Objective
We sought to extend our prior observations and histopathologically characterize key metabolic enzymes ( CYP1A1 ) with markers of oxidative damage in the placental sections from smokers.
Study Design
Placental specimens were collected from term singleton deliveries from smokers (n = 10) and nonsmokers (n = 10) and subjected to a detailed histopathological examination. To quantify the extent of oxidative damage, masked score-graded (0-6) histopathology against 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxydeoxyguanisine (8-OHdG) was performed. Minimal significance ( P < .05) was determined with a Fisher’s exact and a 2-tailed Student t test as appropriate.
Results
We observed a significant increase in the presence of syncytial knots in placentas from smokers (70% vs 10%, P = .02). These gross observations were accompanied by a significant aberrant placental aromatic hydrocarbon metabolism (increased CYP1A1, 4.4 vs 2.1, P = .002) in addition to evidence of oxidative damage (4-HNE 3.4 vs 1.1, P = .00005; 8-OHdG 4.9 vs 3.1, P = .0038).
Conclusion
We observed a strong association between maternal tobacco use and aberrant placental metabolism, syncytial knot formation, and multiple markers of oxidative damage.
Although the concerning effects of maternal tobacco smoke on fetal growth have been well reported for more than 3 decades, it remains today one of the leading preventable causes of fetal growth restriction in developed and developing countries. In the seminal report from Simpson, it was reported that mothers who smoked 10 cigarettes or more per day delivered infants with a decrease in birthweight of approximately 200 g compared with neonates from nonsmoking mothers. However, not all fetuses exposed to maternal tobacco smoke are growth restricted. Susceptibility to tobacco exposure likely involves several factors including, but not limited to, epidemiological, genetic, epigenetic, and socioeconomic.
Nicotine, a principal alkaloid of tobacco smoke, has been shown to mediate constriction of the intrauterine vessels and result in increased proliferation of placental syncytiotrophoblasts. Potentially harmful deoxyribonucleic acid (DNA) adducts (metabolic products of polycyclic aromatic hydrocarbons [PAH]) are known to cross or collect in the placenta of smokers.
PAH compounds, together with nitrosamines, comprise likely carcinogenic species in tobacco smoke. The majority of chemical carcinogens are metabolized in a sequential series of 2-phase enzymatic metabolic reactions ( Figure 1 ) . Phase I enzymes such as CYP1A1 metabolically activate PAH compounds into oxidized derivatives, resulting in reactive oxygen intermediates capable of covalently binding DNA to form adducts. In turn, these reactive electrophilic intermediates can be detoxified by phase II enzymes, such as the glutathione S -transferase (GSTT1), via conjugation with endogenous species to form hydrophilic glutathione conjugates, which are then readily excreted. Thus, the coordinated expression of these enzymes and their relative balance may determine the extent of cellular DNA damage and related development of adverse outcomes.
We have previously demonstrated that in a large matched cohort, deletion of fetal GSTT1 (a phase II pathway gene; Figure 1 ) is associated with birthweight reduction in pregnancies exposed to maternal tobacco use. We have also shown that increased placental CYP1A1 expression was specifically and significantly associated with hypomethylation of XRE-proximal CpG dinucleotides in the CYP1A1 promoter region in smokers compared with nonsmokers. An increase in phase I enzymes without a compensatory increase in phase II enzymes has the potential to create reactive species within the cell. These unprocessed reactive oxygen species (ROSs) have the unmitigated potential to lead to DNA-adduct–mediated damage and lipid oxidation, perpetuating the cycle of modulated cellular and molecular physiology ( Figure 1 ).
In this study, we hypothesized the disrupted metabolic pathways converge at the cellular level to increase markers of oxidative stress in the placenta. To quantify the extent of DNA damage and oxidative damage, we used 2 well-characterized markers: 8-hydroxydeoxyguanisine (8-OHdG; a marker of DNA damage) and 4-hydroxy-2-nonenal (4-HNE; a marker for oxidative lipid damage) as determinates of cellular oxidative stress. We therefore sought to extend our prior observations and histopathologically characterize key metabolic enzymes ( CYP1A1 ) with markers of oxidative damage in placental sections from smokers.
