ORIGINAL E n d o c r i n e

ARTICLE R e s e a r c h

Maternal Smoking and Fetal Sex Significantly Affect Metabolic Enzyme Expression in the Human Fetal Liver
Peter J. OShaughnessy, Ana Monteiro, Siladitya Bhattacharya, and Paul A. Fowler
Institute of Biodiversity, Animal Health, and Comparative Medicine (P.J.O., A.M.), College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G61 1QH, United Kingdom; and Divisions of Applied Health Sciences (S.B.) and Applied Medicine (P.A.F.), Centre for Reproductive Endocrinology and Medicine, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom

Context: Pollutants and toxicants passing from the mother to the fetus may damage developing organ systems. The human fetal liver is both a potential target organ and a critical defense against exposure to such xenochemicals. Objective: The aim of the study was to determine the effects of human fetal toxicant exposure, via maternal smoking, on metabolic enzyme transcripts in the fetal liver. Design and Setting: We conducted an observational study of mRNA transcripts and proteins in livers from second trimester fetuses at the Universities of Aberdeen and Glasgow. Patients/Participants: Liver samples were taken from 55 normal fetuses from women undergoing second trimester elective termination. Main Outcome Measures: Housekeeping genes for normalization were identified by GeNorm and NormFinder. Levels of mRNA transcripts encoding 15 metabolic enzymes and three xenobiotic receptors were measured. Expression of representative proteins was shown by Western blotting. Results: Eighty-nine percent of measured transcripts were detectable in the human fetal liver. Eight transcripts showed significant sex-specific differences in expression levels (EPHX1, GSTA1, GSTT1, AHR, AS3MT, GLRX2, GGT1, CAR). In male fetuses, maternal smoking was associated with a decrease in expression of three transcripts (GGT1, CYP2R1, CAR) and an increase in eight transcripts (CYP1A1, EPHX1, NQO1, GSTP1, GSTT1, AHR, AS3MT, GLRX2). In the female, CYP3A7 and EPHX1 were increased in smoke-exposed fetuses. Conclusions: The human fetal liver expresses a wide array of metabolic enzymes, with sex differences apparent in 44% of the transcripts measured. Exposure of the fetus to pollutants/toxicants is associated with significantly altered transcript expression, with the more marked response in the male potentially affecting levels of endogenous factors involved in fetal growth. (J Clin Endocrinol Metab 96: 28512860, 2011)

uring gestation the human fetus is exposed, via the maternal circulation, to a large number of potential toxicants from the environment and through maternal lifestyle options. Some of these toxicants, especially those

classed as endocrine-disrupting compounds (e.g. phthalates and dioxins), have been implicated in the increasing incidence of reproductive disorders and cancers (1). However, the potential effects on fetal and postnatal developAbbreviations: EPHX1, Epoxide hydrolase; GST, glutathione S-transferase; HKG, housekeeping gene; PAH, polycyclic aromatic hydrocarbon; XRE, xenobiotic response element.

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright 2011 by The Endocrine Society doi: 10.1210/jc.2011-1437 Received May 6, 2011. Accepted June 15, 2011. First Published Online June 29, 2011

J Clin Endocrinol Metab, September 2011, 96(9):28512860

jcem.endojournals.org

2851

2852

OShaughnessy et al.

Maternal Smoking and Fetal Liver Function

J Clin Endocrinol Metab, September 2011, 96(9):28512860

ment of exposure in utero to many toxicants/pollutants remain unclear and give cause for significant concern. Cigarette smoking remains a major health problem in the general population, but it constitutes a particular risk to the fetus if the mother continues to smoke while pregnant (2). Currently, around 20% of women in the United Kingdom and 10% of women in the United States smoke through pregnancy, with the rate significantly higher in some groups (3). Maternal smoking disturbs reproductive development in the fetus and subsequent offspring and also has lasting harmful effects on bone mineralization, the development and function of the brain, lung, and immune systems and leads to increased incidence of postnatal obesity and metabolic syndrome (4 8). Although maternal cigarette smoking per se is of considerable concern in terms of fetal and postnatal health, it does, nevertheless, offer one of the few mechanisms by which we can directly study the effects of environmental pollutants on human fetal health. Over 4800 chemicals are found in tobacco smoke, and many of these are the same as the potential toxicants found in the environment (2). These include heavy metals, polycyclic aromatic hydrocarbons (PAH), volatile hydrocarbons, aldehydes, aromatic amines, and nitrosamines. Fetuses from smoking mothers, therefore, are a unique model of toxicant exposure that can be exploited to generate insight into the general effects of pollutants/toxicants on human development, especially where direct investigation of the fetus is impossible. Protection of the fetus from xenotoxicants in smoke and from environmental sources is through maternal metabolism, placental metabolism, and finally, metabolism by the fetus itself. In the human, the liver starts to form in the fourth week of gestation, and the basic structure is in place at the end of the first trimester (9). During gestation, the fetal liver receives 70% of its blood supply from the umbilical vein and, therefore, directly from the placenta and the fetomaternal interface. The fetal liver is, therefore, exposed to the highest concentrations of maternally derived xenochemicals. Unlike the rodent, the primate fetal liver is active during fetal development and is the most important fetal organ for drug metabolism (10). By the end of the first trimester, several enzymes involved in drug metabolism are already expressed in the fetal liver [e.g. CYP3A7 and FMO1 (11, 12)], and there is evidence from other primates that fetal levels of some metabolites exceed maternal levels (13). The most important metabolic hepatic enzymes are the phase 1 and phase 2 enzymes, which act to detoxify xenobiotics through oxidation, reduction, and hydrolysis reactions [phase 1; typically cytochrome P450 (CYP) enzymes] and through conjugation reactions (phase 2), which normally inactivate the compound and increase excretion rates. The metabolic activity of these

