Elsevier

Biochimie

Volume 97, February 2014, Pages 173-180
Biochimie

Research paper
Protective effects of inhaled carbon monoxide in endotoxin-induced cholestasis is dependent on its kinetics

https://doi.org/10.1016/j.biochi.2013.10.009Get rights and content

Highlights

  • We studied the effect of inhaled CO on endotoxin-induced cholestasis in rats.

  • Detailed kinetic data and tissue distribution of inhaled CO are provided.

  • CO exposure substantially attenuates endotoxin-induced cholestatic liver injury.

  • CO modulates expression of key inflammatory and hepatic transporter genes.

  • Kinetics of CO is directly related to its anticholestatic effect.

Abstract

Carbon monoxide (CO), a product of heme oxygenase (HMOX), has many beneficial biological functions and is a promising therapeutic agent for many pathological conditions. However, the kinetics of inhaled CO and its protective role in endotoxin-induced cholestasis is not fully known. Thus, our objective was to characterize the kinetics of inhaled CO and then investigate its use in early phase experimental endotoxin-induced cholestasis.

Female Wistar rats were randomly divided into 4 groups: CON (control), LPS (lipopolysaccharide, 6 mg/kg), CO (250 ppm COx1h), and CO + LPS. Rats were sacrificed at 0–12 h after LPS administration. Tissues and blood were collected for liver injury markers and tissue CO distribution measurements. Livers were harvested for measurements of Hmox activity, Hmox1 mRNA expression, cytokines (IL10, IL6, TNF), and bile lipid and pigment transporters.

Half-lives of CO in spleen, blood, heart, brain, kidney, liver, and lungs were 2.4 ± 1.5, 2.3 ± 0.8, 1.8 ± 1.6, 1.5 ± 1.2, 1.1 ± 1.1, 0.6 ± 0.3, 0.6 ± 0.2 h, respectively. CO treatment increased liver IL10 mRNA and decreased TNF expression 1 h after LPS treatment and prevented the down-regulation of bile acid and bilirubin hepatic transporters (Slc10a1, Abcb11, and Abcc2, p < 0.05), an effect closely related to the kinetics. The protective effect of CO against cholestatic liver injury persisted even 12 h after CO exposure, as shown by attenuation of serum cholestatic markers in CO-treated animals.

CO exposure substantially attenuated endotoxin-induced cholestatic liver injury and was directly related to the kinetics of inhaled CO. This data underscores the importance of the kinetics of inhaled CO for the proper design of experimental and clinical studies of using CO as a treatment strategy.

Introduction

Carbon monoxide (CO) is a ubiquitous air pollutant and toxic gas, but also is an important endogenous signaling molecule, which regulates many biological functions in the body. This product of the heme catabolic pathway, which is catalyzed by the enzyme heme oxygenase (HMOX), plays an important role in inflammation, cell proliferation and cytoprotection [1], [2] and thus has a considerable therapeutic potential. Inhalation of low doses of CO has been shown to have potent cytoprotective properties in animal models of organ injury and disease [3]. According to the National Institutes of Health clinical trial database, the CO inhalation model is currently being used in studies investigating its use in the treatment of lung and cardiac diseases (ClinicalTrials.gov; identifier: NCT00094406, NCT01727167, accessed 12th March 2013). To evaluate the safety and effectiveness of using of CO inhalation as a treatment modality, it is critical to not only closely monitor blood CO-hemoglobin (COHb) levels in order to prevent CO poisoning; but also, to identify the optimal concentration of CO that needs to be delivered to target tissues. However, current knowledge about the kinetics of inhaled CO is still very limited.

Cholestasis is characterized as an impairment of bile formation and/or outflow. Although it is a serious complication of sepsis, the pathogenesis of cholestasis is still not fully known [4]. However, it has been shown that inflammatory cytokines released by endotoxins can down-regulate expression of hepatobiliary transporters and subsequently impair transport function and bile formation [4], [5].

Anti-inflammatory and pro-inflammatory cytokines have been shown to be regulated by the actions of CO via different mechanisms in both in vivo [6], [7] and in vitro studies [6], [7]. Nevertheless, a direct effect of CO on hepatic transporters in vivo has not been described. Yet, CO has been shown to be an important factor in maintaining the balance between liver sinusoidal perfusion and biliary transport [8], [9]. Furthermore, CO is recognized as a regulator of bile canalicular contractility [10]. Taken together, we believe that endogenous CO appears to play an important role in not only inflammation; but also, in the regulation of bile flow and liver integrity.

Therefore, the objective of this study was to first assess the kinetics of inhaled CO administration and then determine its potential use as a treatment for endotoxin-induced liver injury using a rat model.

Section snippets

Reagents

Bovine serum albumin (BSA), hemin, reduced nicotinamide adenine dinucleotide phosphate (NADPH), sulfosalicylic acid (SSA), ethylenediaminetetraacetic acid (EDTA), RNAlater, lipopolysaccharide from Escherichia coli 0111:B4 (LPS) were all purchased from Sigma–Aldrich (St. Louis, MO, USA). The CO (250 ppm) gas mixture and calibration gas (10 ppm) were purchased from Linde Gas (Prague, Czech Republic).

Animals and in vivo animal studies

Female Wistar rats (190–250 g), obtained from Anlab (Prague, Czech Republic), were allowed water

Kinetic profile of gaseous CO in vivo

COHb levels were 12.8 ± 1.9% of total hemoglobin (or 2116 ± 152 pmol CO/mg FW), a concentration considered to be safe in rats [15].

The kinetic profiles of inhaled CO were biphasic (fast and slow phases) in all the tissues. The rate of distribution to all the tissues occurred with no apparent delay. We noted non-zero Tmax values (n ≤ 2) for liver, spleen, and heart, while there were no non-zero Tmax samples in all the other tissues. CO levels in all selected tissues returned to control

Discussion

In this study, we first characterized in detail the kinetic profile of inhaled CO, and then we correlated these findings to observed changes in expressions of hepatic inflammatory and transporter genes. Initial studies investigating CO kinetics date back to the beginning of 20th century when CO was considered only a waste and toxic product from industrial processes. More than 20 years ago, the beneficial effects of CO were identified and since, CO has been intensively studied for its biological

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Author contribution

KV, LM and LV designed the study, KV, JS, TP, DC performed the experiments, HJV, RJW contributed GC-RGD for the study, KV, OS, HJV, RJW, LV, LM analyzed the data and wrote the paper. All authors approved the final article.

Acknowledgments

The work was supported by grants GAUK 251202, IGA MZ NT 11327-4/2010, and RVO-VFN64165/2013 given by the Grant Agency of the Charles University and Czech Ministry of Health, respectively, as well as by the SCIENTIA Foundation. The authors thank Marie Zadinova, Magdalena Kadlecova, Olina Svejdova and Jana Vanikova for excellent technical assistance during animal experiments.

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