Cigarette smoking during pregnancy has several impacts on fetal development, including teratogenic effects. The objective of this study was to assess whether the toxic substances (cotinine and polycyclic aromatic hydrocarbons) found in pregnant smokers are transmitted to their fetuses. The outcomes were analyzed measuring cotinine and 1-hydroxypyrene in the amniotic fluid and maternal urine, benzopyrene and cotinine in the umbilical cord blood. Through a controlled cross-sectional design, 125 pregnant women were selected and classified according to their smoking status: 37 current smokers, 25 passive smokers and 63 non-smokers (controls). We performed high-performance liquid chromatography to measure substances’ concentrations. A post-hoc Tukey’s test was used to analyze the differences between the groups. All variables were significantly different between controls and smokers. The mean ratios between the concentration of cotinine in smokers compared to controls were as follows: 5.9 [2.5–13.5], p<0.001 in the urine; 25 [11.9–52.9], p<0.001 in the amniotic fluid; and 2.6 [1.0–6.8], p = 0.044 in the umbilical cord blood. The mean ratios of 1-hydroxypyrene concentration between smokers and controls were 7.3 [1.6–29.6], p = 0.003 in the urine and 1.3 [1.0–1.7], p = 0.012 in the amniotic fluid, and of benzopyrene in umbilical cord blood was 2.9 [1.7–4.7], p<0.001. There were no significant differences between controls and passive smokers. When comparing the three groups together, there were statistical differences between all variables. Thus, the fetuses of pregnant smokers are exposed to toxic and carcinogens substances. To our knowledge, this is the first study to measure 1-hydroxypyrene in the amniotic fluid and benzopyrene in umbilical cord blood by high-performance liquid chromatography when considering pregnant women in relation to smoking exposure only.
Citation: Machado JdB, Chatkin JM, Zimmer AR, Goulart APS, Thiesen FV (2014) Cotinine and Polycyclic Aromatic Hydrocarbons Levels in the Amniotic Fluid and Fetal Cord at Birth and in the Urine from Pregnant Smokers. PLoS ONE 9(12): e116293. https://doi.org/10.1371/journal.pone.0116293
Editor: Thomas H. Thatcher, University of Rochester Medical Center, United States of America
Received: July 21, 2014; Accepted: December 8, 2014; Published: December 30, 2014
Copyright: © 2014 Machado et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
Smoking is the leading cause of preventable deaths in the world , , , causing the deaths of approximately 6 million people per year , . Cigarette smoking before and during pregnancy is an important cause of preventable illness and death among mothers and their children and particularly impacts on pregnancies in younger women with lower educational level . Smoke exposure during pregnancy has been repeatedly and consistently related to numerous risks to the fetus, including low birth weight, preterm delivery, increased risk of miscarriage , , , , , , , and increased teratogenic effects on the development of several organs .
The ultrasound pattern of maternal-fetal chronic hypoxia has already been described in pregnant smokers . Placental abruption, which occurs in approximately 1% of pregnancies, is 20 to 30 times more frequent in pregnant smokers . Also, cigarette smoke contains numerous carcinogens that cross the placenta , increasing the risk of childhood cancer, such as brain tumors, leukemia and lymphoma .
Cotinine, nicotine metabolite , , , is widely used as a biological marker to measure tobacco exposure , ,  and is also used in cases of low or intermittent tobacco use . It is also considered the best marker of smoking status, even during pregnancy .
Polycyclic aromatic hydrocarbons (PAHs) are related to the teratogenic and carcinogenic effects of cigarette smoke. PAHs are a class of organic compounds containing two or more fused aromatic rings consisting of carbon atoms and hydrogen . These substances are air pollutants generated by burning tobacco leaves, motor vehicles, and factory combustion and can also be found in some smoked foods . Most PAH compounds have an 18-hour half-life ,  and are metabolized and excreted in the urine. Benzopyrene and 1-hydroxypyrene can be used as biomarkers of recent tobacco exposure .