enzymes and responsiveness to toxicant exposure is likely to be critical in terms of overall metabolism of xenochemicals, with potential consequences for both the fetus itself and for the mother. In the past two decades, considerable progress has been made in our knowledge of human fetal liver activity (14), but our understanding of functional liver development and responsiveness to xenobiotic challenge remains patchy, due largely to a paucity of suitable biological material and relevant model systems. In this study, we have used a cohort of normal second trimester human fetuses to examine the effects of maternal smoking on expression of transcripts encoding metabolic enzymes and xenobiotic receptors in the fetal liver.

Subjects and Methods

Study design
This study was a laboratory-based analysis of mRNA transcripts in livers from 55 fetuses electively terminated from normally progressing pregnancies. The fetuses were divided according to gender and validated maternal cigarette smoking, as shown in Supplemental Fig. 1 (published on The Endocrine Societys Journals Online web site at http://jcem.endojournals.org).

Tissue samples
The collection of fetal material was approved by the National Health Service Grampian Research Ethics Committees (REC 04/ S0802/21) as described previously (1517). Mothers were asked to provide information about smoking and medication during pregnancy; ethical constraints prevented information about recreational drug use being requested. Of the 55 women involved in this study, 12 reported medication use, predominantly asthma inhalers or painkillers, and these 12 women were spread across the four groups. Fetuses were transported to the laboratory within 30 min of delivery, weighed, crown-rump length recorded, and sexed. Fetal livers were removed, snap frozen in liquid nitrogen, and stored at 80 C.

Cotinine assay
Cotinine, a metabolite of nicotine and a marker of smoking, was determined with a commercial kit (Cozart Plc, Abingdon, Kent, UK) giving semiquantitative determination with values between 0 and 12 ng cotinine/ml being considered negative.

RNA extraction, reverse transcription and real-time PCR

Total RNA was extracted from frozen fetal liver samples (10 20 mg) using TRIzol (Life Technologies, Paisley, UK). Reverse transcription, primer design, and real-time PCR were as previously described (15, 18, 19), and the primers used are shown in Supplemental Table 1. Data were normalized against a combination of three housekeeping genes (HKG), as indicated in Results, by expressing the geometric mean of transcript levels relative to each HKG individually.

J Clin Endocrinol Metab, September 2011, 96(9):28512860

jcem.endojournals.org

2853

Cramlington, UK); 5) GSTA1 (1:1000, rabbit, S0796; Epitomics Inc., Burlingame, CA); and 6) CYP3A7 (1 g/ml, mouse, ab55840; Abcam Ltd.). Protein bands were visualized using an Odyssey infrared fluorescence imager (LI-COR), and the resulting electronic images were analyzed using TotalLab TL120 software (v2008.1; Nonlinear Dynamics Ltd., Newcastle-uponTyne, UK) to determine the molecular weights.

Statistical analysis
Pearsons correlation or Spearmans rank correlation coefficient was used to determine whether there was a correlation between transcript expression and age. For most transcripts no significant correlations with fetal age were observed, and so, different ages were combined according to gender and maternal smoking. Normally distributed data were analyzed by two-factor ANOVA (gender maternal smoking), and if group or interaction differences were significant (P 0.05), individual groups were compared by t tests using the pooled error variance. Transcripts that deviated significantly from a normal distribution were analyzed using the Kruskal-Wallis test. Data were analyzed using Minitab (Minitab Ltd., Coventry, UK) and JMP 7.0.2 software (Thomas Learning, London, UK).

FIG. 1. Identification of suitable reference genes for real-time PCR in human fetal liver. GeNorm (A and B) and NormFinder (C and D) analysis of candidate reference genes. A, Expression stability measure, M, of remaining genes during stepwise exclusion of genes with least stability. The lower the value of M, the more stable the gene. B, Determination of the optimal number of reference genes for normalization. Bars indicate the magnitude of the change in normalization factor after the inclusion of an additional reference gene. C, Accumulated SD calculated by adding the next most stable reference gene as shown. The data show that three or four reference genes provide the most stable combination. D, Data analyzed in NormFinder according the gender. The intergroup variation is shown as histogram bars and intragroup variation as error bars. Ideal reference genes have the lowest intergroup and intragroup variation. In AC, n 54; in D, n 30 for males and 24 for females.

Evaluation of candidate reference genes

To identify suitable internal reference genes, we tested seven different HKG in all 55 liver samples using real-time PCR (Supplemental Table 1). Suitability of different HKG was assessed using GeNorm v3.5 and NormFinder algorithms (20, 21).

