Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Hong Kong SAR
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, 1/EF Prince of Wales Hospital, Shatin-New Territories, Hong Kong SAR. e-mail: msrogers{at}cuhk.edu.hk
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Abstract |
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Key words: carbon dioxide/helium/hypoxia/isoprostanes/mesothelial cells
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Introduction |
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The laparoscopic approach induces oxidative stress: we observed that during laparoscopic surgery for benign gynaecological conditions, CO2-pneumoperitoneum increased the levels of 8-iso-prostaglandin F2 (8-iso-PGF2
) in the peritoneum (Bentes de Souza et al., 2003a
). Other lipid peroxidation products have been shown to rise systemically (Glantzounis et al., 2001
). The cause of oxidative stress during laparoscopy may be multifactorial, resulting from elevated intra-abdominal pressure and/or from changes induced directly by CO2 contact or indirectly by desiccation. Abdominal pressure probably induces oxidative stress through disturbance of the splanchnic microcirculation. We have demonstrated in an animal model that pneumoperitoneum increases the levels of 8-iso-PGF2
in a time- and pressure-dependent manner (Bentes de Souza et al., 2003b
). The mechanisms by which CO2 induces oxidative stress are unclear, but may be related to its dry-cold state, its acidification properties, or to its high partial pressures (99%) displacing oxygen to produce a hypoxic condition.
Oxidative stress can damage any cellular constituent, whether composed of lipid, protein or nucleic acids (Davies, 2000). Products of DNA or protein oxidation can be used to quantify oxidative stress, although the conventional approach has been to measure products of lipid peroxidation. Lipid peroxidation results in formation of organic hydroperoxides, degradation products such as alkanes and aldehydes (Esterbauer, 1995
), conjugated dienes such as malondialdehyde, and isoprostanes (Morrow et al., 1992
). 8-iso-PGF2
is a member of the isoprostane family and is generated initially in situ in cell membranes by reactive oxygen species (ROS) attacking arachidonic acid. It is then cleaved, presumably by phospholipases, and circulates in plasma until it is excreted by the kidney (Morrow et al., 1994
). It is detectable in all normal biological fluids and tissues, and has a strong link with hypoxiareperfusion phenomena (Delanty et al., 1997
).
This study was designed to investigate the effect of transient exposure to CO2 on 8-iso-PGF2 levels in human peritoneal mesothelial cells. The duration of CO2 exposure and the influence of pH were also examined.
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Materials and methods |
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Cell isolation and culture
Mesothelial cells were isolated from normal peritoneum of two patients (age 38 and 40 years) undergoing total abdominal hysterectomy for uterine fibroids. One peritoneum sample, 2 x 2 cm2, was obtained from the anterior abdominal wall immediately after reaching the abdominal cavity. Patients were informed about the procedure, and gave written consent to the additional tissue collection. This study was approved by the Human Research Ethics Committee of the Chinese University of Hong Kong.
Harvested tissue was rinsed immediately with pre-warmed DMEM, and placed in a digesting solution containing 0.2% collagenase (type 1) for 3 h at 37°C. The tissue was then gently scraped with a sterile spatula, and the cells and medium were collected and centrifuged for 5 min at 300 g. The cell pellet was suspended in a pre-warmed growth medium (DMEM + 10% FBS + 2% penicillinstreptomycin) and transferred into a 4-well dish, which was placed in a humidified incubator with 5% CO2 maintained at 37°C.
The outgrowth of mesothelial cells took 5 days on average. When confluence was achieved, the cells were transferred to 6-well culture plates. Thereafter, the confluent mesothelial cells were harvested by trypsinization and subcultured (1:3 split ratio) to obtain an adequate number of cells for the experiments.
