.

    Chen Q, Zhao Y, Cheng Z, Xu Y, Yu C. Establishment of a cell-based assay for examining the expression of tumor necrosis factor alpha (TNF-alpha) gene. Appl Microbiol Biotechnol. 2008;80:357–363.

    CAS  Article  Google Scholar 

  19. 19.

    Conkright MD, Guzman E, Flechner L, Su AI, Hogenesch JB, Montminy M. Genome-wide analysis of CREB target genes reveals a core promoter requirement for cAMP responsiveness. Mol Cell. 2003;11:1101–1108.

    CAS  Article  Google Scholar 

  20. 20.

    Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta. 1991;1072:129–157.

    CAS  PubMed  Google Scholar 

  21. 21.

    Shirin H, Aeed H, Alin A, et al. Inhibition of immune-mediated concanavalin a-induced liver damage by free-radical scavengers. Dig Dis Sci. 2010;55:268–275.

    CAS  Article  Google Scholar 

  22. 22.

    Wills ED. Lipid peroxide formation in microsomes. General considerations. Biochem J. 1969;113:315–324.

    CAS  Article  Google Scholar 

  23. 23.

    Batts KP, Ludwig J. Chronic hepatitis. An update on terminology and reporting. Am J Surg Pathol. 1995;19:1409–1417.

    CAS  Article  Google Scholar 

  24. 24.

    Fraser I, Liu W, Rebres R, et al. The use of RNA interference to analyze protein phosphatase function in mammalian cells. Methods Mol Biol. 2007;365:261–286.

    PubMed  Google Scholar 

  25. 25.

    Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8:2281–2308.

    CAS  Article  Google Scholar 

  26. 26.

    Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–823.

    CAS  Article  Google Scholar 

  27. 27.

    Ernst O, Vayttaden SJ, Fraser IDC. Measurement of NF-kappaB activation in TLR-activated macrophages. Methods Mol Biol. 2018;1714:67–78.

    CAS  Article  Google Scholar 

  28. 28.

    Pick E. Cell-free NADPH oxidase activation assays: “in vitro veritas”. Methods Mol Biol. 2014;1124:339–403.

    CAS  Article  Google Scholar 

  29. 29.

    Dickinson BC, Huynh C, Chang CJ. A palette of fluorescent probes with varying emission colors for imaging hydrogen peroxide signaling in living cells. J Am Chem Soc. 2010;132:5906–5915.

    CAS  Article  Google Scholar 

  30. 30.

    Ernst O, Zor T. Linearization of the Bradford protein assay. J Vis Exp. 2010;38:e1918.

    Google Scholar 

  31. 31.

    Zor T, Selinger Z. Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal Biochem. 1996;236:302–308.

    CAS  Article  Google Scholar 

  32. 32.

    Hatano M, Sasaki S, Ohata S, et al. Effects of Kupffer cell-depletion on concanavalin A-induced hepatitis. Cell Immunol. 2008;251:25–30.

    CAS  Article  Google Scholar 

  33. 33.

    Koga K, Takaesu G, Yoshida R, et al. Cyclic adenosine monophosphate suppresses the transcription of proinflammatory cytokines via the phosphorylated c-Fos protein. Immunity. 2009;30:372–383.

    CAS  Article  Google Scholar 

  34. 34.

    Huang B, Yang XD, Lamb A, Chen LF. Posttranslational modifications of NF-kappaB: another layer of regulation for NF-kappaB signaling pathway. Cell Signal. 2010;22:1282–1290.

    CAS  Article  Google Scholar 

  35. 35.

    Mills EL, O’Neill LA. Reprogramming mitochondrial metabolism in macrophages as an anti-inflammatory signal. Eur J Immunol. 2016;46:13–21.

    CAS  Article  Google Scholar 

  36. 36.

    Forman HJ, Torres M. Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. Am J Respir Crit Care Med. 2002;166:S4–S8.

    Article  Google Scholar 

  37. 37.

    Czapski GA, Cakala M, Kopczuk D, Strosznajder JB. Effect of poly(ADP-ribose) polymerase inhibitors on oxidative stress evoked hydroxyl radical level and macromolecules oxidation in cell free system of rat brain cortex. Neurosci Lett. 2004;356:45–48.

    CAS  Article  Google Scholar 

  38. 38.

    Pick E, Keisari Y. Superoxide anion and hydrogen peroxide production by chemically elicited peritoneal macrophages–induction by multiple nonphagocytic stimuli. Cell Immunol. 1981;59:301–318.

    CAS  Article  Google Scholar 

  39. 39.

    Aruoma OI, Halliwell B, Hoey BM, Butler J. The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic Biol Med. 1989;6:593–597.

    CAS  Article  Google Scholar 

  40. 40.