Llop et al.  demonstrated that one of the PAHs, urinary 1-hydroxypyrene, is a good marker for assessing exposure to cigarette smoke and air pollution. When the determination is performed on the blood, the biomarker used is benzopyrene. After inhalation, the PAHs are metabolized to form reactive epoxide and phenolic compounds that have the capacity to bind to DNA, forming complex PAH-DNA adducts . These PAH-DNA adducts increase the probability of genetic mutations and, therefore, might be associated with several forms of cancer .
Smokers excrete significantly higher urine levels of 1-hydroxypyrene than nonsmokers, and these values correlate well with the number of cigarettes smoked . However, only a few studies have investigated the levels of PAHs in pregnant smokers , and the majority of these studies have focused on environmental exposure , . Considering that cigarette smoke is the primary source of exposure to these substances, this area of study requires further investigation.
The metabolism of pregnant smoker is altered, and it is difficult to evaluate the effects of dietary factors and environmental exposure. Thus, it is necessary to compare women from the same population with similar diets and exposures to environmental pollution to try to isolate the true exposure of fetuses to chemicals arising from both active and passive smoking.
The objective of this study was to assess whether cotinine and PAH found in pregnant smokers are transmitted to their fetuses. We measured the amount of 1-hydroxypyrene and cotinine in the amniotic fluid and maternal urine and benzopyrene and cotinine in the umbilical cord blood.
Materials and Methods
In a controlled, cross-sectional design study, we recruited pregnant women in labor admitted at the Obstetric Center Hospital São Lucas (HSL), Pontificia Universidade Catolica do Rio Grande do Sul, Porto Alegre, Brazil, from July 2010 to July 2013. This project was submitted and approved by the PUCRS Research Ethics Committee under the number 10/05066.
The inclusion criteria were signing the informed consent; age 18 to 35 years old; women must had received regular prenatal care at the Department of Obstetrics at the same hospital; and no previous or concomitant gestational diseases. Smokers were defined as pregnant women who had smoked more than 100 cigarettes throughout life and were smoking at the time of the interview . Passive smokers were defined as non-smoking pregnant women who lived with a smoker who smoked inside their home. Non-smokers served as the control groups. The exclusion criteria were patients who declined to participate in the study; women with severe psychopathic disorders; preterm labor; illiteracy; and addiction to other licit or illicit drugs. Former smokers were also excluded.
The volunteers in labor were recruited at the moment of admission to the HSL Obstetric Center. They completed a questionnaire regarding their demographics and smoking habits. Maternal urine was then collected. Upon rupture of the membranes, amniotic fluid was collected. During delivery, the umbilical cord blood sample was collected.
The cotinine in maternal urine and umbilical cord blood were analyzed according to the methods described by Cattaneo et al  and Petersen et al.  respectively. The quantitative methodologies for the determination of cotinine and 1-hydroxypyrene in amniotic fluid, as well as, the 1-hydroxypyrene (1-OHP) in the maternal urine and benzopyrene in the umbilical cord blood were previously validated by our research group in accordance with the International Conference on the Harmonization (ICH) guideline . To assess the specificity of the methodologies blank samples (or drug-free human samples) of urine, amniotic fluid and umbilical cord blood were compared to spiked samples with cotinine, 1-hydroxypyrene or benzopyrene and no interference of the different biological matrices was seen in the peak determination. The calibration curves for each analyte were linear (r>0.99) over the concentration ranges of 10.0–1000 µg/L for cotinine in urine; 5.0–500 µg/L for cotinine in umbilical cord blood; 2.0–600 µg/L for cotinine in amniotic fluid; 0.1–10.0 µg/L for 1-OHP in urine; 0.25–15.0 µg/L for 1-OHP in amniotic fluid; and 0.1–7.5 µg/L for benzopyrene in umbilical cord blood. The limits of quantification (LOQ) were defined as the lowest point of calibration curves, and limits of detection (LOD) were calculated as the minimum concentration providing chromatographic signals 3 times higher than the background noise. The analytical methods fulfilled the acceptance criteria for precision and accuracy showing a coefficient of variation lower than 13.4% and a recovery range of 91.5–110%.
The specimens for the cotinine analysis were alkalinized with 25 µL of sodium hydroxide 10 M and submitted to liquid-liquid extraction with dichloromethane using 2-phenylimidazole as internal standard (IS). For each analysis 2 mL of maternal urine, 600 µL of umbilical cord blood, and 1 mL of amniotic fluid were employed.