Cell characterization
Cultured cells were grown in Lab-Tek chamber slides. To determine the relative contribution of mesothelial and contaminating stromal, fibroblast cells, the primary cultures were subjected to immunocytochemistry. The culture medium was removed and the cells washed twice in PBS. Cells were fixed by incubating at room temperature for 15 min in 1% paraformaldehyde in PBS, which was washed from the cells by three changes of PBS. Excess PBS was removed and 10% horse serum in PBS was added for 20 min at room temperature to block non-specific binding of the primary and secondary antibodies. Excess blocking solution was removed by washing in PBS for 5 min at room temperature. The cells were then incubated with either mouse anti-human cytokeratin (1:50) or mouse anti-human fibroblasts (1:50) in 1.5% horse serum for 2 h at room temperature. Horse serum (1.5%) alone was used as a negative control for the primary antibody. Excess primary antibody was removed by washing three times in PBS. The cells were then incubated with rabbit anti-mouse antibody (1:50) in 1.5% horse serum at room temperature for 45 min. Excess secondary antibody was removed by washing three times in PBS. The cells were counterstained with 0.1% methyl green in 70% glycerol and examined under a fluorescent microscope (Nikon, Yokohama, Japan).
Experiments
The experiments were performed in duplicate using cells from different patients in each trial. Cells from the third passage were seeded in three 6-well culture plates/group at 1 x 106 cells/well, and incubated in DMEM + 5% FBS + 2% penicillinstreptomycin for 12 h before being assigned to one of the following groups: (i) exposure to 100% CO2 for 4 h; (ii) exposure to 100% helium for 4 h; (iii) exposure to 100% CO2 for 24 h; and (iv) parallel cultures kept under standard conditions (5% CO2, 95% relative humidity and 37°C) to act as control samples.
Gas exposure was performed using a modular chamber, which was deoxygenated by a positive infusion of gas (either CO2 or helium) with a continuous flow for 10 min. Both gas input and output were maintained open during this period. Thereafter, they were tightly closed and the chamber was placed inside a 37°C incubator for either 4 or 24 h according to the designated group. To prevent excessive pressure inside the chamber, two precautions were taken: (i) the gas input was closed before the gas output after 10 min of gas infusion; and (ii) the gas output was opened as soon as the temperature inside the chamber reached 37°C (15 min after placing the chamber inside the incubator). Gas humidification was achieved by placing an open 145 cm2 culture dish containing distilled water inside the modular chamber. Preliminary experiments in our laboratory have shown that this procedure results in a relative humidity of 95% inside the chamber when it is placed in the incubator at 37°C.
At the end of the incubation period, the plates were transferred to standard conditions and harvested at three different time points: (i) immediately after gas exposure (T0); (ii) 1 h after gas exposure (T1); and (iii) 3 h after gas exposure (T3).
Cells were harvested using a sterile cell scraper, and 100 µl of the cell suspension was taken for cell viability determination. The remainder were centrifuged at 300 g for 2 min. Cell pellets were snap-frozen in liquid nitrogen, and stored at 80°C for later analysis. Cell viability was assessed by trypan blue dye exclusion at each harvesting time point after gas exposure. The pH of the culture medium was measured to quantify the variations that occurred after transient exposure to the test gases and how quickly these changes come back to normal after returning cultured cells to standard conditions. The pH of the culture medium was assessed before gas exposure, and at each harvesting time point using a calibrated pH probe (Thermo Orion, Beverly, MA).
Three culture plates were used in each experimental group per trial, and one culture plate was harvested per time point. Three to four readings (each containing 1 x 106 cells) were performed on each culture plate. The data represent the median of 68 readings per experimental group.
