Early Human Development (2007) 83, 699–706 a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m w w w. e l s e v i e r. c o m / l o c a t e / e a r l h u m d e v BEST PRACTICE GUIDELINE ARTICLE Morphological and biological effects of maternal exposure to tobacco smoke on the feto-placental unit Eric Jauniaux ⁎, Graham J. Burton Academic Department of Obstetrics and Gynaecology, Royal Free and University College London Medical School, London, UK Department of Physiology, Development and Neuroscience, University of Cambridge, UK KEYWORDS Placenta; Fetus; Maternal smoking; Anatomy; Biology; Tobacco Abstract Active and passive maternal smoking has a damaging effect in every trimester of human pregnancy. Cigarette smoke contains scores of toxins which exert a direct effect on the placental and fetal cell proliferation and differentiation and can explain the increased risk of miscarriage, fetal growth restriction (FGR) stillbirth, preterm birth and placental abruption reported by epidemiological studies. In the placenta, smoking is associated from early in pregnancy, with a thickening of the trophoblastic basement membrane, an increase in collagen content of the villous mesenchyme and a decrease in vascularisation. These anatomical changes are associated with changes in placental enzymatic and synthetic functions. In particular, nicotine depresses active amino-acid (AA) uptake by human placental villi and trophoblast invasion and cadmium decreases the expression and activity of 11 beta-hydroxysteroid dehydrogenase type 2 which is causally linked to FGR. Maternal smoking also dysregulates trophoblast expression of molecules that govern cellular responses to oxygen tension. In the fetus, smoking is associated with a reduction of weight, fat mass and most anthropometric parameters and as in the placenta with alterations in protein metabolism and enzyme activity. These alterations are the results of a direct toxic effect on the fetal cells or an indirect effect through damage to, and/or functional disturbances of the placenta. In particular, smoking interferes strongly with the fetal brain and pancreas biological parameters and induces chromosomal instability, which is associated with an increase in the risk of cancer, especially childhood malignancies. © 2007 Elsevier Ireland Ltd. All rights reserved. Contents 1. 2. 3. 4. Introduction . . . . . . . . . . . . . . . . . . . . . Effect of maternal smoking on placental anatomy Effect of tobacco smoke on placental biology . . Effect of tobacco smoke on fetal anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 700 700 701 703 ⁎ Corresponding author. Academic Department of Obstetrics and Gynaecology, University College London Medical School, 86-96 Chenies Mews, London WC1E, 6HX, UK. Tel.: +44 207 2096057; fax: +44 207 3837429. E-mail address: e.jauniaux@ucl.ac.uk (E. Jauniaux). 0378-3782/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2007.07.016 700 5. Effect of tobacco smoke on fetal biology . 6. Key guidelines . . . . . . . . . . . . . . . . 7. Conclusion . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . E. Jauniaux, G.J. Burton . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction Chronic exposure of the fetus to the effects of tobacco smoke is recognized as the most important preventable risk factor for a complicated pregnancy outcome in all developed, and an increasing number of developing, countries. Active maternal cigarette smoking has a damaging effect in every trimester of pregnancy (Table 1). There is undisputed evidence from large epidemiological studies that maternal smoking is associated with fetal growth restriction (FGR), and with increased risks of stillbirth, preterm birth and placental abruption [1–4]. Smoking around the time of implantation and establishment of the placenta is generally associated with an increased risk of miscarriage, ectopic pregnancies, and placenta previa [4–9]. Maternal smoking can also be a risk factor for orofacial clefts, particularly among fetuses lacking enzymes involved in the detoxification of tobacco-derived chemicals [10,11]. Epidemiological studies have also indicated that passive maternal smoking can have a negative impact on fetal growth [12,13]. Cigarette smoke contains scores of toxins, including cyanide, sulphides, cadmium, carcinogenic hydrocarbons and nicotine, all of which are capable of inducing direct cellular damage [14]. Most tobacco toxins have a low molecular weight and high water solubility, and therefore readily cross the placenta [15–17]. In particular, nicotine and cotinine pass freely across to the fetus, which as a result is exposed to relatively