Cigarette smoking is associated with systemic oxidative stress leading to an upregulation of antioxidant systems [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and heme oxygenase (HO)] in some tissues, but the response in the human placenta is unknown. The aim of this study was to determine the effect of cigarette smoke exposure on placental antioxidant expression in vivo, as well as the effect on antioxidant expression in the human trophoblast choriocarcinoma (HTR)-8SVNeo cell line. In the in vivo experiment, normal-term placentas were obtained following elective caesarean section. The chorionic villi (CV), anchoring villi (AV), and basal plate (BP) were dissected, and Western blot analysis was carried out for HO-1, HO-2, SOD, CAT, and GPx. In vitro experiment, a cigarette smoke extract (CSE) was prepared by bubbling the smoke form three cigarettes through 15 ml of RPMI. This 100% CSE was syringe filtered and diluted to 0.1, 0.5, 1, 2, 5, and 10% concentrations. HTR-8SVNeo cells were cultured with the CSE for 48 h. The cells were harvested, protein was extracted, and run on SDS-PAGE gels, and Western blot analysis was carried out for HO-1, HO-2, SOD, and CAT. Immunofluorescence for HTR-8SVNeo cells HO-1 was carried out following increasing concentrations of CSE. In the in vivo experiment, HO-1 and HO-2 expression was increased in the BP of placentas from smokers compared with nonsmokers. CAT, GPx, and SOD levels in all placental regions, as well as HO-1 and HO-2 expression in the AV and CV were unchanged. In the in vitro experiment, The 5%, 10%, and 20% dilutions were toxic to the cells. The 0.1% CSE solution did not significantly alter HO-1 expression. Treatment with the 0.5%, 1% and 2% CSE solutions resulted in a dose-dependent increase in HO-1 expression. None of the CSE treatments resulted in a significant alteration in HO-2, SOD, GPx, or CAT expression. HO-1 immunoflourescence confirmed the HO-1 expression studies. Cigarette smoke exposure increases HO-1 and HO-2 expression in the placental basal plate and increases HO-1 expression in the HTR-8SVNeo cell line. Increased HO-1 and HO-2 protein expression may increase the production of the antioxidants biliverdin and bilirubin, which are products of heme metabolism. This could function to reduce the oxidative load that is released into the maternal plasma from the preeclamptic placenta and may contribute to the observed decreased incidence of preeclampsia in smokers.
- heme oxygenase
- cigarette smoking
- cell culture
cigarette smoking during pregnancy is a major public health concern that is associated with an increased risk of premature birth, spontaneous abortion, placenta previa, and intrauterine fetal growth restriction (13). The effect of fetal growth is dose dependent, with birth weight decreasing 250 g·pack−1·day−1 smoked (28). Paradoxically, women who smoke during pregnancy exhibit a 32% decreased incidence in preeclampsia (PE) (6, 31). In order for this effect to occur, the woman must smoke for the duration of the pregnancy (7), and the incidence of PE is inversely correlated with the number of cigarettes smoked per day (6). A recent review of the subject (2) hypothesized that the effect is likely multifactorial due in part to increased circulating levels of carbon monoxide (CO), which acts as a placental vasodilator (2) and decreases placental apoptosis (1), as well as possible upregulation of placental antioxidant systems.
Placental oxidative stress appears to be critical for the development of PE (16, 20, 21, 22, 27). Therefore, any increase in the levels of endogenous antioxidants may prevent the development of PE. Several antioxidant systems have been identified within the placenta, including copper/zinc superoxide dismutase [Cu/Zn superoxide dismutase (SOD); inactivates O2•−] (29), catalase (CAT; inactivates H2O2) (29), glutathione peroxidase (GPx; inactivates H2O2 and lipid peroxides) (29) and the heme oxygenase enzymes heme oxygenase (HO)-1 and HO-2 (18). HO enzymes act as antioxidants by degrading heme (a prooxidant) into equimolar quantities of CO, biliverdin (bilirubin), and Fe2+ (3, 4); biliverdin and bilirubin are potent physiological antioxidants. HO-1 (32 kDa) is the inducible form of the enzyme, while HO-2 (36 kDa) is constitutively active (17).