Oxidative metabolite measurement
Esterified 8-iso-PGF2 was assayed by enzyme-linked immunosorbent assay (ELISA) using a modified method previously described (Bentes de Souza et al., 2003a
). Briefly, each cell pellet (
1 x 106 cells) was homogenized with 1 ml of ice-cold 100% methanol, containing 0.005% BHT, using a 2.5 ml syringe and 2527 gauge needle. Homogenized pellets were vortexed and stored on ice for 5 min. A 50 µl aliquot was taken for phospholipid determination according to a method described by others (Mrsny et al., 1986
). A 1 ml aliquot of 15% potassium hydroxide was added to the lysate, which was kept at 40°C for 30 min. The samples were diluted with Ultra pure water (pH 3), and then eluted through Sep-Pak C18 and Sep-Pak Silica cartridges. The elution was developed in an ELISA kit and analysed on a spectrophotometric microplate reader (Spectramax, Bio-Tek Instruments, Winooski, USA) at 412 nm wavelength. Cross-reactivity for the enzyme immunoassay was 100% with 8-iso-PGF2
, 20% with 8-iso-PGF3
, and <1% with other prostaglandin metabolites. In this study, [3H]8-iso-PGF2
was used as internal standard for method validation and calibration. The 8-iso-PGF2
concentration in mesothelial cells is presented in pg/µg phospholipid.
Statistical analysis
The variability between patients was not significant. Non-parametric tests were used because 8-iso-PGF2 values were not symmetrically distributed even after logarithmic transformation. Between-group comparisons were performed using KruskalWallis and MannWhitney U-tests, and within-group comparisons were performed using Wilcoxon signed rank test. They were conducted using SPSS, version 11. Statistical significance was determined at P < 0.05.
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Results |
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Effects of CO2 and helium on pH
Exposure to both CO2 and helium induced marked changes in the culture medium pH. Within-group comparisons (Wilcoxon signed rank test) showed significant differences between pH values before and after (T0, T1 and T3) gas incubation in all three treated groups. The biggest changes were observed at T0 where exposure to 100% CO2 caused a significant decrease in the culture medium pH from 7.43 and 7.42 to 6.34 and 6.29 (4 and 24 h, respectively), and exposure to 100% helium increased the pH from 7.42 to 8.33. These values (for both CO2 groups) gradually returned to baseline levels after placing the plates under standard conditions. A similar trend occurred in the 4 h helium group, but significant differences were still observed at T3.
Between-group comparisons (KruskalWallis test) showed significant differences in culture medium pH at all time points. Further analyses between control and treated groups (100% helium, 4 and 24 h 100% CO2, MannWhitney U-test) revealed significant differences at T0 and T1 (P < 0.001), but only the 4 h helium group differed significantly from the control group at T3 (P < 0.001). The duration of CO2 exposure augmented the pH drop, as a significant difference was found between 4 and 24 h groups at T0 (P < 0.001). Table I shows the variations of culture medium pH during the experimental period in the four groups.
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Prolongation of the CO2 incubation period from 4 to 24 h had a more pronounced effect on the 8-iso-PGF2 levels (Table II). Between-group comparisons (KruskalWallis test) showed significant differences (24 h > 4 h > control) at T0 (P < 0.01) and T1 (P < 0.001), but not at T3 (P > 0.05). Further analyses using two group comparisons (MannWhitney U-test) demonstrated that the differences at T0 were predominantly between 24 h CO2 and control groups (P < 0.001), as no significant changes were observed between either the 24 and 4 h groups (P > 0.05) or between the 4 h and control groups. At T1, the 8-iso-PGF2
levels in the 24 h CO2 group were significantly higher than in the control and 4 h CO2 groups (P < 0.001 and P < 0.01, respectively).
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Effects of helium on 8-iso-PGF2
In this study, 100% helium was used as an alternative gas to create hypoxic conditions. Comparison of the 4 h helium group with the control group (MannWhitney U-test) showed that 8-iso-PGF2 levels were significantly higher in the 4 h helium group at all time points (Table II): T0 (P < 0.001), T1 (P < 0.001) and T3 (P < 0.001). Comparison of the 4 h helium and 4 h CO2 groups also showed significant differences at all time points (T0, P < 0.001; T1, P < 0.01; and T3, P < 0.01). No significant changes were observed over time within the 4 h helium group (P > 0.05, Wilcoxon signed rank test).