    Scott CL, Swisher EM, Kaufmann SH. Poly(ADP-ribose) polymerase inhibitors: recent advances and future development. J Clin Oncol. 2015;33:1397–1406.

    CAS  Article  Google Scholar 

  41. 41.

    Biro A, Vaknine H, Cohen-Armon M, et al. The effect of poly(ADP-ribose) polymerase inhibition on aminoglycoside-induced acute tubular necrosis in rats. Clin Nephrol. 2016;85:226–234.

    CAS  Article  Google Scholar 

  42. 42.

    Cover C, Fickert P, Knight TR, et al. Pathophysiological role of poly(ADP-ribose) polymerase (PARP) activation during acetaminophen-induced liver cell necrosis in mice. Toxicol Sci. 2005;84:201–208.

    CAS  Article  Google Scholar 

  43. 43.

    Lakatos P, Szabo E, Hegedus C, et al. 3-Aminobenzamide protects primary human keratinocytes from UV-induced cell death by a poly(ADP-ribosyl)ation independent mechanism. Biochim Biophys Acta. 2013;1833:743–751.

    CAS  Article  Google Scholar 

  44. 44.

    Jamil I, Symonds A, Lynch S, Alalami O, Smyth M, Martin J. Divergent effects of paracetamol on reactive oxygen intermediate and reactive nitrogen intermediate production by U937 cells. Int J Mol Med. 1999;4:309–312.

    CAS  PubMed  Google Scholar 

  45. 45.

    Mittal M, Siddiqui MR, Tran K, Reddy SP, Malik AB. Reactive oxygen species in inflammation and tissue injury. Antioxid Redox Signal. 2014;20:1126–1167.

    CAS  Article  Google Scholar 

  46. 46.

    Abd Elmageed ZY, Naura AS, Errami Y, Zerfaoui M. The poly(ADP-ribose) polymerases (PARPs): new roles in intracellular transport. Cell Signal. 2012;24:1–8.

    CAS  Article  Google Scholar 

  47. 47.

    Matsuzawa A, Saegusa K, Noguchi T, et al. ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity. Nat Immunol. 2005;6:587–592.

    CAS  Article  Google Scholar 

  48. 48.

    Le Page C, Sanceau J, Drapier JC, Wietzerbin J. Inhibitors of ADP-ribosylation impair inducible nitric oxide synthase gene transcription through inhibition of NF kappa B activation. Biochem Biophys Res Commun. 1998;243:451–457.

    Article  Google Scholar 

  49. 49.

    Zerfaoui M, Errami Y, Naura AS, et al. Poly(ADP-ribose) polymerase-1 is a determining factor in Crm1-mediated nuclear export and retention of p65 NF-kappa B upon TLR4 stimulation. J Immunol. 2010;185:1894–1902.

    CAS  Article  Google Scholar 

  50. 50.

    Hassa PO, Hottiger MO. The functional role of poly(ADP-ribose)polymerase 1 as novel coactivator of NF-kappaB in inflammatory disorders. Cell Mol Life Sci. 2002;59:1534–1553.

    CAS  Article  Google Scholar 

  51. 51.

    Masmoudi A, Mandel P. ADP-ribosyl transferase and NAD glycohydrolase activities in rat liver mitochondria. Biochemistry. 1987;26:1965–1969.

    CAS  Article  Google Scholar 

  52. 52.

    Ha HC, Hester LD,  Snyder SH. Poly(ADP-ribose) polymerase-1 dependence of stress-induced transcription factors and associated gene expression in glia. Proc Natl Acad Sci USA. 2002;99:3270–3275.

    CAS  Article  Google Scholar 

  53. 53.

    Hassa PO, Haenni SS, Buerki C, et al. Acetylation of poly(ADP-ribose) polymerase-1 by p300/CREB-binding protein regulates coactivation of NF-kappaB-dependent transcription. J Biol Chem. 2005;280:40450–40464.

    CAS  Article  Google Scholar 

  54. 54.

    Oliver FJ, Menissier-de Murcia J, Nacci C, et al. Resistance to endotoxic shock as a consequence of defective NF-kappaB activation in poly(ADP-ribose) polymerase-1 deficient mice. EMBO J. 1999;18:4446–4454.

    CAS  Article  Google Scholar 

  55. 55.

    Wei J, Dong S, Bowser RK, et al. Regulation of the ubiquitylation and deubiquitylation of CREB-binding protein modulates histone acetylation and lung inflammation. Sci Signal. 2017;10:eaak9660.

    Article  Google Scholar 

  56. 56.

    Menon D, Coll R, O’Neill LA, Board PG. Glutathione transferase omega 1 is required for the lipopolysaccharide-stimulated induction of NADPH oxidase 1 and the production of reactive oxygen species in macrophages. Free Radic Biol Med. 2014;73:318–327.