Materials and Methods
Study population
Placental samples (n = 20) for this study were obtained from subjects selected from a well-described cohort of 20 self-reported smokers in addition to 53 nonsmoking controls; this has been previously validated as an accurate measure of maternal tobacco exposure.
The Institutional Review Board of Baylor College of Medicine and its affiliated institutions approved this study, and written informed consent was obtained from each participant at the time of enrollment. Data collected from each patient included age, ethnicity, height and weight, past obstetrical history, gestational age at delivery, and potential maternal comorbidities. Data collected from the newborns included sex, Apgar scores, weight and length, and level of resuscitation interventions if any.
Exclusion criteria included multiple gestations; known fetal anomalies; and maternal hepatic, hypertensive, or endocrine disorders. For the analysis reported herein, subjects were matched in a nested cohort design by virtue of maternal age (±3 years), race/ethnicity, body mass index (BMI), and gestational age (±1 week). Consistent with a nested cohort design, matching was performed prior to knowledge of the primary outcomes (ie, histopathology and immunohistochemistry) and without consideration of fetal factors (beyond gestational age) including fetal weight or length or neonatal outcome. In such a manner, an initial 20 matched subjects were analyzed with minimized potential for selection bias. This is as noted in Table 1 .
| Characteristic | Nonsmokers (n = 10) | Smokers (n = 10) | P value |
|---|---|---|---|
| Maternal age, y | 29.7 ± 1.8 | 27.8 ± 2.1 | .504 |
| Maternal BMI, kg/m 2 | 26.5 ± 0.9 | 22.7 ± 3.1 | .251 |
| Gestational age, wks | 39.3 ± 0.7 | 39.0 ± 0.4 | .719 |
| Infant weight, g | 3619 ± 128 | 3159 ± 144 | .029 a |
| Infant length, cm | 49.8 ± 0.7 | 48.2 ± 0.9 | .226 |
a In our nested cohort design, after matching for maternal characteristics and gestational age, we observed a statistically significant association between infant birth weight and maternal smoking.
Collection and standardized processing of placental samples
Placental specimens were collected immediately after delivery, systematically stored, and processed for histopathology within 12 hours. Standardized collection and section methodology included uniform triplicate 3 cm excisional blocks at a prescribed 4 cm trinary distance from the umbilical cord insertion along with a section from the insertion point and random 3 marginal sections. All sections collected were full thickness. The excised sections were embedded into paraffin blocks and stained with hematoxylin and eosin (H&E) for microscopic examination. In addition, unstained sections were prepared for use in immunohistochemistry.
Placental histopathology analysis
All H&E-stained sections were examined by reviewers masked to maternal cohort. Pathological changes were recorded as present or absent, and the prevalence of abnormalities observed (eg, infarcts, inflammation, syncytial knots) was compared between the 2 groups and analyzed with the statistical software package SPSS version 11.5 (SPSS Inc, Chicago, IL) using a Fisher’s exact test with a minimal P < .05 denoting significance.
Immunohistochemistry
Primary antibodies employed were 4-HNE mouse monoclonal (Abcam, Cambridge, UK), CYP1A1 rabbit polyclonal (Millipore, Bisley, UK), and 8-OHdG goat polyclonal (Millipore). For immunostaining, unstained paraffin sections were deparaffinized in 4 changes of xylene for 5 minutes each and then rehydrated through a series of graded alcohols with a final rinse in distilled water. Endogenous peroxidase was quenched by soaking sections in 2 changes of 3% H 2 O 2 in methanol for 10 minutes at room temperature.