To determine 1-hydroxypyrene, 2.5 mL of urine samples were treated with acetate buffer (5 mL, pH 5) and β-glucuronidase/arylsulfatase (10 µL), to promote the hydrolysis of urinary 1-hydroxypyrene, and subsequently submitted to solid phase extraction (SPE) using a C18 cartridge. For the analysis of 1-hydroxypyrene in the amniotic fluid 2 mL of sample was submitted to liquid-liquid extraction with ethyl ether. To the determination of benzopyrene in the umbilical cord blood 150 µL of sample was extracted with cyclohexane. The organic phase was separated and dried under nitrogen flow at room temperature. Then, the samples were recovered with mobile phase and injected into the HPLC system.
Cotinine analysis was performed using an high-performance liquid chromatograph equipped with a UV detector isocratic pump, manual injection and software. The chromatographic separation was performed (4.6×150 mm×5 µm), column protected (4.6×12.5 mm, 5 µM). The mobile phase consisted of a mixture of water:methanol:sodium acetate (0.1 M):acetonitrile (50∶15∶25∶10, v/v/v/v), and the flow was maintained at 0.5 mL/minute with UV detection at 260 nm, yielding a total run time of 10 minutes.
A high-efficiency liquid chromatograph equipped with a fluorescence detector, isocratic pump and manual injector was used to analyze the presence of 1-hydroxypyrene and benzopyrene. The chromatographic separation was performed with (4.6×150 mm, 5 µM) column protected by a guard column (4.6×12.5 mm, 5 µM). The mobile phase consisted of a methanol:acetonitrile:water (35∶35∶30, v/v/v) mixture for 1-hydroxypyrene, and acetonitrile:water (75∶25, v/v) for benzopyrene analysis. A 1.0 mL/min flow rate was maintained isocratically with fluorescence detector set at 242 nm (excitation) and 388 nm (emission) for 1-hydroxypyrene, and 290 nm (excitation) and 430 nm (emission) for benzopyrene (see also S1 Table).
To characterize the patients and fetal birth we used means and standard deviations. For analyses the group comparisons of cotinine and PAHs concentration according to smoking habit, the quantitative variables were logarithmically transformed and presented as geometric means and geometric mean deviation. The difference between the 3 groups together was calculated using ANOVA.
To estimate the proportional difference of asymmetrical variables between groups, we used the mean ratio (MR) and 95% confidence interval (CI). In relative terms, the MR expresses how many times the average of a group is larger than the other. The MR was obtained in a model of analysis of covariance, and a robust standard error was applied to the logarithms of the measures. The post-hoc Tukey’s test was used to determine the group differences, letters a & b denote non-significant or significant difference between groups, respectively. The groups with no significant difference were labeled as (a) and the groups, with significant difference as (b). The significance level was 0.05. The data were analyzed using SPSS version 21.0.
A total of 125 patients were enrolled in the study: 37 (29.6%) were smokers; 25 (20%) were passive smokers; and 63 (50.4%) were non-smokers. There were no significant differences in age or obstetric aspects between the groups (Table 1).
Table 2 presents the cotinine and 1-hydroxypyrene levels in the maternal urine and amniotic fluid and the benzopyrene and cotinine levels in the umbilical cord blood.
When compared the three groups together were statistical difference between all variables (P in table 2). When compared in pair all variables also were significantly different between the control and smoker groups, represented by letter b in Table 2. There were no significant differences between controls and passive smokers, represented by letter a in Table 2: urinary cotinine P = 0.57, amniotic fluid cotinine P = 0.92, umbilical cord cotinine P = 0.99, 1-hydroxypyrene in maternal urine P = 0.39, 1-hydroxypyrene in amniotic fluid P = 0.86 and benzopyrene in umbilical cord P = 0.47.
The number is not the same in all groups because some samples not able to be analyzed.