higher nicotine concentrations than its mother. Although placental xenobiotic-metabolizing enzymes can detoxify foreign chemicals, tobacco constituents exert direct effects on the villous cytotrophoblast proliferation and differentiation [18,19]. These can explain the negative effects of smoking on placentation and formation of the placental membranes, and on feto-placental growth and development. We have reviewed the effects of chronic maternal exposure to tobacco smoke on feto-placental development and the associated impact on fetal organ biology. Table 1 Placental and fetal side-effects of maternal tobacco smoking in early and late pregnancy Early pregnancy Miscarriage Ectopic pregnancy Placenta previa and placenta previa-accreta Fetal orofacial clefts Late pregnancy Fetal growth restriction Placental insufficiency Placental abruption Premature rupture of the placental membranes and preterm delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703 704 704 704 2. Effect of maternal smoking on placental anatomy Placental morphological damage related to heavy chronic maternal smoking can be identified as early as the first trimester of pregnancy [19]. It is well established that the mean placental weight in smokers is decreased, depending on the number of cigarettes smoked by the mother per day throughout pregnancy [20]. Other gross morphological changes associated with maternal smoking are more controversial. For example, most authors have found no alteration in the pattern of lobation or the incidence of infarcts [21,22], whereas some have suggested that the incidence of such infarcts is lower amongst smokers [23] and others have reported the opposite [24]. Similar controversy surrounds the incidence of calcification observed in the region of the basal plate [21]. The clinical significance of these gross morphological findings remains to be established as no correlation has been observed between the extent of these changes and the occurrence of FGR (Tables 1 and 2). Initial ultrastructural studies showed that chronic exposure of placental tissues to tobacco smoke is associated with changes similar to those found in adult organs from heavy smokers. The characteristic features include thickening of the trophoblastic basement membrane, an increase in collagen content of the villous mesenchyme, a decrease in vascularisation and pronounced intimal oedema in the villous arterioles [25,26]. The placental villi from smokers display abnormalities of the microvilli, focal syncytial necrosis, decreased syncytial pinocytotic activity, and degenerate cytoplasmic organelles [20]. Some of these changes, such as excessive syncytial necrosis, can be observed from 9 weeks of gestation [19]. Histomorphometric studies have also yielded contrasting results, most likely due to the considerable methodological variations employed. An early study suggested that the placentas of mothers who smoked are microscopically similar to those of nonsmokers. There was a tendency, however, for the placentas in the smokers group to contain proportionally more nonparenchymal and less parenchymal tissue than the control group, mainly in terms of a relative reduction in the volumes of the intervillous space and the peripheral villous tree [27]. The findings of this study supported the hypothesis that the increased perinatal morbidity associated with cigarette smoking during pregnancy is more related to the ischemic and/or toxic effects of several compounds in tobacco smoke, partly on placental function and also directly on the fetus, than to significant alterations in the functional structure of the placenta. Subsequent studies using computerised histomorphometric techniques demonstrated that placentas of smokers and of those who stopped smoking after conception exhibit a reduced capillary volume, surface area and length compared with the placentas of nonsmokers and of those who stopped before pregnancy [22,28]. Furthermore, there was no evidence of a dose-dependent response, suggesting that any smoking at any stage of pregnancy has Effects of maternal exposure to tobacco smoke on the fetoQplacental unit 701 Table 2 Placental morphological and biological changes associated with maternal smoking Increased Trophoblastic and villous membrane thickness. Syncytiotrophoblastic necrosis. Thickness of the trophoblast basal membrane. Apoptosis in the syncytiotrophoblast. Expression of pVHL, HIFs, and VEGFs. Simulation of nicotinic acetylcholine receptors. Level of metallothionein. Vasoconstrictive response to endothelin 1 Decreased Decrease in villous capillary volume fraction. Decrease in total surfaces of