Results
Maternal and fetal characteristics of the samples used in this study are shown in Supplemental Fig. 1A. For most parameters, the groups were well balanced, and there were no significant differences in maternal characteristics, fetal age, or fetal crown-rump length. The female fetuses were significantly (P 0.034) heavier than the male fetuses (Supplemental Fig. 1A), although this was due largely to inclusion of one older (21-wk) female fetus (Supplemental Fig. 1B). In both fetal genders, the average intensity of TABLE 1. Most stable HKG in fetal human liver in descending order
GeNorm PMM1/B2Ma TBP SDHA HMBS UBC SFRS4 NormFinder B2M SDHA PMM1 TBP HMBS UBC SFRS4

Western blot
Proteins were extracted from fetal liver slices ( 30 mg) with QIAGEN AllPrep DNA/RNA/Protein mini kits (QIAGEN Ltd., Crawley, UK). Tissue was homogenized in the presence of protease inhibitors (Protease Inhibitor Cocktail; Sigma-Aldrich Company Ltd., Gillingham, UK), and protein pellets were suspended in Modified Re-swell Solution [7 M urea, 2 M thiourea, 4% (wt/vol) CHAPS, 0.3% (wt/vol) DL-dithiothreitol]. Odyssey Two-Color molecular weight markers (LI-COR Biosciences UK Ltd., Cambridge, UK) were electrophoresed in every third lane. Representative male and female protein extracts were electrophoresed (30 g protein/lane) on 1-DE 4 12% Bis-Tris gels (Invitrogen Ltd., Paisley, UK) under reducing conditions and transferred to Immobilon-FL membrane (Millipore UK Ltd., Watford, UK) as described previously (22). The membranes were blocked overnight at 4 C with Odyssey Blocking Buffer (9274000; LI-COR) and were then incubated overnight with primary antibodies at 4 C. The antibodies used were: 1) CYP2R1 (1:50, rabbit, ab80101; Abcam Ltd., Cambridge, UK); 2) CYP1A1 (1: 1000, rabbit, sc-2072; Santa Cruz Biotechnology Inc., Santa Cruz, CA); 3) EPXH1 (1:1000, rabbit, ab96695; Abcam Ltd.); 4) AHR (1:250, mouse, MA1514; Thermo Fisher Scientific,

a GeNorm does not distinguish between the two most stable transcripts.

2854

OShaughnessy et al.

Maternal Smoking and Fetal Liver Function

J Clin Endocrinol Metab, September 2011, 96(9):28512860

GeNorm (Fig. 1B) and the accumulation of SD using NormFinder (Fig. 1C) showed that using four HKG would provide the most stable normalization, although stability using three HKG was very similar. NormFinder analysis of the data according to gender (Fig. 1D) showed that B2M had the lowest intragroup and intergroup variation, whereas TBP, PMM1, and SDHA were similar. For subsequent studies, data were therefore normalized against a combination of B2M, PMM1, and TBP. Expression of fetal liver transcripts and proteins Phase 1 enzyme transcripts Transcripts encoding CYP3A7 were highly expressed in all liver samples (Fig. 2). Other P450 enzyme transcripts were more variably expressed, with CYP2R1 detected in most samples, CYP1A1 and CYP2B6 expressed at low but detectable levels in 44 and 30% of samples, respectively (Fig. 2), and CYP1A2 undetectable in all samples (data not shown). The microsomal epoxide hydrolase (EPHX1) transcript was highly expressed in most FIG. 2. Expression of selected transcripts encoding phase 1 enzymes in the human fetal liver and effects of gender and maternal smoking. Real-time PCR was used to measure specific samples, and transcripts encoding the flatranscript levels relative to three HKG as described in the text. For each enzyme, data from vin-containing monooxygenase FMO1 individual fetuses (1116 fetuses per group) are shown, and the horizontal bar represents the were detectable in all samples. In female mean of the group. Data were analyzed using parametric or nonparametric tests as fetuses, there was a significant (P 0.05) appropriate. Significant differences between groups are indicated by letters in the shaded boxes above each graph. Within each transcript, groups that do not share a letter are gestational age-dependent increase in significantly (P 0.05) different. NSm, Nonsmoking group; Sm, smoking group. CYP3A7 and EPHX1, whereas CYP1A1 decreased with age (Supplemental Fig. 2). maternal cigarette smoking was heavy (10 20 cigaThere were no age-dependent changes in male fetuses. rettes per day). EPHX1 showed the only significant gender difference in the phase 1 transcripts with levels in the female fetuses higher Identification of suitable reference genes To identify suitable internal reference genes for the fetal than in the males (P 0.01; Fig. 2). Maternal smoking was liver samples used in this study, we examined expression associated with significant (P 0.05) increases in transcripts of seven HKG using GeNorm and NormFinder (20, 21). encoding CYP1A1 and EPHX1 in male fetuses and CYP3A7 Both algorithms rank transcripts according to their ex- and EPHX1 in female fetuses. Levels of CYP2R1 were repression stability, and results in Fig. 1 and Table 1 show duced in smoke-exposed male fetuses. that the HKG identified as least stable were identical in both algorithms (HMBS, UBC, and SFRS4). There were Phase 2 enzyme transcripts Transcripts encoding GSTA1 were present at very high some differences in the order of the most stable HKG (Table 1), but B2M was identified as the most stable by Norm- levels in almost all fetal liver samples (Fig. 3). GSTP1 Finder and one of the two most stable by GeNorm. Both transcripts were also detected at a high level in most samalgorithms can also provide a measure of the number and ples, whereas GSTT1 was detected at a lower level in 85% best combination of transcripts required for optimal nor- of samples and GSTM1 in 47% of samples. Transcripts malization. Analysis of the pairwise variation using encoding NQO1 were present in 84% of samples (Fig. 3),