The MRs of the cotinine concentrations of pregnant smokers compared to controls were 5.9 [95%CI 2.5 to 13.5] (p<0.001) in the urine; 25 [95%CI 11.9–52.9] (p<0.001) in the amniotic fluid; and 2.6 [95%CI 1.0–6.8] (p = 0.044) in the umbilical cord blood.
The MRs of the 1-hydroxypyrene concentrations in pregnant smokers compared to controls were 7.3 [95%CI 1.6–29.6] (p = 0.003) in the urine and 1.3 [95%CI 1.0–1.7] (p = 0.012) in the amniotic fluid. The concentration of benzopyrene in the umbilical cord blood of fetuses from mothers who smoked had an MR of 2.9 [95%CI 1.7–4.7] (p<0.001) compared to controls.
In Table 3 data of groups are presented as geometric mean (GM) and geometric standard deviation (GSD).
Cotinine and polycyclic aromatic hydrocarbons in the urine and amniotic fluid of pregnant smokers and umbilical cord blood of their fetuses had significant higher levels compared to the non-smokers (controls). There were no significant differences between passive smokers and controls. When comparing the three groups together were statistical differences between all variables.
To our knowledge, this study is the first to measure 1-hydroxypyrene in the amniotic fluid and benzopyrene in umbilical cord blood by high-performance liquid chromatography when considering pregnant women in relation to smoking exposure only.
Our results demonstrate that pregnant smokers expose their fetuses to several substances in cigarette smoke, such as nicotine/cotinine and PAHs.
The cotinine concentrations in pregnant smokers were approximately 6 times higher in the urine, 25 times higher in the amniotic fluid, and 2.6 times higher in the cord blood when compared to non-smokers (control group).
The concentrations of 1-hydroxypyrene in pregnant smokers were seven times higher in the urine and 30% higher in the amniotic fluid compared to the control group. The benzopyrene concentration was approximately 3 times higher in cord blood of fetuses of the smokers compared to the control group.
Thus, these substances found in the urine of pregnant smokers pass to the fetus through the amniotic fluid and umbilical cord blood. The process of amniotic fluid formation, in which the fetus urinates and swallows his own urine, forms a cycle in which they are more exposed to these substances. The higher concentration of cotinine in the amniotic fluid compared to the concentration of 1-hydroxypyrene can be explained by its longer half-life (36 hours versus 18 hours, respectively) , . The non-significant difference among passive pregnant smokers and controls is probably related that they do not inhale enough cigarette smoke to transmit to the fetus such toxic substances.
We classified the patient smoking status according to their self–report because many smokers were abstinent for many hours before labor. Thus, the smoking status could be affected if it were based on the cotinine levels only (due to the half-life rating). Also, many participants did not smoke when the pain of labor contractions began. Some of the collected samples could not be analyzed by having insufficient amount of material for analysis.
For the statistical analyses, we calculated the mean and median, but the results were not satisfactory due to the intense asymmetry data. We then used the geometric mean and geometric mean ratios, more appropriate tools for studying asymmetric data and values close to zero. We also calculated the geometric standard deviation, but to interpret the values is necessary perform logarithmic transformation and then Gauss (mean +/−1.96 SD).
Our results confirms that nicotine cross the placenta and is already known that it can cause extensive damage to the fetus, since nicotine is clearly neuroteratogenic and impacts the fetal brain at critical stages of development in pregnant women using tobacco . This may explain the cognitive, emotional and behavioral problems observed in these children. Furthermore, exposure to cigarette smoke throughout prenatal and postnatal development increases the likelihood of dependence on licit and illicit drugs in adolescence or adult age . The development of other organs, including the lungs, is also adversely affected by nicotine , .
We also found an increased fetal exposure to 1-hydroxypyrene, a known carcinogenic substance. The carcinogenic pathway for PAHs or their metabolites involves the production of reactive oxygen species, which generate oxidative stress, lipid peroxidation, protein modifications, and DNA damage, and may influence birth outcomes and child health in later life , , , .
Perera et al.  showed that a range of relevant environmental exposures to certain carcinogenic PAHs during pregnancy may damage the fetal DNA through histone modification, which may result in fetal chromosomal abnormalities. PAH-DNA adducts were detected in the umbilical cord blood and maternal blood after exposure to ambient air PAHs  and predisposes the fetus to aberrations in the cord blood. Thus, prenatal exposure to PAHs may increase the risk of cancer in humans .