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Discussion |
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There are thought to be two major sources of ROS generated during hypoxiareoxygenation: xanthine oxidase and the mitochondrial respiratory chain. During hypoxia, the breakdown of ATP results in accumulation of the purine metabolite hypoxanthine, and the enzyme xanthine dehydrogenase is transformed into a xanthine oxidase (Kehrer et al, 1987). Under normoxic conditions, hypoxanthine is converted sequentially to xanthine and uric acid by xanthine dehydrogenase without generating free radicals. Xanthine oxidase catalyses the same reaction, but yields superoxide radicals in the presence of molecular oxygen, which acts as an electron acceptor (Nishino, 1994
).
Hypoxia also brings oxidative phosphorylation to a halt, thus inhibiting aerobic ATP synthesis. This condition leaves the mitochondrial carriers (ubiquinone) in a more reduced state, which in turn results in an increase in electron leakage from the respiratory chain (Freeman and Crapo, 1982). These electrons react with the residual molecular oxygen trapped within the inner mitochondrial membrane, leading to the formation of superoxide radicals. Reintroduction of oxygen re-energizes the mitochondria, but electron efflux through cytochrome oxidase is reduced because of the lack of ADP. As a result, the percentage of electron leakage further increases, enabling more reactions with molecular oxygen, and consequently more production of superoxide radicals (Liu et al., 1996
).
We therefore presume that during the 4 h CO2 incubation, some disarrangement in cellular metabolism occurred, but the production of ROS did not overcome the cellular anti-oxidant capacity as no significant change in 8-iso-PGF2 was observed immediately after gas exposure. The rise in 8-iso-PGF2
levels observed 1 h after gas incubation supports our hypothesis that it was the return to standard conditions (reoxygenation period) that led to overproduction of ROS in this group.
In this study, we also verified that the duration of gas exposure influences the overproduction of ROS in mesothelial cells. Increasing the CO2 incubation period from 4 to 24 h produced higher 8-iso-PGF2 levels than 4 h of exposure. In the 24 h exposed cells, a significant rise was observed immediately following gas incubation, which implies that overproduction of ROS occurred during the hypoxic period, and not only following reoxygenation. A further increase in 8-iso-PGF2
level was observed after returning the cells to normoxic conditions (T1) but, as with the 4 h exposed cells, this rise was transient, with 8-iso-PGF2
levels returning to normal within 3 h of the reintroduction of oxygen. The findings of these two experiments (4 and 24 h) show that exposure to 100% CO2 impacts on the mesothelial cell oxidative status in a time-dependent manner. They also imply that the effect of the oxidant insult was temporary regardless of the duration of CO2 exposure, as no difference was found between control, 4 h CO2 and 24 h CO2 groups at T3 (KruskalWallis test, P > 0.05). The decline in 8-iso-PGF2
levels at T3 in both groups (4 and 24 h CO2) may be a result of its removal from cell membranes. Previous observations have shown that 8-iso-PGF2
once formed is hydrolysed, presumably by phospholipases (Basu, 1998
), and accumulates in its free form until it is metabolized and excreted by the kidney (Morrow et al., 1992
). Since only esterified 8-iso-PGF2
was quantified in the present study, the return of 8-iso-PGF2
levels to baseline values may have been due to its cleavage and subsequent diffusion into the culture medium.
CO2 is the standard gas used for abdominal insufflation during laparoscopy. We have demonstrated previously that the laparoscopic approach, when compared with laparotomy, significantly increased the peritoneal levels of 8-iso-PGF2 (Bentes de Souza et al., 2003a
). The mechanisms involved in the process were unclear, but were thought to be related to intra-abdominal pressure and/or the gas used. The findings of the present investigation in association with those of an animal study (Bentes de Souza et al., 2003b
) support this hypothesis. Furthermore, the rise of 8-iso-PGF2
in the 4 h CO2 group at T1, but not T0, in this study explains the observation that in animals submitted to low intra-abdominal pressure 8-iso-PGF2
only rose after pneumoperitoneum release (reoxygenation) (Bentes de Souza et al., 2003b
). Both findings suggest that although exposure to 100% CO2 under mild conditions induces changes in cellular metabolism during the hypoxic period, it is only following reoxygenation that the overproduction of ROS occurs.