J Clin Endocrinol Metab, September 2011, 96(9):28512860

jcem.endojournals.org

2855

from nonsmoking mothers (Fig. 4). Expression of AS3MT and GLRX2 was age-dependent in males (Supplemental Fig. 2). Other transcripts in this group showed no age-dependent changes. Levels AS3MT and GLRX2 were significantly higher in livers from female fetuses, whereas GGT1 was higher in males (Fig. 4). In this group, maternal smoking was associated with significantly increased AS3MT and GLRX2 and decreased GGT1 in male fetuses without any significant associations in female fetuses. Xenobiotic receptor transcripts Transcripts encoding the xenobiotic receptors AHR and CAR were consistently detected in all samples (Fig. 5). Expression of PXR was lower than the other xenosensors but was detected in all samples with the exception of three control (nonexposed) males. There were no significant age-dependent changes in expression in this group. Levels of AHR were significantly higher in livers from male fetuses, whereas CAR was significantly higher in females. Smoking was associated with increased AHR and decreased CAR in males, but not in females.

FIG. 3. Expression of selected transcripts encoding phase 2 enzymes in the human fetal liver and effects of gender and maternal smoking. Real-time PCR was used to measure specific transcript levels relative to three HKG as described in the text. For each enzyme, data from individual fetuses (1116 fetuses per group) are shown, and the horizontal bar represents the mean of the group. Data were analyzed using parametric or nonparametric tests as appropriate. Significant differences between groups are indicated by letters in the shaded boxes above each graph. Within each transcript, groups that do not share a letter are significantly (P 0.05) different. NSm, Nonsmoking group; Sm, smoking group.

whereas UGT1A9 was undetectable in all samples (data not shown). Expression of GSTP1 decreased significantly with gestational age in males only (Supplemental Fig. 2). Other transcripts did not show age-dependent effects. Levels of GSTT1 were significantly higher in livers from female fetuses, whereas GSTA1 was significantly higher in males (Fig. 3). Maternal cigarette smoking was associated with increased GSTT1, GSTP1, and NQO1 levels in male fetuses without being associated with any alterations in transcript levels in female fetuses (Fig. 3). Other enzyme transcripts Transcripts encoding AS3MT and GGT1 were consistently detected in all samples (Fig. 4). All fetuses also expressed the glutaredoxin GLRX2 except for some males

Protein expression Protein expression of representative transcripts was examined qualitatively by Western blotting (Fig. 6). Proteins were selected for study because of their likely importance to liver function, levels of expression, and availability of suitable antibodies. CYP1A1 was selected because there is ongoing controversy about its expression in the fetal liver (see Discussion). For all proteins, bands of the expected molecular mass were seen in both male and female liver samples, indicating that transcripts quantified above are translated into the mature protein. Although CYP2R1 and AHR (Fig. 6, AF) immunoblots showed at least one other band outside the expected molecular mass range, these were less abundant and were likely to be due to nonspecific antibody cross-reactivity. The CYP1A1 band was faint, suggesting low abundance, and while it was 4 kDa lighter than expected (Fig. 6E), this was within 10% of the expected mass.

2856

OShaughnessy et al.

Maternal Smoking and Fetal Liver Function

J Clin Endocrinol Metab, September 2011, 96(9):28512860

FIG. 4. Expression of selected transcripts encoding other metabolic enzymes in the human fetal liver and effects of gender and maternal smoking. Real-time PCR was used to measure specific transcript levels relative to three HKG as described in the text. For each enzyme, data from individual fetuses (1116 fetuses per group) are shown, and the horizontal bar represents the mean of the group. Data were analyzed using parametric or nonparametric tests as appropriate. Significant differences between groups are indicated by letters in the shaded boxes above each graph. Within each transcript, groups that do not share a letter are significantly (P 0.05) different. NSm, Nonsmoking group; Sm, smoking group.

FIG. 5. Expression of selected transcripts encoding xenobiotic receptors in the human fetal liver and effects of gender and maternal smoking. Real-time PCR was used to measure specific transcript levels relative to three HKG as described in the text. For each receptor, data from individual fetuses (1116 fetuses per group) are shown, and the horizontal bar represents the mean of the group. Data were analyzed using parametric or nonparametric tests as appropriate. Significant differences between groups are indicated by letters in the shaded boxes above each graph. Within each transcript, groups that do not share a letter are significantly (P 0.05) different. NSm, Nonsmoking group; Sm, smoking group.