A better understanding of the mechanism(s) by which cigarette smoking decreases the risk of developing PE will increase our understanding of the PE disease process and may lead to therapeutic options; delivery is currently the only known cure for PE. The objective of this study was to determine in vivo the effect of smoking on placental antioxidant enzyme expression, as well as the in vitro effect of cigarette smoke extract (CSE) treatment on trophoblast antioxidant systems. We hypothesized that exposure to cigarette smoke increases the expression of antioxidants within placental tissue and that this increase in placental antioxidant enzymes is linked to the decreased incidence of PE in smokers.
Term placentas (n = 6 smokers, n = 6 nonsmokers) from uncomplicated pregnancies were obtained immediately following elective caesarian section at the Kingston General Hospital and transported to the laboratory on ice. Inclusion criteria for smokers/nonsmokers was based on maternal end-tidal CO measured using a Micro CO meter (Micro Direct, Lewiston, ME) (nonsmokers: <5 ppm CO, smokers: >15 ppm CO).
Tissue and cell preparation.
Placental tissues were blunt dissected and random samples from the basal plate, anchoring villi (AV) and chorionic villi (CV) were obtained. The tissue was flash frozen in liquid nitrogen and stored at −80°C for future analysis.
Cells of the immortalized human trophoblast choriocarcinoma cell line HTR-8SVneo were a gift from Dr. C. H. Graham (Queen's University). The cells were maintained at 37°C in 75 cm2 flasks containing RPMI (Sigma-Aldrich, Oakville, Ontario, Canada) + 5% FBS (Sigma-Aldrich) (10). Confluent monolayers were trypsinized (Sigma-Aldrich), and then 5 × 105 cells were plated in 60-mm dishes for experimental procedures.
Cigarette smoke extract preparation and cell reatment.
A vacuum-driven smoking device was adapted from the protocol used by Lee et al. (15). Briefly, the smoke from three cigarettes was drawn through 15 ml of RPMI media. This was established as 100% CSE. To ensure reproducibility from one batch of CSE to the next, dissolved CO2 levels were measured using the ABL5 gas monitor (Radiometer, London, Ontario, Canada) The 100% CSE was then diluted using RPMI containing 5% FBS.
Previous work with pulmonary artery endothelial cells demonstrated that treatment with CSE concentrations of greater than 10% resulted in significant cell death (15). The HTR-8SVneo cells were incubated for 48 h with 0.1%, 0.5%, 1%, 2%, 5%, 10%, and 20% dilutions of CSE or control RPMI.
Placental samples were thawed and washed in PBS to remove excess blood. Four milliliters of protein extraction buffer (2% SDS, 10 mM Tris, 0.15 M NaCl, pH 7.5) was added for every 1 g of tissue; the tissue was homogenized, and DNA was sheared by passing the homogenate 12 times through an 18-gauge needle. Protein was extracted from the HTR-8SVneo cells using 70 μl of a protein extraction buffer (2% SDS, 10 mM Tris, 0.15 M NaCl, pH 7.5). The samples were then sonicated for 15 s to denature nucleic acids. Tissue and cell culture homogenates were boiled for 5 min and then centrifuged at 14,000 g at 22°C for 15 min to separate out cellular fragments. The supernatant was collected and stored at −80°C. The protein concentration of each sample was determined using the DC protein assay (Bio-Rad Laboratories, Hercules, CA).
Western Blot analysis.
The protein extraction samples (30–60 μg) were separated by electrophoresis on 12% SDS-PAGE gels at 165V for 2.5 h. The separated proteins were then transferred onto PVDF membranes (Millipore, Billerica, MA) at 110 V for 1 h. The membranes were blocked overnight in PBS-Tween (PBST) with 5% skim milk and 1% bovine serum albumin to prevent nonspecific binding. Membranes were then incubated for 1 h with primary antibody (HO-1, RCH Antibodies, Sydenham, Ontario, Canada; HO-2, and GPx, Stressgen, Victoria, British Columbia, Canada; SOD, Santa Cruz Biotechnology, Santa Cruz, CA; and CAT, Sigma-Aldrich). Following incubation with the primary antibody, the membranes were washed five times for 5 min in PBST and then incubated in the appropriate secondary antibody [1:15,000 goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) for HO-1, HO-2, and SOD; and 1:25,000 goat anti-mouse IgG (Bio-Rad Laboratories, Hercules, CA) for CAT and GPx] for 1 h, and washed as after the primary antibody. Following the second set of washes, secondary antibodies were detected by enhanced chemiluminescence (PerkinElmer Life Sciences, Boston, MA) and exposed onto X-OMAT Blue XB-1 film (Kodak, Rochester, NY).