The early embryonic period, when the rates of DNA synthesis are high and the patterns of DNA methylation are established , may be a particularly sensitive phase for epigenetic dysregulation due to environmental exposures.
Perera et al.  proposed that the transplacental transfer of PAHs to the fetus could have significant impacts on fetal development. Several studies found a reduction in head circumference at birth, which is correlated with a lower intelligence quotient and poor cognitive functioning and school performance in childhood , . Furthermore, PAHs are associated with restricted intrauterine growth , small gestational age  and preterm delivery . When a group of newborns monitored prenatally was followed through school age, there was a decline in neurological development  and an increased likelihood of asthma-related symptoms .
Jules et al.  suggests that intrauterine exposure to benzopyrene predisposes newborn rats to functional deficits in cardiovascular development, which may contribute to cardiac dysfunction throughout life. Rundle et al.  suggests that prenatal exposure to PAHs causes increased fat mass gains during childhood and an increased risk of obesity. Langlois et al.  showed an association between maternal occupational exposure to PAHs and increased risk of cleft lip with or without cleft palate.
Stejskalova and Pavek  confirmed that PAH-DNA adducts in the placenta also led to pregnancy complications, such as preterm labor, intrauterine growth restriction, structural abnormalities, fetal death, placental abruption, low birth weight, and small birth length.
The measurement of PAH and its fetal effects have been extensively studied, as mentioned above. However, most of these studies focused on environmental exposures.
What this study adds is that we studied the fetal exposure to PAHs solely related to smoking, which is the primary means of exposure, in a population with a similar diet, living in the same geographic area, and without significant variations in pollution exposure. We measured benzopyrene levels in the umbilical cord blood by high-performance liquid chromatography, a simple and inexpensive method. Our results confirmed that benzopyrene also passes to the fetus from cigarette smoke exposure. We also showed that PAHS were also transmitted to the fetus through the amniotic fluid.
Thus, the fetuses of pregnant women who smoke are exposed to notoriously toxic and carcinogenic substances.
Conceived and designed the experiments: JDBM JMC APSG ARZ FVT. Performed the experiments: JDBM JMC APSG ARZ FVT. Analyzed the data: JDBM JMC APSG ARZ FVT. Contributed reagents/materials/analysis tools: JDBM JMC APSG ARZ FVT. Contributed to the writing of the manuscript: JDBM JMC APSG ARZ FVT.
- 1. Centers for Disease Control and Prevention (2004) State medicaid coverage for tobacco-dependence treatments–United States, 1994–2002. MMWR Morb Mortal Wkly Rep 53:54–57.
- 2. Centers for Disease Control and Prevention (2005) Tobacco use, access, and exposure to tobacco in media among middle and high school students–United States, 2004. MMWR Morb Mortal Wkly Rep 54:297–301.
- 3. (2011) WHO urges more countries to require large, graphic health warnings on tobacco packaging: the WHO report on the global tobacco epidemic, 2011 examines anti-tobacco mass-media campaigns. Cent Eur J Public Health 19: 133, 151.
- 4. World Health Organization (2011) Implementing tobacco control.
- 5. WHO (2013) report on the global tobacco epidemic Enforcing bans on tobacco advertising, promotion and sponsors Hip.
- 6. Centers for Disease Control and Prevention (2007) Fact sheet: preventing smoking and exposure to secondhand smoke before, during and after pregnancy.
- 7. Gilboa SM, Mendola P, Olshan AF, Langlois PH, Savitz DA, et al. (2005) Relation between ambient air quality and selected birth defects, seven county study, Texas, 1997–2000. Am J Epidemiol 162:238–252.
- 8. Mannes T, Jalaludin B, Morgan G, Lincoln D, Sheppeard V, et al. (2005) Impact of ambient air pollution on birth weight in Sydney, Australia. Occup Environ Med 62:524–530.
- 9. Wilhelm M, Ritz B (2005) Local variations in CO and particulate air pollution and adverse birth outcomes in Los Angeles County, California, USA. Environ Health Perspect 113:1212–1221.