Prior to immunohistochemistry, antigen retrieval was performed to facilitate antibody binding to antigen. Slides were incubated at 99¼C for 20 minutes in either Target retrieval citrate solution (pH 6.0) (Dako Corp, Carpinteria, CA) for CYP1A1 and 4-HNE or for 28 minutes in Diva Decloaker retrieval solution (pH 6.2) (Biocare Medical, Concord, CA). Slides were then allowed to cool down for 10 minutes in the same solution, rinsed in 3 changes of distilled water, and placed in Tris-buffered saline with Tween 20 (pH 7.4) (Signet Pathology Systems, Inc, Dedham, MA) for 5 minutes to decrease surface tension and facilitate coating by the subsequent reagents. The PolyVue horseradish peroxidase (HRP)/diaminobenzidine (DAB) nonbiotin polymer detection system (Diagnostic Biosystems, Pleasanton, CA) was used in the immunostaining protocols for CYP1A1 and 8-OHdG, whereas the MACH 4 HRP biotin-free universal polymer detection system (Biocare Medical) was used for 4-HNE.
Incubations occurred at room temperature unless otherwise specified, and for each step the sections were coated with 200 μL of reagent. Tris-buffered saline with Tween 20 (pH 7.4) was used to rinse the sections between each of the immunohistochemistry steps. Background Sniper solution (Biocare Medical) was used to block nonspecific staining for 10 minutes at room temperature. The primary antibody was diluted using the solution provided with the detection kit, using a dilution of 1:200 for CYP1A1 and 8-OHdG or 1:50 for 4-HNE.
Slides were incubated with the primary antibody solution for 30 minutes at room temperature (CYP1A1), 60 minutes at room temperature (4-HNE), or overnight at 4¼C (8-OHdG). Sections were then incubated in the universal secondary antibody provided with the kit for 15 minutes, followed by the HRP label reagent. Afterward, Stable DAB Plus (Diagnostic Biosystems) was applied for 5 minutes as chromagen. The slides were rinsed in distilled water and manually counterstained with Harris hematoxylin (Fisher Scientific, Fair Lawn, NJ) for 15-30 seconds and then rinsed in distilled water. Coverslips were then applied to each slide, using synthetic glass and permount mounting media. Negative controls and nonspecific antibodies were included in each immunostaining procedure.
Immunohistochemistry (IHC) analysis
Immunostained slides were examined by 2 independent reviewers masked to whether the case was a smoker or nonsmoker. For each slide examined, 10 random high-power fields were graded using a 0-6 scale in which 0 indicated the absence of positive staining and 6 indicated intense and diffuse positive staining. The location of positive staining areas was also recorded. The average of all grades was calculated for each slide, and IHC grades of smokers were compared with those of nonsmokers using the independent-sample Student t test, after equal variance test was performed, using the statistical software package SPSS version 11.5 with minimal significance designated at P < .05.
Results
Study subjects
Our nested cohort design of singleton gestations at term (longer than 37 weeks’ gestation) yielded matched cohorts, which were designated to differ by virtue of maternal smoking, but manifest a significant decrease in infant birthweight in smokers (3159 ± 144 vs g 3619 ± 128 g, P = .028; Table 1 ). By design, gestational age as well as maternal age, BMI, race/ethnicity, and maternal comorbities did not differ significantly in the 2 groups ( Table 1 ). There was no observed difference among infant length or neonatal outcome among cohorts ( Table 1 ).
Histopathology
No significant differences in gross pathological abnormalities (ie, placental abruption, subchorionic hematoma, or umbilical cord abnormalities) were observed among the cohorts; a single case of chorioamnionitis was observed in our smoking cohort. Meticulous standardized examination of 6-8 H&E-stained placental sections from subject triplicate samples was undertaken. In 7 of 10 smokers, all sections of villous parenchyma were remarkable for prominent syncytiotrophoblastic knots (clusters of syncytial nuclei that form on the surface of a terminal villus characterized by a display of highly condensed chromatin); conversely, this feature was observed only in 1 of 10 nonsmokers and was statistically significant ( P = .020; Figure 2 , A and B) .