As an internal control, all membranes were stripped of bound antibody (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris, pH 6.7) at 50°C for 30 min and reprobed for the housekeeping protein β-actin (1:8,000, Sigma-Aldrich). The membranes were processed as above.
Immunofluorescence staining of CSE-treated HTR8 SVneo cells.
HTR8 SVneo cells were seeded on glass coverslips in 35-mm cell culture dishes and allowed to adhere overnight in 37°C incubator at 5% CO2 in complete RPMI (RPMI +10% FBS) (Sigma-Aldrich). Media were replaced with complete RPMI, RPMI + 0.1%, or RPMI + 0.5% CSE, and cells were incubated for a further 24 h. Cells were fixed in 4% PFA on ice for 15 min, washed twice in cold PBS, and permeabilized in 0.5% Triton × 100 PBS for 15 min on ice. Cells were washed twice in PBS 01% Triton × 100 and incubated in 200 μg/ml RNAse A (Sigma-Aldrich) at 37°C for 1 h. Cells were then blocked in 5% normal goat serun PBST for 1 h, incubated with anti HO-1 chicken antibody(RCH Antibodies) for 1 h followed by incubation for 1 h in anti-chicken Alexa 488 secondary antibody (Molecular Probes Eugene OR), and washed and incubated in propidium iodide 2 μg/ml. (Sigma-Aldrich). The cells were mounted on slides and photographed on a Leica TCS SP2 Confocal Microscope.
The band densities were determined using the AlphaEase software (Alpha Innotech, San Leandro, CA). The data are expressed as the ratio of the protein density of each lane to the β-actin density value of the same lane.
Results were expressed as the means ± SE. The human placenta data were analyzed using a two-tailed paired Students t-test, and results were considered statistically significant when P < 0.05. The HTR-8SVneo data were analyzed using a one-way ANOVA, and the data were subjected to a Tukey post hoc test.
The smoking group exhibited significantly higher exhaled CO than the nonsmokers (19.7 ± 2.2 vs. 3 ± 0.45 P = 0.001). Exhaled CO is correlated with number of cigarettes smoked .
The HO-1 (Fig. 1, A–C) and HO-2 (Fig. 2, A and C) expression was increased (P < 0.05) in the smoking group compared with the nonsmoking group in the basal plate but not (P > 0.05) in either the AV or CV. Placental HO-1 protein expression was lower than that of HO-2. 50 μg/ul of protein was required to visualize the 32 kDa band corresponding to HO-1, while 30 μg/ul of protein was sufficient for the visualization of HO-2, CAT, SOD, and GPx.
In all regions of the placenta, CAT, GPx, and SOD expression in smokers was not different (P > 0.05) compared with nonsmokers (data not shown).
Cell culture data.
A reproducible CO2 concentration of 80–100 ppm was used to demonstrate consistency and accepted for use in CSE preparations. A “fresh” pack of cigarettes was used for each experiment as using “stale” cigarettes resulted in elevated CO2 concentrations that were not within this 80–100 ppm working range. Although it is not known which component(s) of cigarette smoke are captured in this CSE, CO levels are undetectable as measured by gas chromatography (data not shown).
All concentrations of CSE above 2% were associated with 100% HTR-8SVneo human cytotrophoblast cell death. The 2% dilution was also associated with cell death, although sufficient protein could still be collected to allow for Western blot analysis. Concentrations less than 2% did not alter the rate of cell growth and division nor were they associated with visible morphological changes in the HTR-8SVneo cell line.
HO-1 levels were increased in a dose-dependent manner following treatment with increasing concentrations of CSE (0.1%, 0.5%, 1.0% and 2.0%) (Fig. 3). HO-2, CAT, and SOD levels were unaffected by the CSE treatment (data not shown). Immunostaining for HO-1 confirmed the dose-dependent effect of increasing CSE treatment (representative data Fig. 4).