syncytial knots. Synthesis and activation of the 92 kDa type IV collagenase. Cytotrophoblast expression of l-selectin and TRA-1-81-reactive carbohydrate ligands. Expression and activity of 11 beta-hydroxysteroid dehydrogenase type 2. Progesterone synthesis. Enzymatic activity of complex III and mitochondrial DNA. pVHL = von Hippel–Lindau tumor suppressor protein; HIFs = hypoxia-inducible transcription factors; VEGFs = vascular endothelial growth factors. equivalent effects on the vasculature. When perfusion-fixed under physiological pressures the terminal villi from all subjects, even women who smoked 30 cigarettes/day, showed large dilated fetal capillaries, with smooth luminal outlines, and no evidence of widespread trophoblastic damage (Figs. 1 and 2). In addition, most histological and histomorphometric studies have found that the villous membrane is significantly thicker among smokers compared to nonsmokers [20,28–30]. Unlike the changes in the terminal villous capillaries, changes in trophoblast and overall villous membrane thickness can be observed from Figure 2 Terminal villi from a mature placenta of a mother who smoked 20 cigarettes per day throughout pregnancy perfusion-fixed at the same physiological pressure as used in Fig. 1. The fetal capillaries are less distended than in placentas from nonsmoking mothers. As a result they represent a lower fraction of the villous volume, and the villous membrane is thicker than in nonsmokers. The impact on fetal blood flow coupled with the poorer diffusion characteristics will impair oxygen exchange to the fetus. Note also the increased collagen content of the villous core. Mag. ×160. the end of the first trimester of pregnancy [19]. The increased thickness of the villous membrane in smokers might be expected to compromise gas transfer to the fetus from an early stage in pregnancy and therefore explain the subsequent FGR [20,22,28]. However, no differences were found when the total placental conductance for oxygen was calculated for term placentas [31], although this may reflect the fact that birthweight was not significantly reduced in this sample group. A recent study of the deposition of fibrin-type fibrinoid in placentas from smokers showed that the total surface area of syncytial knots is reduced in smokers, consistent with reports that smoking reduces the incidence of trophoblast apoptosis [32,33]. On the other hand, the surface area of syncytial bridges is increased, consistent with enhanced branching angiogenesis [32]. In heavy smokers there are reduced deposits of perivillous fibrin at syncytial knots. In all placentas, the greatest deposits occurred where there was trophoblast denudation, suggesting a haemostatic response to loss of what effectively constitutes the endothelial lining of the intervillous space. Overall these data indicate that smoking perturbs the normal pattern of fibrin deposition, that the impact is greater in heavy smokers and that the placental site is privileged or active in terms of fibrinolytic or anticoagulatory activity. This activity seems to reside in thin regions of syncytium [32]. 3. Effect of tobacco smoke on placental biology Figure 1 Terminal villi from a mature placenta of a nonsmoking mother perfusion-fixed at physiological pressure. Note the distended fetal capillaries which are closely approximated to the trophoblast covering of the villi, resulting in a very thin membrane separating the two circulations. Mag. × 160. Maternal smoking during pregnancy is associated with a decrease in all placental biological functions, which leads progressively to intrauterine placental growth restriction. Overall, tobacco smoke chemicals can have a direct impact by altering trophoblast proliferation and differentiation, and indirectly by altering the mechanical properties of the villous vasculature with subsequent reduction of blood flow 702 in the umbilico-placental circulation. Maternal smoking has been known to be associated with changes in placental enzymatic and synthetic functions for several decades [21] but the precise role of tobacco smoke on these functions is difficult to establish as the early studies varied widely in their methodology. Nicotine is one of the main pathogenic compounds of cigarette smoke, and is known to depress active amino-acid (AA) uptake by human placental villi. In particular, it binds to the acetylcholine (ACh) binding site of the alpha-subunits of nicotinic acetylcholine receptors (nAChR) [34]. ACh has been suggested to be an important placental signalling molecule that, through stimulation of nAChR, controls the uptake of nutrients, blood flow and fluid volume