Discussion
Maternal smoking exposes the fetus to a wide array of potential toxicants, although the mechanisms by which these toxicants act to affect fetal growth and development are unclear. Results from this study now show, however,

that the xenochemicals introduced to the fetus through maternal smoking are associated with significant effects on the expression of key fetal liver transcripts. This is likely to affect liver function and to have consequential effects on other organ systems through changes in the amount and types of toxicants in the fetal circulation. In addition, our data show, unexpectedly, that a gender difference exists both in control (nonexposed) fetal livers and in the re-

J Clin Endocrinol Metab, September 2011, 96(9):28512860

jcem.endojournals.org

2857

FIG. 6. Expression of selected proteins in the human fetal liver. Protein was extracted from male and female fetal livers and immunoblotted. For each blot, the arrow indicates the molecular mass of the protein as determined using TotalLab software.

sponse of the fetal liver to maternal cigarette smoke chemicals. Overall, eight transcripts showed a gender difference, while maternal smoking was associated with increased levels of eight transcripts in male fetuses but only two transcripts in females. The implications of such constitutive and induced gender-specific differences in transcript expression may be highly significant with respect to xenochemical metabolism. Lower expression of some transcripts such as EPHX1 in male fetuses may mean, for example, that they are more susceptible to xenotoxicant exposure and, at the very least, the data implies that fetal exposure/susceptibility to some toxicants is likely to be sex-specific. Gender differences in the adult human liver are well established and are related to differences in the pattern of GH release established at puberty (23). In the fetus, the major hormonal difference between genders is the presence of high androgens in the male secreted by the developing testis. It is possible that these androgens may act to establish a male pattern of fetal liver activity because the androgen receptor is expressed in the human fetal liver (24). Pertinently, androgens can induce gender-specific patterns of metabolic enzyme activity in other tissues (23). Other evidence for gender differences in human fetal liver function is scanty, although such differences have been reported in other species (2528). Furthermore, maternal smoking has been shown to have gender-specific effects in the human fetus (29). Cigarette smoke contains known inducers of adult hepatic enzymes. Induction of enzyme transcription by xenobiotics is through the xenobiotic receptors (the xenosensors), AHR, CAR and PXR, which act on specific xenobiotic response elements (XRE) on the enzyme promoter (30). Expression of all three xenosensors in our samples, together with the likely inductive effects of maternal smoking, suggests that smoking acts through the canonical pathways responsible for xenobiotic alteration of hepatic activity in the second trimester human fetus.

The principal phase 1 enzymes known to be induced through the AHR are the CYP1 family (31). In adult humans, smoking has been shown to induce protein or transcript levels of CYP1A1 and CYP1B1 in the lungs (32, 33) and CYP1A2 and CYP1B1 in the liver (34 36). It is notable that CYP1A1 does not appear to be expressed in the adult human liver (37), and its presence in the fetal liver has been a matter of some debate (14). We show here that CYP1A1, and its product, were expressed in 30% of fetal livers from nonsmoking mothers, and this rose significantly to 80% of male fetal livers from smoking mothers. This induction may contribute to the variability in detection of CYP1A1, as may the apparent age-dependent decline in CYP1A1 in female fetuses in the early second trimester, as suggested previously (14). CYP1A1 can activate PAH to more toxic metabolites (38, 39), and its presence in the early fetal liver, its apparent induction by maternal smoking, and its absence in the adult liver may reflect important age differences in liver susceptibility to toxicants. The principal phase 1 enzymes induced by CAR and PXR include the CYP3A family (40), and there is evidence from previous studies that CYP3A5 is induced in adult lungs by smoking (33). This is consistent with the apparent induction of CYP3A7 by maternal smoking in female fetal livers reported here. CYP3A7 is the predominant phase 1 enzyme in the fetal liver, but it is replaced fairly rapidly after birth by CYP3A4 (41). Induction of CYP3A7 is of significance because the CYP3A enzymes metabolize around 50% of xenobiotics in the liver (14) and may, therefore, play a role in generation of harmful metabolites. Maternal smoking is associated with a reduction in active vitamin D levels and bone mineral acquisition in the fetus (42, 43). Formation of active 1 ,25-dihydroxyvitamin D3 is dependent on the critical 25-hydroxylation of vitamin D3, and this is mediated primarily by CYP2R1 (44, 45). Interestingly, maternal smoking was associated with a significant reduction in CYP2R1 levels in male fetuses, although the effect in females was not significant. This inhibition of hepatic CYP2R1 may contribute to a reduction in fetal synthesis of active vitamin D and reduced overall levels, particularly if maternal levels are also affected (46). The effect of lowered CYP2R1 may be most acute in the neonate, which can no longer rely on a maternal supply of active vitamin D (43). The toxicity and/or carcinogenesis of many xenochemicals is linked to their metabolism to active intermediates such as epoxides. These epoxides are deactivated primarily by microsomal EPHX1 and cytosolic glutathione Stransferases (GST). Levels of EPHX1, GSTP1, and particularly GSTA1 were high in both male and female livers, emphasizing their detoxification role in the fetus. In addition, maternal smoking was associated with induction

2858

OShaughnessy et al.