The present study characterized the effect of cigarette smoking in vivo during pregnancy on placental antioxidant systems and in vitro using CSE on the HTR-8SVneo antioxidant profile. Smoking throughout pregnancy resulted in elevated expression of the heme oxygenase enzymes HO-1 and HO-2 in the placental basal plate region. Smoking did not alter the expression of CAT, SOD, or GPx, in any of the placental regions studied. HTR-8SVneo cell line exhibited a dose-dependent increase in HO-1 expression and immunofluorescence following treatment with CSE. CSE treatment did not alter the expression of HO-2, SOD, or CAT in this cell line.
Smoking constitutes a systemic oxidative stress, as each inhalation of cigarette smoke contains 1014 oxidants in the tar phase and 1015 oxidants in the gas phase (11). Although acute oxidative stress (i.e., ischemia/reperfusion injury) is associated with tissue damage, chronic oxidative stresses such as that experienced by smokers may lead to an upregulation of the antioxidant enzymes. HO-1 is the inducible isoform of HO, and its expression is known to be increased in response to oxidative stress in the heart, kidneys, and liver (12).
While HO-1 is known to be induced by cigarette smoke in other tissues, the increased expression of HO-2 in the basal plate of smokers was unexpected as this isoform is constitutively active. However, HO-2 has recently been shown to be upregulated in nervous tissue following hyperthermic injury (9, 24, 25), which can be attenuated by antioxidant pretreatment (23), suggesting that this upregulation is at least in part due to oxidative stress.
The observed dose-dependent increase in HO-1 expression and immunoflourescence following CSE treatment in the HTR-8SVneo cells mimics the increased HO-1 expression seen in the placental basal plate of smoking women. However, the in vivo experiments did not examine a possible dose-dependent effect but compared HO expression in the placentas from nonsmokers with that from moderate to heavy smokers.
The in vivo effect of cigarette smoke on HO-1 and HO-2 expression was limited to the basal plate. The cells of the basal plate are mainly extravillous cytotrophoblast, while the AV and CV are composed of syncytiotrophoblast with scattered cytotrophoblast cells (Langerhans' cells) found only in 20% of the villous surface at term. The different structural and functional properties of the subdivisions of trophoblast could result in different responses to the stress of maternal cigarette smoking. The HTR-8SVneo cell line employed in this study is believed to be extravillous cytotrophoblast-like in that it possesses numerous characteristics that are limited to this trophoblast cell population, including a highly invasive phenotype (10) and an inability to fuse to form a syncytium.
Decreased HO expression has been observed in several pathological conditions in pregnancy, including spontaneous abortion, hydatiform mole, choriocarcinoma, and PE (30, 32). The decreased expression of HO in PE placentas may be the result of tissue damage, as HO-2 levels are decreased in infarcted and periinfarcted regions of the placenta (14), and the placentas from women with PE exhibit more areas of infarction. This decreased HO activity could contribute to the widespread oxidative stress that is present in PE.
Increased HO expression would result in increased biliverdin and bilirubin production locally. At physiological oxygen tensions, bilirubin has been found to be significantly more potent than vitamin E at protecting against lipid peroxidation (26); as little as 10 nM bilirubin is capable of protecting against a 10,000 fold higher concentration of H2O2 (5). Farrera et al. (8) compared antioxidants and found that bilirubin was the most potent superoxide and peroxyl radical scavenger. Bilirubin is also capable of preventing peroxynitrite-induced protein oxidation (19). Therefore, any upregulation in HO-1 expression in the basal plate of smokers could potentially lead to the generation of more bilirubin, which could then function at the maternal-fetal interface to decrease oxidative stress.
In summary, smoking throughout pregnancy is associated with an increase in placental basal plate HO activity, which is supported by the in vitro CSE-mediated dose-dependent increase in HO-1 in the placental cell line HTR-8SVneo. It is speculated that this upregulation of HO may explain, in part, the decreased incidence of PE in smokers by facilitating trophoblast invasion of the spiral arteries and decreasing placental oxidative damage, especially as the relationship between smoking and the development of PE is also dose dependent. The “protective” effect of smoking is likely multifactorial, representing a combination of the antiapoptotic and vasodilatory effects of CO, as well as the antioxidant actions of HO-1. Investigations into how cigarette smoking alters the HO enzyme system and the effects this has on endogenous oxidant/antioxidant balance and the development of PE could lead to the therapeutic use of inducers of this system in the prevention and/or treatment of PE.
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