in placental vessels, and vascularisation during placental development. Chronic stimulation of nAChR by nicotine might result in unbalanced receptor activation or functional desensitisation, leading to the known pathological effects of smoking. Cadmium, a common environmental pollutant, is also a major constituent of tobacco smoke. It has been identified as a new class of endocrine disruptors with a wide range of detrimental effects on mammalian reproduction [35]. Cadmium concentrations are of the same order of magnitude in both cord and maternal serum [22,36], indicating that cadmium, like most tobacco chemical constituents, is transferred easily from the mother to her fetus through the placenta. A substantial amount of evidence suggests that cadmium may affect fetal growth indirectly via the placenta. However, the mechanisms linking placental cadmium accumulation and decreased fetal growth remain poorly understood. Previous data have shown a link between placental cadmium and lower progesterone levels in smokers than in nonsmokers [37]. More recent data have shown that the expression and activity of 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2), which is causally linked to FGR, is decreased in cultured human trophoblast cells exposed to cadmium, and that the decrease is both time- and concentration-dependent [35]. The metal-binding protein, metallothionein (MT), is involved in the protection of human trophoblastic cells from heavy metal-induced, and oxidative stress-induced, apoptosis [38]. Higher levels of MT have been found in placentas of smokers compared to nonsmokers suggesting that MTs are in excess to bind all cadmium ions present in placentas exposed to maternal smoking. By contrast, most placental zinc remains unbound to MTs, although twice as much zinc ions could theoretically be bound to MT in smokers [38]. MT-2 is the main isoform induced by smoking indicating that this isoform could be involved in placental cadmium and zinc retention. This may reduce the transference of zinc to the fetus, contributing to the detrimental effects on fetal growth and development [39]. Maternal smoking harms human placental development by changing the balance between cytotrophoblast (CTB) proliferation and differentiation [40]. Nicotine inhibits normal first trimester cytotrophoblast invasion, apparently by reducing the ability of treated cells to synthesize and activate the 92 kDa type IV collagenase, an important mediator of invasion in vitro [41]. Exposure to tobacco smoke also dysregulates CTB expression of molecules that govern cellular responses to oxygen tension such as the von Hippel–Lindau tumor suppressor protein (pVHL), the hypoxia-inducible transcription factors (HIFs), and the vascular endothelial growth factors (VEGFs) which are key E. Jauniaux, G.J. Burton mediators of placental development [42]. A subset of these effects can be detected in samples obtained from women who are passively exposed to cigarette smoke during pregnancy [43]. In addition, maternal smoking downregulates in a dosedependent manner cytotrophoblast expression of l-selectin, and its TRA-1-81-reactive carbohydrate ligands. Cell islands, cell columns that fail to make uterine attachments and are often more numerous in the placentas of smokers, exhibit an even greater downregulation of the l-selectin adhesion system. These effects are attributable to nicotine, since exposure of villous explants to the drug in vitro reproduces the effects [43]. These results suggest that nicotine, acting through the l-selectin adhesion system, impairs the development of cell columns that connect the fetal portion of the placenta to the uterus. This is one possible reason why women who smoke experience more difficulty achieving and sustaining a pregnancy than their nonsmoking counterparts. Tobacco usage is known to alter mitochondrial respiratory function in cardiomyocytes and lung tissue. A reduction in the enzymatic activity of complex III (mitochondrial membranebound cytochrome bc1 proton pump complex) of approximately 30% has recently been demonstrated in placental mitochondria from smokers compared with nonsmokers [44]. The enzymatic activity of complex III and mitochondrial DNA (mtDNA) content are inversely related to the daily consumption of cigarettes indicating that maternal smoking is associated with placental mitochondrial dysfunction, which might contribute to restricted fetal growth by limiting energy availability in cells. Changes in the pattern of apoptosis have also been studied in the placenta of smokers, and conflicting results have been presented [33,45,46]. Vogt compared placentas from