Maternal Smoking and Fetal Liver Function

J Clin Endocrinol Metab, September 2011, 96(9):28512860

of EPHX1, GSTP1, and GSTT1 expression in the fetal liver. This is consistent with previous reports that PAH can increase EPHX1 and GST protein and transcript levels in adult human liver (47, 48). Similarly, smoking has been shown to increase EPHX1 and GSTP1 in adult bronchoalveolar cells (33). The potential involvement of PAH indicates that these effects may be mediated through the AHR (47), and this is supported by the presence of eight XRE in the human EPHX1 promoter (49). Similarly, the promoter of NQO1 also contains seven XRE (49), and maternal smoking was associated with induced transcript levels of this enzyme in male fetuses. This is consistent with previous studies that have shown that condensates from cigarette smoke will induce NQO1 in isolated human epithelial airway cells (50). The challenges facing toxicity testing and chemical safety testing strategies (e.g. of candidate endocrine-disrupting compounds) are considerable, and the results of this and previous studies suggest that recent legislation, such as the European Unions REACH regulation (51), may require further consideration. Previous studies have shown that metabolic enzyme activity in the human liver is developmentally regulated, with a unique expression pattern in the developing fetus (14). It is also clear that there are marked species differences in liver enzyme activity, and these are particularly pronounced during fetal development, partly due to differences in the length of gestation (5254). Added to that, we can now combine gender differences in fetal liver function, as well as the induction of fetal liver activity by prior exposure to xenochemicals. Our data suggest, for example, that current safety testing programs (55) may fail to detect effects that might be specific to one gender at particular life stages. With respect to the health and safety of the developing human fetus in utero, therefore, these findings demonstrate not only that the species used to test chemical safety must be appropriate and relevant to human health, but also that the appropriate developmental stage and fetal gender must be identified. Overall, our studies show that there are gender differences in constitutive hepatic transcript levels in the human fetus and that maternal smoking is associated with significant changes in the expression pattern of these transcripts, particularly in the male. Although these results emphasize that maternal smoking per se should be strongly discouraged, they also show that fetal exposure to environmental toxicants which act through similar mechanisms to cigarette smoke toxicants will probably affect fetal liver activity, irrespective of whether the mother herself smokes. This change in activity will have consequences for the fetus, through both changes in the nature of the

toxicant exposure and alterations in endogenous metabolic pathways.

Acknowledgments
The help of the staff of the Pregnancy Counseling Service, Aberdeen Maternity Hospital, was essential for the collection of the fetuses. We are grateful to Ms. Margaret Fraser for her expert technical assistance. Address all correspondence and requests for reprints to: Professor P. J. OShaughnessy, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Garscube Estate, Bearsden Road, Glasgow G61 1QH, United Kingdom. E-mail: Peter.OShaughnessy@glasgow.ac.uk. The study was partly supported by grants from the Chief Scientist Office (Scottish Executive, CZG/1/109), the European Communitys Seventh Framework Program (FP7/2007-2013) under Grant Agreement no. 212885, and National Health Service Grampian Endowments Grants 08/02 and 09/12. The study sponsors had no role in study design, collection, analysis and interpretation of data, writing of the paper, or the decision to submit the paper for publication. Disclosure Summary: The authors have nothing to disclose.

References
1. Wigle DT, Arbuckle TE, Walker M, Wade MG, Liu S, Krewski D 2007 Environmental hazards: evidence for effects on child health. J Toxicol Environ Health B Crit Rev 10:339 2. Rogers JM 2008 Tobacco and pregnancy: overview of exposures and effects. Birth Defects Res C Embryo Today 84:115 3. Tappin DM, MacAskill S, Bauld L, Eadie D, Shipton D, Galbraith L 2010 Smoking prevalence and smoking cessation services for pregnant women in Scotland. Subst Abuse Treat Prev Policy 5:1 4. Weinberg CR, Wilcox AJ, Baird DD 1989 Reduced fecundability in women with prenatal exposure to cigarette smoking. Am J Epidemiol 129:10721078 5. Montgomery SM, Ekbom A 2002 Smoking during pregnancy and diabetes mellitus in a British longitudinal birth cohort. BMJ 324: 26 27 6. Oken E, Levitan EB, Gillman MW 2008 Maternal smoking during pregnancy and child overweight: systematic review and meta-analysis. Int J Obes (Lond) 32:201210 7. Hylkema MN, Blacquiere MJ 2009 Intrauterine effects of maternal ` smoking on sensitization, asthma, and chronic obstructive pulmonary disease. Proc Am Thorac Soc 6:660 662 8. Cornelius MD, Day NL 2009 Developmental consequences of prenatal tobacco exposure. Curr Opin Neurol 22:121125 9. Ring JA, Ghabrial H, Ching MS, Smallwood RA, Morgan DJ 1999 Fetal hepatic drug elimination. Pharmacol Ther 84:429 445 10. Krauer B, Dayer P 1991 Fetal drug metabolism and its possible clinical implications. Clin Pharmacokinet 21:70 80 11. Koukouritaki SB, Simpson P, Yeung CK, Rettie AE, Hines RN 2002 Human hepatic flavin-containing monooxygenases 1 (FMO1) and 3 (FMO3) developmental expression. Pediatr Res 51:236 243 12. Stevens JC, Hines RN, Gu C, Koukouritaki SB, Manro JR, Tandler PJ, Zaya MJ 2003 Developmental expression of the major human hepatic CYP3A enzymes. J Pharmacol Exp Ther 307:573582 13. Garland M, Abildskov KM, Kiu TW, Daniel SS, Stark RI 2005 The