- 10. Ziaei S, Nouri K, Kazemnejad A (2005) Effects of carbon monoxide air pollution in pregnancy on neonatal nucleated red blood cells. Paediatr Perinat Epidemiol 19:27–30.
- 11. Leem JH, Kaplan BM, Shim YK, Pohl HR, Gotway CA, et al. (2006) Exposures to air pollutants during pregnancy and preterm delivery. Environ Health Perspect 114:905–910.
- 12. Ritz B, Wilhelm M, Hoggatt KJ, Ghosh JK (2007) Ambient air pollution and preterm birth in the environment and pregnancy outcomes study at the University of California, Los Angeles. Am J Epidemiol 166:1045–1052.
- 13. Liu S, Krewski D, Shi Y, Chen Y, Burnett RT (2007) Association between maternal exposure to ambient air pollutants during pregnancy and fetal growth restriction. J Expo Sci Environ Epidemiol 17:426–432.
- 14. Shi M, Wehby GL, Murray JC (2008) Review on genetic variants and maternal smoking in the etiology of oral clefts and other birth defects. Birth Defects Res C Embryo Today 84:16–29.
- 15. Machado Jde B, Plinio Filho VM, Petersen GO, Chatkin JM (2011) Quantitative effects of tobacco smoking exposure on the maternal-fetal circulation. BMC Pregnancy Childbirth 11:24.
- 16. Ananth CV, Smulian JC, Vintzileos AM (1999) Incidence of placental abruption in relation to cigarette smoking and hypertensive disorders during pregnancy: a meta-analysis of observational studies. Obstet Gynecol 93:622–628.
- 17. Sasco AJ, Vainio H (1999) From in utero and childhood exposure to parental smoking to childhood cancer: a possible link and the need for action. Hum Exp Toxicol 18:192–201.
- 18. Rogers JM (2009) Tobacco and pregnancy. Reprod Toxicol 28:152–160.
- 19. Benowitz NL (1996) Cotinine as a biomarker of environmental tobacco smoke exposure. Epidemiol Rev 18:188–204.
- 20. Binnie V, McHugh S, Macpherson L, Borland B, Moir K, et al. (2004) The validation of self-reported smoking status by analysing cotinine levels in stimulated and unstimulated saliva, serum and urine. Oral Dis 10:287–293.
- 21. Pichini S, Basagana XB, Pacifici R, Garcia O, Puig C, et al. (2000) Cord serum cotinine as a biomarker of fetal exposure to cigarette smoke at the end of pregnancy. Environ Health Perspect 108:1079–1083.
- 22. Kohler E, Bretschneider D, Rabsilber A, Weise W, Jorch G (2001) Assessment of prenatal smoke exposure by determining nicotine and its metabolites in maternal and neonatal urine. Hum Exp Toxicol 20:1–7.
- 23. Kohler E, Avenarius S, Rabsilber A, Gerloff C, Jorch G (2007) Assessment of prenatal tobacco smoke exposure by determining nicotine and its metabolites in meconium. Hum Exp Toxicol 26:535–544.
- 24. Ostrea EM Jr, Knapp DK, Romero A, Montes M, Ostrea AR (1994) Meconium analysis to assess fetal exposure to nicotine by active and passive maternal smoking. J Pediatr 124:471–476.
- 25. Haley NJ, Sepkovic DW, Hoffmann D (1989) Elimination of cotinine from body fluids: disposition in smokers and nonsmokers. Am J Public Health 79:1046–1048.
- 26. Klebanoff MA LR, Clemens JD, DerSimonian R, Wilkins DG. (1998) Serum cotinine concentration and self-reported smoking during pregnancy. Am J Epidemiol 148:259–262.
- 27. World Health Organization, International Programme on Chemical Safety (1998) Selected non-heterocyclic polyciclic aromatic hydrocarbons. Geneva: World Health Organization. 883 p.
- 28. Bostrom CE, Gerde P, Hanberg A, Jernstrom B, Johansson C, et al. (2002) Cancer risk assessment, indicators, and guidelines for polycyclic aromatic hydrocarbons in the ambient air. Environ Health Perspect 110 Suppl 3: 451–488.