mothers who smoked and had growth-restricted babies with those from nonsmokers with infants of appropriate weight. Although there was an increased incidence of apoptosis in the syncytiotrophoblast as assessed by terminal transferase dUTP nick end labelling (TUNEL) and M30 staining [46], it is not clear whether this was due to the smoking or some other underlying pathology that induced growth restriction. By contrast, in a small study investigating 12 placentas, Marana et al. found a highly significant reduction in apoptosis in smokers [33]. Their assessment was based only on the TUNEL staining method and the rate that they reported for the normal placenta appears very high compared to other studies employing more reliable markers. Gruslin et al. also reported a reduction in trophoblast apoptosis in smokers' placentas, but only at term [45]. They reported that concentrations of the X-linked inhibitor of apoptosis protein (Xiap) decrease significantly throughout development in nonsmokers but remain elevated in smokers. Pro-apoptotic type II transmembrane homotimer protein Fas and Fas ligand (FasL) levels do not vary significantly throughout development or between groups, but levels of procaspase-3 are significantly increased in smokers at term. Apoptosis is determined by the balance of various pro-and anti-apoptotic factors, and so it is possible that maternal smoking has different effects at different stages of trophoblast differentiation. In an attempt to investigate the mechanisms underlying apoptosis placental villous explants have been subjected to hypoxia/reoxygenation in vitro. Interestingly, treatment with carbon monoxide reduces apoptosis by 60% in the differentiated syncytiotrophoblast layer compared with Effects of maternal exposure to tobacco smoke on the fetoQplacental unit untreated explants undergoing a similar insult [47]. In addition, retention of intact syncytial membranes is observed in carbon monoxide-treated explants, which could partially explain why women who smoke cigarettes throughout pregnancy are less likely to develop pre-eclampsia than nonsmoking women [48]. Stem villous arteries of heavy smokers have altered mechanical properties and a greater vasoconstrictive response to endothelin 1 than do those from nonsmokers. These changes may compromise fetal placental blood flow and thereby contribute to the lower birth weights seen among infants born to heavy smokers [49]. 4. Effect of tobacco smoke on fetal anatomy The main effect of maternal smoking on fetal morphology is on the fetal growth. The association between cigarette smoke and FGR has been known for five decades [50]. Epidemiological data indicate that the risk of FGR is 2.07 times higher in mothers who smoked, and that smoking by the mother's partner also increased the risk of FGR [51]. Neonates born to women who reported smoking from the first trimester had a 0.6–1.9% reduction in most neonatal anthropometric measurements, resulting in an overall reduction of birth weight of 110–130 g (4%) compared to neonates born to mother who never smoked and were not exposed to passive smoking during pregnancy [52–56]. In mothers heavily exposed to passive smoking both at home and at work, the neonates' birthweight is lower by more than 180 g in comparison with the group of nonexposed [53]. Overall most studies have shown that the fetuses of women who stopped smoking during the first trimester have a similar risk of growth restriction to that of nonsmokers. The yield Tar content of cigarette is more closely related to the reduction in fetal growth than to the number of cigarettes smoked, and there is strong evidence of an interaction between smoking and alcohol consumption [50]. Cigarette smoke appears to have a selective effect within lean body mass compartments including fat mass [57] and peripheral fetal muscle [58]. Exposure of the fetus to passive and/or light active smoking involves a reduction of not only weight and fat mass and but also most anthropometric parameters [58]. Maternal exposure to tobacco smoke in early pregnancy, as measured by serum cotinine concentrations at 20–24 weeks of gestation, is know to adversely affects fetal head development as assessed by the ultrasound biparietal (BPD) measurements [59]. Data from a longitudinal ultrasound study of fetal growth in utero have shown that maternal smoking is associated with early growth acceleration in head and abdominal diameters before 27 weeks of gestation [60]. This is followed by altered head shape and a proximal/distal growth gradient as proportionately long arms and short legs with a significant reduction in the tibia/femur ratio