    CAS  Article  Google Scholar 

  57. 57.

    Wang K. Molecular mechanisms of hepatic apoptosis. Cell Death Dis. 2014;5:e996.

    CAS  Article  Google Scholar 

  58. 58.

    Kanno S, Ishikawa M, Takayanagi M, Takayanagi Y, Sasaki K. Characterization of hydrogen peroxide-induced apoptosis in mouse primary cultured hepatocytes. Biol Pharm Bull. 2000;23:37–42.

    CAS  Article  Google Scholar 

  59. 59.

    Shin SM, Cho IJ, Kim SG. Resveratrol protects mitochondria against oxidative stress through AMP-activated protein kinase-mediated glycogen synthase kinase-3beta inhibition downstream of poly(ADP-ribose)polymerase-LKB1 pathway. Mol Pharmacol. 2009;76:884–895.

    CAS  Article  Google Scholar 

  60. 60.

    Jouan-Lanhouet S, Arshad MI, Piquet-Pellorce C, et al. TRAIL induces necroptosis involving RIPK1/RIPK3-dependent PARP-1 activation. Cell Death Differ. 2012;19:2003–2014.

    CAS  Article  Google Scholar 

  61. 61.

    Liu Z, Li X, Ding X, Yang Y. In silico and experimental studies of concanavalin A: insights into its antiproliferative activity and apoptotic mechanism. Appl Biochem Biotechnol. 2010;162:134–145.

    CAS  Article  Google Scholar 

  62. 62.

    Imose M, Nagaki M, Naiki T, et al. Inhibition of nuclear factor kappaB and phosphatidylinositol 3-kinase/Akt is essential for massive hepatocyte apoptosis induced by tumor necrosis factor alpha in mice. Liver Int. 2003;23:386–396.

    CAS  Article  Google Scholar 

  63. 63.

    Wang K. Molecular mechanisms of hepatic apoptosis regulated by nuclear factors. Cell Signal. 2015;27:729–738.

    CAS  Article  Google Scholar 

  64. 64.

    Kanno S, Ishikawa M, Takayanagi M, Takayanagi Y, Sasaki K. Combination acetaminophen and doxapram potentiated hepatotoxicity in mouse primary cultured hepatocytes. Methods Find Exp Clin Pharmacol. 1999;21:647–652.

    CAS  Article  Google Scholar 

  65. 65.

    Shen W, Kamendulis LM, Ray SD, Corcoran GB. Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: effects of Ca(2+)-endonuclease, DNA repair, and glutathione depletion inhibitors on DNA fragmentation and cell death. Toxicol Appl Pharmacol. 1992;112:32–40.

    CAS  Article  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr. C. Daniels and M. Athamna for critical reading of the manuscript, to Dr. M. Cohen-Armon for various reagents and helpful discussions, to Dr. E. Pick and Dr. E. Bechor for the help with the superoxide assay and insightful discussions; to Dr. D. Shabat, N. Hananya and O. Green for help with fluorescence measurement; to Dr. C. Yu (Xiamen University, Xiamen, Fujian, China) for the TNFα promoter luciferase plasmid; to Dr. M. Montminy (Salk Institute, La-Jolla, CA) for the CRE-luciferase plasmid; to Dr. P. Angel (German Cancer Research Center, Heidelberg, Germany) for the AP-1-luciferase plasmid; to Dr. Ariel Munitz (TAU, Israel) for the iNOS antibody; and to Dr. Y. Ebenstein (TAU, Israel) for the EL-4 cell line.

Funding

This work was supported by the United States – Israel Binational Science Foundation [Grant 2011360 to TZ]. IF is supported by the Intramural Research Program of NIAID, NIH.

Author information

Affiliations

Authors

Contributions

JW, AB and TZ conceived and designed the study; OE, AL, HA, SK, IBN, IBD, DK, and KR performed the experiments; JW, OE, AL, HA, SK, DK, OB, KR, IF, RW, AB, and TZ analyzed the data; JW, OE, AB, and TZ wrote the manuscript.

Corresponding authors

Correspondence to Joram Wardi or Alexander Biro or Tsaffrir Zor.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval and consent to participate

All animal experiments were performed in accordance with the guidelines of the Care and Use of Laboratory Animals and have been approved by the research ethics committee at Wolfson medical center.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wardi, J., Ernst, O., Lilja, A. et al. 3-Aminobenzamide Prevents Concanavalin A-Induced Acute Hepatitis by an Anti-inflammatory and Anti-oxidative Mechanism. Dig Dis Sci 63, 3382–3397 (2018). https://doi.org/10.1007/s10620-018-5267-1

Download citation

Keywords

  • Liver failure
  • Inflammation
  • Macrophages
  • TNFα
  • Reactive oxygen species
  • NFκB