- 29. Buchet JP, Gennart JP, Mercado-Calderon F, Delavignette JP, Cupers L, et al. (1992) Evaluation of exposure to polycyclic aromatic hydrocarbons in a coke production and a graphite electrode manufacturing plant: assessment of urinary excretion of 1-hydroxypyrene as a biological indicator of exposure. Br J Ind Med 49:761–768.
- 30. Jongeneelen FJ (2001) Benchmark guideline for urinary 1-hydroxypyrene as biomarker of occupational exposure to polycyclic aromatic hydrocarbons. Ann Occup Hyg 45:3–13.
- 31. Ramesh A, Walker SA, Hood DB, Guillen MD, Schneider K, et al. (2004) Bioavailability and risk assessment of orally ingested polycyclic aromatic hydrocarbons. Int J Toxicol 23:301–333.
- 32. Llop S, Ballester F, Estarlich M, Ibarluzea J, Manrique A, et al. (2008) Urinary 1-hydroxypyrene, air pollution exposure and associated life style factors in pregnant women. Sci Total Environ 407:97–104.
- 33. Whyatt RM, Bell DA, Jedrychowski W, Santella RM, Garte SJ, et al. (1998) Polycyclic aromatic hydrocarbon-DNA adducts in human placenta and modulation by CYP1A1 induction and genotype. Carcinogenesis 19:1389–1392.
- 34. Kriek E, Rojas M, Alexandrov K, Bartsch H (1998) Polycyclic aromatic hydrocarbon-DNA adducts in humans: relevance as biomarkers for exposure and cancer risk. Mutat Res 400:215–231.
- 35. Polanska K, Hanke W, Sobala W, Brzeznicki S, Ligocka D (2011) Predictors of environmental exposure to polycyclic aromatic hydrocarbons among pregnant women–prospective cohort study in Poland. Int J Occup Med Environ Health 24:8–17.
- 36. Suzuki Y, Niwa M, Yoshinaga J, Mizumoto Y, Serizawa S, et al. (2010) Prenatal exposure to phthalate esters and PAHs and birth outcomes. Environ Int 36:699–704.
- 37. (2009) State-specific secondhand smoke exposure and current cigarette smoking among adults―United States, 2008. MMWR Morb Mortal Wkly Rep 58:1232–1235.
- 38. Cattaneo RA. A P, Sagebina FR, Abreuc CM, Petersen GO, Chatkin JM, Thiesen FV (2007) Validação de método para determinação de cotinina em urina por cromatografia líquida de alta eficiência. Brazilian Journal of Toxicology 19:6.
- 39. Petersen GO, Leite CE, Chatkin JM, Thiesen FV (2010) Cotinine as a biomarker of tobacco exposure: development of a HPLC method and comparison of matrices. J Sep Sci 33:516–521.
- 40. Harmonization ICot (November, 2005) Validation of Analytical Procedures: Text and Methodology, ICH, Geneva.
- 41. Santos S, Studart F, Lamont V (2008) Marcadores biológicos do tabagismo. Pneumologia Paulista: Órgão Informativo da Sociedade Paulista de Pneumologia e Tisiologia 21.
- 42. Dwyer JB, Broide RS, Leslie FM (2008) Nicotine and brain development. Birth Defects Res C Embryo Today 84:30–44.
- 43. Hellstrom-Lindahl E, Nordberg A (2002) Smoking during pregnancy: a way to transfer the addiction to the next generation? Respiration 69:289–293.
- 44. Maritz GS (2008) Nicotine and lung development. Birth Defects Res C Embryo Today 84:45–53.
- 45. Wang L, Pinkerton KE (2008) Detrimental effects of tobacco smoke exposure during development on postnatal lung function and asthma. Birth Defects Res C Embryo Today 84:54–60.
- 46. Rossner P Jr, Milcova A, Libalova H, Novakova Z, Topinka J, et al. (2009) Biomarkers of exposure to tobacco smoke and environmental pollutants in mothers and their transplacental transfer to the foetus. Part II. Oxidative damage. Mutat Res 669:20–26.