which becomes apparent by 32 weeks. These fetal body growth patterns, expressed in terms of size and proportionality, are consistent with the presence of chronic hypoxia associated with maternal smoking. These growth pattern differences suggest that prenatal smoking is not merely an insult resulting in consistent size and growth rate restriction across all developmental ages. These growth patterns can be linked to the unique pattern of fetal blood flow favouring 703 upper body oxygen distribution and extraction [61], together with genetically based adaptive strategies that permit the fetus to adjust the timing and magnitude of its growth to local environmental resources. The effect of maternal smoking on fetal cells could be modified by genes involved in the biotransformation of toxic compounds derived from tobacco. Polymorphic variants of fetal acetyl-N-transferases 1 (NAT1) and 2 (NAT2) and glutathione S-transferase (GSTM) enzymes interact with tobacco smoke during early pregnancy. Fetuses with NAT1 1088 and 1095 polymorphisms have a higher risk of orofacial clefts if their mother smokes [12]. Similarly, fetuses that are homozygous null for GSTM1and whose mothers smoked N 20 cigarettes per day, present with a 7-fold increased risk of orofacial clefts [13]. The combined absence of GSTM1 and GSTT1 enzymes among the offspring of smoking mothers is associated with a nearly 6-fold increased risk for cleft lip. 5. Effect of tobacco smoke on fetal biology Similar to the effects of maternal smoking on placental biology, one or more of the many constituents of tobacco smoke can have a direct toxic effect on the fetal cells or an indirect effect through damage to, or functional disturbances of the placenta. Smoking may also affect fetal development by influencing maternal nutritional intake and metabolism [62]. For example, fetal birth weight is inversely correlated with maternal and cord blood cadmium concentrations suggesting that birth weight might be negatively influenced by cadmium levels as a result of the toxic effects of the metal on the placenta [36]. However, at term, cadmium and nicotine concentrations are of the same order of magnitude both in cord and maternal serum of smokers, indicating that like most tobacco components, cadmium is transferred easily from the mother to the fetus through the placenta. The placenta has the potential to inactivate carcinogens locally and to regulate the transfer of metabolized toxic agents into the fetal compartments [63]. Those mechanisms might protect the fetus to some extent, until the trophoblast detoxification function is overwhelmed by long-term exposure to the same toxins. The best example is probably that of cadmium, which accumulates mainly in villous tissue, and only when the placenta is saturated does cadmium leak through to the fetus. This suggest that fetal growth is first affected indirectly by the effect of tobacco toxins on trophoblast biological functions subsequently directly by the effect of these toxins on the developing fetal cells. Chronic maternal smoking is mainly associated with alterations in protein metabolism and enzyme activity in the fetus. Most of these metabolic alterations have been investigated in cord blood and related to fetal growth in utero or birth weight. For example, cord blood concentrations of thyroxine [64], high density lipoprotein cholesterol [65], osteocalcin and bone isoenzyme of alkaline phosphatase [66], ascorbic acid [67], IGF-I and IGFBP-3 [68], betacarotene [69] are lower in smokers whereas leptin [70], carbonyl group and lipid peroxides [71] concentrations are higher in the cord blood of smokers. The influence of other factors such as maternal diet and alcohol intake on the cord blood composition of fetuses exposed to tobacco smoke remains to be determined in most studies. 704 Nicotine and its main metabolite cotinine readily cross the placenta, and the fetuses of mothers who smoke are exposed to relatively higher cotinine concentrations than their mothers [17]. The feto-maternal cotinine ratio is lower in second trimester pregnancies than in term pregnancies. Throughout pregnancy, positive linear correlations are found between maternal and fetal serum or amniotic fluid cotinine concentrations, indicating that placental cotinine transfer increases with advancing gestation [17,72]. This may be secondary to increased placental permeability in the third trimester, which could be linked to progressive placental damage in heavy chronic smokers. Nicotine was not known to have a distinctive effect on fetal cells until recently. Nicotine treatment of microvesicles prepared from term