J Clin Endocrinol Metab, September 2011, 96(9):28512860

jcem.endojournals.org

2859

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

contribution of fetal metabolism to the disposition of morphine. Drug Metab Dispos 33:68 76 Hines RN 2008 The ontogeny of drug metabolism enzymes and implications for adverse drug events. Pharmacol Ther 118:250 267 OShaughnessy PJ, Baker PJ, Monteiro A, Cassie S, Bhattacharya S, Fowler PA 2007 Developmental changes in human fetal testicular cell numbers and messenger ribonucleic acid levels during the second trimester. J Clin Endocrinol Metab 92:4792 4801 Fowler PA, Cassie S, Rhind SM, Brewer MJ, Collinson JM, Lea RG, Baker PJ, Bhattacharya S, OShaughnessy PJ 2008 Maternal smoking during pregnancy specifically reduces human fetal desert hedgehog gene expression during testis development. J Clin Endocrinol Metab 93:619 626 Fowler PA, Bhattacharya S, Gromoll J, Monteiro A, OShaughnessy PJ 2009 Maternal smoking and developmental changes in luteinizing hormone (LH) and the LH receptor in the fetal testis. J Clin Endocrinol Metab 94:4688 4695 OShaughnessy PJ, Willerton L, Baker PJ 2002 Changes in Leydig cell gene expression during development in the mouse. Biol Reprod 66:966 975 Baker PJ, OShaughnessy PJ 2001 Expression of prostaglandin D synthetase during development in the mouse testis. Reproduction 122:553559 Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F 2002 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034 Andersen CL, Jensen JL, rntoft TF 2004 Normalization of realtime quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:52455250 Fowler PA, Flannigan S, Mathers A, Gillanders K, Lea RG, Wood MJ, Maheshwari A, Bhattacharya S, Collie-Duguid ES, Baker PJ, Monteiro A, OShaughnessy PJ 2009 Gene expression analysis of human fetal ovarian primordial follicle formation. J Clin Endocrinol Metab 94:14271435 Waxman DJ, Holloway MG 2009 Sex differences in the expression of hepatic drug metabolizing enzymes. Mol Pharmacol 76: 215228 Stubbs AP, Engelman JL, Walker JI, Faik P, Murphy GM, Wilkinson ML 1994 Measurement of androgen receptor expression in adult liver, fetal liver, and Hep-G2 cells by the polymerase chain reaction. Gut 35:683 686 Kwong WY, Wild AE, Roberts P, Willis AC, Fleming TP 2000 Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension. Development 127:4195 4202 Lange IG, Daxenberger A, Meyer HH, Rajpert-De Meyts E, Skakkebaek NE, Veeramachaneni DN 2002 Quantitative assessment of foetal exposure to trenbolone acetate, zeranol and melengestrol acetate, following maternal dosing in rabbits. Xenobiotica 32:641 651 ORegan D, Kenyon CJ, Seckl JR, Holmes MC 2004 Glucocorticoid exposure in late gestation in the rat permanently programs genderspecific differences in adult cardiovascular and metabolic physiology. Am J Physiol Endocrinol Metab 287:E863E870 Kwong WY, Miller DJ, Wilkins AP, Dear MS, Wright JN, Osmond C, Zhang J, Fleming TP 2007 Maternal low protein diet restricted to the preimplantation period induces a gender-specific change on hepatic gene expression in rat fetuses. Mol Reprod Dev 74:48 56 Bahado-Singh RO, Schenone M, Cordoba M, Shieh WS, Maulik D, Kruger M, Reece EA 2011 Male gender significantly increases risk of oxidative stress related congenital anomalies in the non-diabetic population. J Matern Fetal Neonatal Med 24:687 691 Kohle C, Bock KW 2007 Coordinate regulation of phase I and II

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45. 46.

47.

48.

