Elsevier

Toxicology in Vitro

Volume 27, Issue 1, February 2013, Pages 378-386
Toxicology in Vitro

Histamine at low concentrations aggravates rat liver BRL-3A cell injury induced by hypoxia/reoxygenation through histamine H2 receptor in vitro

https://doi.org/10.1016/j.tiv.2012.07.014Get rights and content

Abstract

Aim

Histamine released from mast cell degranulation participates in the pathogenesis of ischemia/reperfusion injury. The purpose of our study was to define the role of histamine in hypoxia/reoxygenation mediated liver cell injury and to elucidate the underlying mechanism in vitro.

Methods

Histamine alone or in combination with H1 receptor antagonist (pyrilamine), H2 receptor antagonist (cimetidine) or H3/4 receptor antagonist (thioperamide) at different concentrations before hypoxia was added to rat liver BRL-3A cell which was subjected to 24 h hypoxia followed by 4 h reoxygenation. Cell proliferation, apoptosis and the changes of ultrastructure were assessed, and MDA contents, SOD activities and ALT levels were quantified as well.

Results

Histamine (from 10−3 to 10−9 M) did not affect the growth of BRL-3A cells without hypoxia treatment. However, histamine 10−8 M significantly lowered the growth of BRL-3A cells challenged by hypoxia/reoxygenation, accompanied with concomitant elevations in MDA contents and decreases in SOD activities, all these changes were blocked by cimetidine, not by pyrilamine or thioperamide. However, histamine (above 10−6 M) did not show exacerbating effects in BRL-3A cell subjected to hypoxia/reoxygenation.

Conclusion

Histamine at low concentrations (10−7–10−9 M) aggravates hypoxia/reoxygenation mediated BRL-3A damage through histamine H2 receptor.

Highlights

► Histamine below 10−7 M aggravates BRL-3A cell damages triggered by hypoxia/reoxygenation. ► The deleterious effects of histamine are through histamine H2 receptor. ► Histamine alone does not affect the viability rates of BRL-3A cell under normal condition.

Introduction

Hepatic inflow occlusion (pringle’s maneuver) offers an effective strategy to reduce blood loss in liver surgery (Park et al., 2012), however, this strategy often leads to hepatic ischemia/reperfusion injury (HIRI) which is associated with postoperative liver dysfunction (Talmon and Wisecarver, 2010, Heizmann et al., 2008). Therefore, it is imperative to explore the underlying mechanisms of HIRI. Several lines of evidence so far have demonstrated that reactive oxygen species, mitochondrial dysfunction, calcium overload, leukocyte infiltration, cytokine release, and microcirculation failure are implicated in HIRI (Zhang et al., 2007, Puhl et al., 2005, Abu-Amara et al., 2010, Montalvo-Jave et al., 2008).

Mast cells are widely present in the skin, lung, gastrointestinal tract and liver. There are increasing interests to define the role of mast cells in the process of ischemia/reperfusion injury. Previous studies have revealed that mast cells are activated to release numerous mediators during ischemia/reperfusion (Akdis and Simons, 2006, Jin et al., 2009), and mast cell inhibition has provided protective effects against ischemia reperfusion injury (Hei et al., 2008, Jaggi et al., 2007). In addition, accumulation of hepatic mast cells has been proved to play an central role in the experimental liver diseases (Francis and Meininger, 2010), meanwhile, the concomitant increases of intestinal mucosal mast cells in rats undergoing partial hepatectomy have also been confirmed (Moquillaza et al., 2010). Hence, mast cell degranulation could be considered as a pathogenic factor in the development of HIRI.

Histamine, one of characteristic makers released from mast cell degranulation, contributes to many disorders (Schneider et al., 2010), it should be noted that the histamine levels in the portal vein were dramatically enhanced during liver transplantation (Hansen et al., 1988). A very recent study have demonstrated that subcutaneous injecting histamine with 10 days markedly resulted in liver dysfunction in rabbit (Tripathi et al., 2011). Therein, histamine may contribute to HIRI based on the previous findings. The histamine mediated effects are through four types of histamine receptors: H1, H2, H3 and H4 receptors. However, the precise mechanisms by which histamine confers HIRI remain largely unknown.

In order to define the role of histamine and its signal pathway in liver ischemia/reperfusion injury, the present study was designed to establish a hypoxia/reoxygenation model in vitro by using hepatocyte (BRL-3A cells), one rat liver derived cell line, which has been found to express four histamine receptors, and to investigate the effects of histamine alone or combined with histamine receptor antagonists on cell viability, apoptosis, oxidative stress and changes of ultrastructure.

Section snippets

Reagents

Histamine, pyrilamine, cimetidine and thioperamide were purchased from sigma Chemical (St. Louis, MO). Cell Counting Kit-8 and SOD assay kit-WST were from Dojindo company (Japan), ALT/GPT kit and MDA kit were purchased from Jiancheng biochemical company (China), and Annexin V-FITC apoptosis detection kit was from Multisciences Biotech (China), PrimeScript RT Reagent Kit and SYBP Premix Ex Taq were obtained from Takara Biosciences (Japan). All other chemicals used were of the highest grade

Different duration of hypoxia/reoxygenation on viability rate of BRL-3A

The first experiment was to adjust an appropriate model of hypoxia/reoxygenation in vitro. BRL-3A cells (5 × 103/well) were seeded into 96-well plates and randomly divided into seven groups: control group, H2R24, H4R24, H8R24, H24R4, H24R8, and H24R24 groups. The last six groups were received hypoxia for 2 h, 4 h, 8 h and 24 h followed by reoxgenation for 4 h, 8 h or 24 h.

The results (Fig. 1A) showed that a progressive decrease of viability rate induced by hypoxia/reoxygenation was found in a

Discussion

It has been noted that histamine participates in hepatic ischemia–reperfusion injury (Adachi et al., 2006), and the histamine mediated effects have been demonstrated through specific histamine receptors, including H1, H2, H3 and H4 subtypes. In the current study, we found that histamine at the concentrations ranging from 10−7 to 10−9 M enhanced the injuries in BRL-3A rat liver cell under the challenge of 24 h hypoxia followed by 4 h rexygenation, of note, those deleterious effects were blocked by

Acknowledgment

This work was supported by National Natural Science Foundation of China (81170449 and 30901408).

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