- 47. Perera FP, Li Z, Whyatt R, Hoepner L, Wang S, et al. (2009) Prenatal airborne polycyclic aromatic hydrocarbon exposure and child IQ at age 5 years. Pediatrics 124:e195–202.
- 48. Obolenskaya MY, Teplyuk NM, Divi RL, Poirier MC, Filimonova NB, et al. (2010) Human placental glutathione S-transferase activity and polycyclic aromatic hydrocarbon DNA adducts as biomarkers for environmental oxidative stress in placentas from pregnant women living in radioactivity- and chemically-polluted regions. Toxicol Lett 196:80–86.
- 49. Kim H, Hwang JY, Ha EH, Park H, Ha M, et al. (2011) Fruit and vegetable intake influences the association between exposure to polycyclic aromatic hydrocarbons and a marker of oxidative stress in pregnant women. Eur J Clin Nutr 65:1118–1125.
- 50. Perera F, Tang D, Whyatt R, Lederman SA, Jedrychowski W (2005) DNA damage from polycyclic aromatic hydrocarbons measured by benzo[a]pyrene-DNA adducts in mothers and newborns from Northern Manhattan, the World Trade Center Area, Poland, and China. Cancer Epidemiol Biomarkers Prev 14:709–714.
- 51. Bocskay KA, Tang D, Orjuela MA, Liu X, Warburton DP, et al. (2005) Chromosomal aberrations in cord blood are associated with prenatal exposure to carcinogenic polycyclic aromatic hydrocarbons. Cancer Epidemiol Biomarkers Prev 14:506–511.
- 52. Dolinoy DC, Weidman JR, Jirtle RL (2007) Epigenetic gene regulation: linking early developmental environment to adult disease. Reproductive Toxicology 23:297–307.
- 53. Jedrychowski W, Perera FP, Tang D, Stigter L, Mroz E, et al. (2012) Impact of barbecued meat consumed in pregnancy on birth outcomes accounting for personal prenatal exposure to airborne polycyclic aromatic hydrocarbons: Birth cohort study in Poland. Nutrition 28:372–377.
- 54. Edwards SC, Jedrychowski W, Butscher M, Camann D, Kieltyka A, et al. (2010) Prenatal exposure to airborne polycyclic aromatic hydrocarbons and children’s intelligence at 5 years of age in a prospective cohort study in Poland. Environ Health Perspect 118:1326–1331.
- 55. Dejmek J, Solansky I, Benes I, Lenicek J, Sram RJ (2000) The impact of polycyclic aromatic hydrocarbons and fine particles on pregnancy outcome. Environ Health Perspect 108:1159–1164.
- 56. Choi H, Rauh V, Garfinkel R, Tu Y, Perera FP (2008) Prenatal exposure to airborne polycyclic aromatic hydrocarbons and risk of intrauterine growth restriction. Environ Health Perspect 116:658–665.
- 57. Perera F, Tang WY, Herbstman J, Tang D, Levin L, et al. (2009) Relation of DNA methylation of 5′-CpG island of ACSL3 to transplacental exposure to airborne polycyclic aromatic hydrocarbons and childhood asthma. PLoS One 4:e4488.
- 58. Jules GE, Pratap S, Ramesh A, Hood DB (2012) In utero exposure to benzo(a)pyrene predisposes offspring to cardiovascular dysfunction in later-life. Toxicology 295:56–67.
- 59. Rundle A, Hoepner L, Hassoun A, Oberfield S, Freyer G, et al. (2012) Association of childhood obesity with maternal exposure to ambient air polycyclic aromatic hydrocarbons during pregnancy. Am J Epidemiol 175:1163–1172.
- 60. Langlois PH, Hoyt AT, Lupo PJ, Lawson CC, Waters MA, et al. (2013) Maternal occupational exposure to polycyclic aromatic hydrocarbons and risk of oral cleft-affected pregnancies. Cleft Palate Craniofac J 50:337–346.
- 61. Stejskalova L, Pavek P (2011) The function of cytochrome P450 1A1 enzyme (CYP1A1) and aryl hydrocarbon receptor (AhR) in the placenta. Curr Pharm Biotechnol 12:715–730.