xenobiotic metabolisms by the Ah receptor and Nrf2. Biochem Pharmacol 73:18531862 Waxman DJ 1999 P450 gene induction by structurally diverse xenochemicals: central role of nuclear receptors CAR, PXR, and PPAR. Arch Biochem Biophys 369:1123 McLemore TL, Adelberg S, Czerwinski M, Hubbard WC, Yu SJ, Storeng R, Wood TG, Hines RN, Boyd MR 1989 Altered regulation of the cytochrome P4501A1 gene: novel inducer-independent gene expression in pulmonary carcinoma cell lines. J Natl Cancer Inst 81:17871794 Thum T, Erpenbeck VJ, Moeller J, Hohlfeld JM, Krug N, Borlak J 2006 Expression of xenobiotic metabolizing enzymes in different lung compartments of smokers and nonsmokers. Environ Health Perspect 114:16551661 Baker JR, Satarug S, Reilly PE, Edwards RJ, Ariyoshi N, Kamataki T, Moore MR, Williams DJ 2001 Relationships between non-occupational cadmium exposure and expression of nine cytochrome P450 forms in human liver and kidney cortex samples. Biochem Pharmacol 62:713721 Rasmussen BB, Brix TH, Kyvik KO, Brsen K 2002 The interindividual differences in the 3-demthylation of caffeine alias CYP1A2 is determined by both genetic and environmental factors. Pharmacogenetics 12:473 478 Chang TK, Chen J, Pillay V, Ho JY, Bandiera SM 2003 Real-time polymerase chain reaction analysis of CYP1B1 gene expression in human liver. Toxicol Sci 71:1119 Edwards RJ, Adams DA, Watts PS, Davies DS, Boobis AR 1998 Development of a comprehensive panel of antibodies against the major xenobiotic metabolising forms of cytochrome P450 in humans. Biochem Pharmacol 56:377387 Roberts-Thomson SJ, McManus ME, Tukey RH, Gonzalez FF, Holder GM 1993 The catalytic activity of four expressed human cytochrome P450s towards benzo[a]pyrene and the isomers of its proximate carcinogen. Biochem Biophys Res Commun 192:1373 1379 Shou M, Korzekwa KR, Crespi CL, Gonzalez FJ, Gelboin HV 1994 The role of 12 cDNA-expressed human, rodent, and rabbit cytochromes P450 in the metabolism of benzo[a]pyrene and benzo[a]pyrene trans-7,8-dihydrodiol. Mol Carcinog 10:159 168 Vyhlidal CA, Gaedigk R, Leeder JS 2006 Nuclear receptor expression in fetal and pediatric liver: correlation with CYP3A expression. Drug Metab Dispos 34:131137 Lacroix D, Sonnier M, Moncion A, Cheron G, Cresteil T 1997 Expression of CYP3A in the human liver evidence that the shift between CYP3A7 and CYP3A4 occurs immediately after birth. Eur J Biochem 247:625 634 Cooper C, Javaid K, Westlake S, Harvey N, Dennison E 2005 Developmental origins of osteoporotic fracture: the role of maternal vitamin D insufficiency. J Nutr 135:2728S2734S Daz-Gomez NM, Mendoza C, Gonzalez-Gonzalez NL, Barroso F, Jimenez-Sosa A, Domenech E, Clemente I, Barrios Y, Moya M 2007 Maternal smoking and the vitamin D-parathyroid hormone system during the perinatal period. J Pediatr 151:618 623 Cheng JB, Motola DL, Mangelsdorf DJ, Russell DW 2003 De-orphanization of cytochrome P450 2R1: a microsomal vitamin D 25hydroxilase. J Biol Chem 278:38084 38093 Schuster I 2011 Cytochromes P450 are essential players in the vitamin D signaling system. Biochim Biophys Acta 1814:186 199 Javaid MK, Crozier SR, Harvey NC, Gale CR, Dennison EM, Boucher BJ, Arden NK, Godfrey KM, Cooper C 2006 Maternal vitamin D status during pregnancy and childhood bone mass at age 9 years: a longitudinal study. Lancet 367:36 43 Pushparajah DS, Umachandran M, Plant KE, Plant N, Ioannides C 2008 Up-regulation of the glutathione S-transferase system in human liver by polycyclic aromatic hydrocarbons; comparison with rat liver and lung. Mutagenesis 23:299 308 Pushparajah DS, Umachandran M, Plant KE, Plant N, Ioannides C 2008 Differential response of human and rat epoxide hydrolase to

2860

OShaughnessy et al.

Maternal Smoking and Fetal Liver Function

J Clin Endocrinol Metab, September 2011, 96(9):28512860

polycyclic aromatic hydrocarbon exposure: studies using precisioncut tissue slices. Mutat Res 640:153161 49. Burchiel SW, Thompson TA, Lauer FT, Oprea TI 2007 Activation of dioxin response element (DRE)-associated genes by benzo (a) pyrene 3,6-quinone and benzo (a) pyrene 1,6-quinone in MCF-10A human mammary epithelial cells. Toxicol Appl Pharmacol 221: 203214 50. Pickett G, Seagrave J, Boggs S, Polzin G, Richter P, Tesfaigzi Y 2010 Effects of 10 cigarette smoke condensates on primary human airway epithelial cells by comparative gene and cytokine expression studies. Toxicol Sci 114:79 89

51. Williams ES, Panko J, Paustenbach DJ 2009 The European Unions REACH regulation: a review of its history and requirements. Crit Rev Toxicol 39:553575 52. Juchau MR, Chao ST, Omiecinski CJ 1980 Drug metabolism by the human fetus. Clin Pharmacokinet 5:320 339 53. Pelkonen O 1980 Biotransformation of xenobiotics in the fetus. Pharmacol Ther 10:261281 54. Klinger W 2005 Developmental pharmacology and toxicology: biotransformation of drugs and other xenobiotics during postnatal development. Eur J Drug Metab Pharmacokinet 30:317 55. Cooper RL 2009 Current developments in reproductive toxicity testing of pesticides. Reprod Toxicol 28:180 187

Members have FREE online access to the journal Hormones & Cancer.
www.endo-society.org/hc