Helium is considered an alternative gas for the creation of a pneumoperitoneum. It is both inert and minimally absorbed; and causes less hypercapnoea and acidaemia than CO2 (Bongard et al., 1993). In this study, we used 100% helium as an option to create a hypoxic environment without acidification of the culture medium. We expected that induction of hypoxia without extracellular acidification would indicate whether low pH is involved in the overproduction of ROS, or whether this is purely a hypoxiareoxygenation phenomenon. Unfortunately, following helium exposure, not only were 8-iso-PGF2
levels significantly higher than in both control and CO2 groups at all time points (T0, T1 and T3) but the pH of the culture medium also became alkaline, rising to 8.33 (T0). In both CO2 and helium groups, the pH medium returned to baseline levels by T1 (Table I). These results indicate that a 4 h hypoxic insult induces more oxidative stress (as indicated by 8-iso-PGF2
concentrations) under alkaline than under acidic conditions.
Alkaline conditions increase the mitochondrial transmembrane potential, altering various mitochondrial functions (Bolwell et al., 1995). Under alkaline conditions, protons diffuse away from the mitochondria, which compensate by increasing respiration, which in turn produces more superoxide radicals (Majima et al., 1998
). In our study, alkalinization of the culture medium may therefore have been responsible for the higher levels of 8-iso-PGF2
observed after exposure to helium. The persistence of these high levels of 8-iso-PGF2
at T3 could be explained by inhibition of phospholipase activity under alkaline conditions; preventing cleavage of the molecule from the cell membrane (Grotendorst and Hessinger, 1999
).
The variation in the pH of the mesothelial cell culture medium observed in this study may be explained by the buffering system of the medium used. DMEM has a CO2/HCO3 buffering system, which means that its pH is maintained by an exogenous supply of CO2 in conjunction with a HCO3 ion (in the form of NaHCO3) present in the medium. The CO2/HCO3 buffering system is indispensable for maintaining the pH of culture media for long-term cell culture. However, it has some disadvantages such as the need to maintain a CO2-enriched atmosphere continuously, and suboptimal buffering in the physiological pH range due to the 6.1 pKa of NaHCO3 (Chakrabarti and Chakrabarti, 2001). Standard culture systems use 5% CO2 + 95% relative humidity to keep the pH of the culture medium at physiological levels (
7.4) as CO2 reacts with H2O to form H+ and HCO3. This concentration of CO2 produces the ideal amount of H+ to buffer the medium. However, exposure to high pCO2, as in this experiment, increases the concentration of H+ in the medium, leading to a decrease of the pH. Helium has the opposite effect: as it is an inert gas and does not react with H2O to yield H+, the lack of H+ supply results in alkalinization of the pH of the medium. The CO2/HCO3 buffering system is very sensitive and quickly equilibrates with the environmental concentration of CO2. This fact explains the normal pH levels observed in both groups upon returning the cells to standard conditions.
Conclusion
The present study investigated the effect of transient exposure to CO2 on 8-iso-PGF2 levels in human peritoneal mesothelial cells. The following conclusions can be drawn from the results. (i) In vitro exposure to 100% CO2 increased the levels of 8-iso-PGF2
in human peritoneal mesothelial cells. This rise occurred in a time-dependent manner, and seemed to be associated with hypoxiareoxygenation phenomena. (ii) 8-Iso-PGF2
was also influenced by the pH of the culture medium as alkaline pH (100% helium group) produced higher levels of 8-iso-PGF2
than acid pH (4 h 100% CO2 group) under similar levels of hypoxia. Also, in the two CO2 groups, the rise in 8-iso-PGF2
was transient regardless of the duration of exposure, whereas the levels of 8-iso-PGF2
were persistently high at T3 following exposure to helium.
The role of low pH in this process is not clear: did it augment or blunt the overproduction of ROS during hypoxiareoxygenation? Further studies using different gas mixtures are in progress to answer this question.
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Submitted on October 30, 2003; accepted on January 8, 2004.
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