Oxidative DNA damage and repair in skeletal muscle of humans exposed to high-altitude hypoxia

Abstract

Recent research suggests that high-altitude hypoxia may serve as a model for prolonged oxidative stress in healthy humans. In this study, we investigated the consequences of prolonged high-altitude hypoxia on the basal level of oxidative damage to nuclear DNA in muscle cells, a major oxygen-consuming tissue. Muscle biopsies from seven healthy humans were obtained at sea level and after 2 and 8 weeks of hypoxia at 4100 m.a.s.l. We found increased levels of strand breaks and endonuclease III-sensitive sites after 2 weeks of hypoxia, whereas oxidative DNA damage detected by formamidopyrimidine DNA glycosylase (FPG) protein was unaltered. The expression of 8-oxoguanine DNA glycosylase 1 (OGG1), determined by quantitative RT-PCR of mRNA levels did not significantly change during high-altitude hypoxia, although the data could not exclude a minor upregulation. The expression of heme oxygenase-1 (HO-1) was unaltered by prolonged hypoxia, in accordance with the notion that HO-1 is an acute stress response protein. In conclusion, our data indicate high-altitude hypoxia may serve as a good model for oxidative stress and that antioxidant genes are not upregulated in muscle tissue by prolonged hypoxia despite increased generation of oxidative DNA damage.

Introduction

Oxidative stress is a well-known toxicological mechanism that may cause damage to cellular macromolecules. Short-term oxidative stress exposures can be studied in humans, e.g. after exhaustive exercise (Møller et al., 1996), hyperbar oxygen treatment (Dennog et al., 1996), or after surgery involving ischemia and reperfusion (Willy et al., 2000). However, the effect of prolonged oxidative stress is less investigated in healthy humans. This could be because most models of oxidative stress are not applicable to prolonged exposures, e.g. prolonged hyperbaric oxygen exposure may cause lung damage and death. We have investigated high-altitude hypoxia as a model of prolonged oxidative stress. Several lines of evidence suggest that high-altitude hypoxia is associated with oxidative stress (Askew, 2002). The lack of oxygen supply relative to the metabolic need (hypoxia) appears paradoxically to be associated with increased production of reactive oxygen species (ROS) and oxidative stress (Li and Jackson, 2002). Hypoxia appears to increase mitochondrial production of ROS (Li and Jackson, 2002), and prolonged hypoxia also seems to activate the immune system (Møller et al., 2001). Moreover, environmental factors other than hypoxia, such as the cold climate, ultraviolet light, and physical demands may also contribute to the oxidative stress observed by high-altitude hypoxia (Askew, 2002).

The consequences of ROS generation can be detected by formation of oxidatively altered biomolecules, including DNA, lipids, and protein. Oxidative DNA damage encompasses a wide range of lesions, including base damage and strand breaks (SB) (Møller and Wallin, 1998). Previously we have shown that high-altitude hypoxia was associated with increased steady-state level of oxidative DNA damage in circulating mononuclear blood cells and higher urinary excretion of the specific oxidative DNA base damage, 7-hydro-8-oxo-2′deoxyguanine (8-oxodG) (Møller et al., 2001). Other studies have shown increased lipid peroxidation in the circulatory compartment of humans (Joanny et al., 2001; Bailey et al., 2001a, Bailey et al., 2001b; Wozniak et al., 2001), and of rats following exposure to hypoxia (Yoshikawa et al., 1982, Nakanishi et al., 1995, Kumar et al., 1999, Ilavazhagan et al., 2001). As a measurement of whole-body lipid peroxidation, breath pentane was increased among subjects in high-altitude hypoxia (Simon-Schnass and Pabst, 1988). Also, exercising in hypoxia appears to be associated with increased lipid peroxidation in the circulatory compartment (Vasankari et al., 1997, Chao et al., 1999, Pfeiffer et al., 1999, Wozniak et al., 2001), and excretion of 8-oxodG in urine (Chao et al., 1999, Schmidt et al., 2002). These data suggest that high-altitude hypoxia induces a general oxidative stress situation in the whole body.

The aim of this study was to investigate the effect of prolonged high-altitude hypoxia in human skeletal muscle, a major oxygen-consuming organ. In a resting state, approximately 20% of the blood flow is distributed to skeletal muscles. Thus, it is likely that muscle tissue produces ROS in hypoxia. Determined from indices of lipid damage intermittent exposure to hypoxia simulated to 4000 m.a.s.l. increased rat skeletal muscle lipid peroxidation in the soleus (Radak et al., 1994) but not in the quadriceps and gastrocnemius muscles (Radak et al., 1997, Nakanishi et al., 1995). Reactive carbonyl derivates, a marker for protein oxidation also was increased in rat skeletal muscle in hypoxia (Radak et al., 1997). To the best of our knowledge, human muscle tissue has not been examined for oxidative DNA damage in prolonged high-altitude-exposed subjects.

The level of oxidative damage in nuclear DNA was determined in muscle biopsies from healthy human subjects at sea level, and after 2 and 8 weeks of hypoxia by single-cell gel electrophoresis (comet) assay. The comet assay detects SB, and by including DNA glycosylase enzymes oxidatively altered purines can be detected by formamidopyrimidine DNA glycosylase (FPG), and oxidative pyrimidines by endonuclease III (ENDOIII). In addition, we determined the expression of the mRNAs of 8-oxoguanine DNA glycosylase (OGG1) and heme oxygenase-1 (HO-1) by real-time quantitative RT-PCR. OGG1 encodes a DNA glycosylase that removes 8-oxodG (Roldan-Arjona et al., 1997). HO-1 catalyzes the first and the rate-limiting step in the oxidative degradation of heme to bilirubin, and is often observed as an oxidative stress response protein. Recent results suggest that HO-1 is involved in adaptive protection to oxidative stress, e.g. HO-1 protects against induction of oxidative DNA damage in human lymphocytes following hyperbaric oxygen treatment (Rothfuss et al., 2001).