placentas decreases activity of amino acid transport system A, a key sodium-dependent transporter of neutral amino acids such as alanine, glutamine, and glycine. In particular, it blocks the trophoblast acetylcholine-facilitated amino acid transport [73,74]. In vivo at 12– 17 weeks' gestation, the fetal plasma of smokers contains lower concentrations of serine, proline, α-aminobutyric acid, leucine, and arginine than the plasma of fetuses not exposed to tobacco smoke [75]. At term, lower concentrations of aspartic acid, hydroxyproline, threonine, alanine, αaminobutyric acid, methionine, tyrosine, phenylalanine, and lysine are found in the venous cord plasma of the smokers compared with nonsmokers [72]. The level of aminobutyric acid in fetal blood is decreased in similar proportions in smokers at 12–17 weeks and at term. In vitro, 15 cigarette smoke induces the formation of new trophoblastic carriers for the uptake of aminobutyric acid, suggesting that part of the fetal amino acid deficit induced by maternal smoking may be compensated for by the induction of new amino acid transport systems [73]. The above data suggest that this upregulation is a temporary phenomenon and indicate that chronic maternal smoking induces major alterations in the placental metabolism of some amino acids from as early as the first month of pregnancy. Nicotine induces also cytoplasmic vacuolation and cellular edema in the adult rat pancreas, and has been implicated in the etiology of pancreatitis and pancreatic carcinoma [76]. In humans, smoking enhances the secretion of amylase by the exocrine pancreas and higher fetal plasma amylase activity in mothers who smoked compared with nonsmokers, indicates that nicotine or its metabolites might affect the fetal pancreas as early as 12 weeks' gestation [75]. In the fetal brain, nicotine can activate nicotinic receptors, which play an important role during development of the brain [77]. Maternal smoking interferes strongly with brain biological parameters, giving rise not only to structural developmental abnormalities of the arcuate nucleus, but also to a decrease of noradrenergic activity in the LC, of EN2 gene expression in the ArcN and of SS in the HypoglN [78]. Nicotine targets specific neurotransmitter receptors in the fetal brain, eliciting abnormalities of cell proliferation and differentiation, leading to shortfalls in the number of cells, and eventually to altered synaptic activity [79]. Because of the close regulatory association of the cholinergic and catecholaminergic systems, adverse effects of nicotine involve multiple transmitter pathways and influence not only the immediate developmental events in fetal brain, but also the eventual programming of synaptic competence. A direct specific E. Jauniaux, G.J. Burton action on the developing 16 human brain is plausible during the major part of the prenatal life, since the nicotinic receptors are already present in the brain during the first trimester. Benzo[a]pyrene is a compound of cigarette smoke that is metabolically activated to the diol-epoxide derivative: benzo [a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide, a carcinogen (BPDE-I). This derivative is covalently fixed on DNA and gives BPDE-I-DNA adducts. Maternal tobacco consumption is linked to the accumulation of BPDE-I-DNA adducts in the placenta and in smaller quantities in the umbilical cord blood [80]. Both active maternal smoking and secondary maternal exposure produce a quantitatively and qualitatively increase in fetal HPRT mutations [81]. An increase in illegitimate V(D)J recombinase-mediated deletion of HPRT exons 2–3 has also been found in cord blood T-lymphocytes of newborns of mothers who smoked during pregnancy [82]. Smoking 10 or more cigarettes per day for at least 10 years and during pregnancy is associated with increased chromosomal instability in amniocytes. Band 11q23, known to be involved in leukemogenesis, seems especially sensitive to genotoxic compounds contained in tobacco [83]. These recent data suggest that maternal smoking contains geneotoxicants capable of inducing chromosomal instability, which is associated with an increase in the risk of cancer, especially childhood malignancies. 6. Key guidelines • Maternal smoking is associated with placental damage in all 3 trimester of pregnancy. • Tobacco toxins dysregulate trophoblastic and fetal cells biological functions mainly protein metabolism and enzyme activity. • The main impact of antenatal smoking exposure is on fetal growth with a reduction of weight, fat mass and most anthropometric parameters. 7. 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