Elsevier

Toxicology

Volume 267, Issues 1–3, 12 January 2010, Pages 147-153
Toxicology

Effect of astaxanthin on hepatocellular injury following ischemia/reperfusion

https://doi.org/10.1016/j.tox.2009.11.003Get rights and content

Abstract

This study investigated the effect of astaxanthin (ASX; 3,3-dihydroxybeta, beta-carotene-4,4-dione), a water-dispersible synthetic carotenoid, on liver ischemia–reperfusion (IR) injury. Astaxanthin (5 mg/kg/day) or olive oil was administered to rats via intragastric intubation for 14 consecutive days before the induction of hepatic IR. On the 15th day, blood vessels supplying the median and left lateral hepatic lobes were occluded with an arterial clamp for 60 min, followed by 60 min reperfusion. At the end of the experimental period, blood samples were obtained from the right ventricule to determine plasma alanine aminotransferase (ALT) and xanthine oxidase (XO) activities and animals were sacrificed to obtain samples of nonischemic and postischemic liver tissue. The effects of ASX on IR injury were evaluated by assessing hepatic ultrastructure via transmission electron microscopy and by histopathological scoring. Hepatic conversion of xanthine dehygrogenase (XDH) to XO, total GSH and protein carbonyl levels were also measured as markers of oxidative stress. Expression of NOS2 was determined by immunohistochemistry and Western blot analysis while nitrate/nitrite levels were measured via spectral analysis. Total histopathological scoring of cellular damage was significantly decreased in hepatic IR injury following ASX treatment. Electron microscopy of postischemic tissue demonstrated parenchymal cell damage, swelling of mitochondria, disarrangement of rough endoplasmatic reticulum which was also partially reduced by ASX treatment. Astaxanthine treatment significantly decreased hepatic conversion of XDH to XO and tissue protein carbonyl levels following IR injury. The current results suggest that the mechanisms of action by which ASX reduces IR damage may include antioxidant protection against oxidative injury.

Introduction

Ischemia–reperfusion injury of the liver is an important clinical problem in many clinical conditions such as liver transplantation, hepatic surgery for tumor excision, trauma and hepatic failure after hemorrhagic shock (Lemasters and Thurman, 1997, Olthoff, 2001). Partial or, mostly, total interruption of hepatic flow is often necessary when liver surgery is performed. This interruption of blood flow is termed as “warm ischemia” and upon revascularization, when molecular oxygen is reintroduced, the organ undergoes a process called “reperfusion injury” that causes deterioration of organ function (Hasselgren, 1987). Although the mechanisms by which organ damage occurs in I/R injury are incompletely understood, it has been suggested that reperfusion of the liver following ischemia, triggers the activation of kupffer cells causing oxygen free radical formation, production of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) (McCord, 1985, Adkison et al., 1986, Carden and Granger, 2000). Elevated levels of the pro-inflammatory cytokines TNF-α and IL-1 promote PMN recruitment and activation which also generates reactive oxygen species (ROS) and leads to the release of proteases (Colletti et al., 1996, Jaeschke et al., 1990). Although increased expression of NOS2 and elevated tissue nitrite (NO2) and nitrate (NO3) occurs in animal models of I/R (Isobe et al., 2000) formation of ROS can scavange NO produced by the sinusoidal endothelial cells and lead to vasoconstriction and narrowing of the sinusoidal lumen (Aslan and Freeman, 2002).

Reports showing that antioxidant deficiency exacerbate I/R injury and the beneficial outcome of pharmacological interventions such as superoxide dismutase, catalase, vitamin E, or desferrioxamine, support ROS as a major pathophysiological component of I/R (Atalla et al., 1985, Marubayashi et al., 1986, Drugas et al., 1991).

Astaxanthin is a naturally occurring carotenoid pigment and is a powerful biological antioxidant (Palozza and Krinsky, 1992). It exhibits free radical scavenging activity and protects against lipid peroxidation and oxidative damage to cell membranes, cells, and tissues (Lim et al., 1992). Astaxanthine has a molecular structure similar to that of β-carotene. However, it has 13 conjugated double bonds, in contrast to 11 in β-carotene, which gives it significantly greater antioxidant capacity (Shibata et al., 2001). Moreover, ASX has hydroxyl groups in the 3 and 3′ positions, making the molecule highly polar (Fig. 1) and dramatically enhancing its membrane function to protect against degenerative conditions (Shibata et al., 2001). Because of its polar end groups, ASX spans the cell membrane bilayer allowing it to sit near the lipid/water interface, where free radical attack first occurs and contributes to cell membrane mechanical strength (Palozza and Krinsky, 1992). Astaxanthin stabilizes free radicals by adding them to its long double-bond chain rather than donating an atom or electron to the radical. Consequently, it can resist chain reactions that occur when a fatty acid is oxidized, thus allowing it to scavenge or quench longer than antioxidants that cannot stop this chain reaction (Kurashige et al., 1990).

The liver has effective mechanisms for inactivating and then excreting foreign substances through biotransformation. These functions can lead to significant release of free radicals and oxidation byproducts and therefore it is important to have mechanisms that protect liver cells against oxidative damage. It has been shown that astaxanthin is much more effective than vitamin E in protecting mitochondria from rat liver cells against lipid peroxidation (Guerin et al., 2003, Kurashige et al., 1990).

Previous studies on animal models of IR-induced myocardial injury have shown a linear correlation between the plasma concentrations of astaxanthin and the extent of infarct size reduction (Gross and Lockwood, 2004, Gross and Lockwood, 2005) Based on this information, the current experimental protocol was designed to determine whether the tissue protective effect of ASX could be observed in a rat model of liver IR injury. We aimed to determine whether ASX, a potent antioxidant, had any effect on plasma levels of liver enzymes, tissue markers of oxidative stress and histopathologic alterations in an experimental rat model of hepatic I/R.

Section snippets

Animals

All experimental protocols conducted on rats were performed in accordance with the standards established by the Institutional Animal Care and Use Committee at Akdeniz University Medical School. Male Wistar rats weighing 350–450 g were housed in stainless steel cages and given food and water ad libitum. Animals were maintained at 12 h light–dark cycles and a constant temperature of 23 ± 1 °C at all times. Rats were randomly divided into two groups of 12 animals each which received olive oil or 5 

ALT, XO and XDH enzyme activity

Plasma ALT and XO levels were significantly increased in all IR groups. Treatment of ASX significantly decreased liver XO levels under basal conditions and increased XDH/XO ratio when compared to both control and IR groups. XDH/XO ratio was greater in I/R livers treated with ASX as compared to the non-treated IR group (Table 1).

Histological analysis

Congestion, intracellular edema and necrosis were significantly greater (*p < 0.05) in IR and ASX + IR groups as compared to ASX and control (Fig. 2, Table 2). Although

Discussion

This study examined the effect of ASX treatment on liver injury resulting from I/R. Astaxanthin was dissolved in olive oil and administered at a dose 5 mg/kg/day via oral gavage for 14 consecutive days before the induction of hepatic I/R. Plasma appearance and tissue accumulation of ASX was studied previously in rodents after single- and multiple-dose regimens (Showalter et al., 2004). One time dosing at 500 mg/kg resulted in significant appearance of ASX in plasma (0.2 mg/L) and liver (0.9 mg/L).

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

This study was supported by a grant (No: 2007.04.0103.002) from Akdeniz University Research Foundation.

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