Toxicology and Applied Pharmacology 275 (2014) 1–11

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Toxicology and Applied Pharmacology

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Fetal and neonatal exposure to nicotine leads to augmented hepatic andcirculating triglycerides in adult male offspring due to increasedexpression of fatty acid synthase☆

Noelle Ma a,b,d, Catherine J. Nicholson e, Michael Wong a,b,d, Alison C. Holloway e, Daniel B. Hardy a,b,c,d,⁎a Department of Physiology & Pharmacology, The University of Western Ontario, Canadab Department of Obstetrics & Gynecology, The University of Western Ontario, Canadac The Children's Health Research Institute, The University of Western Ontario, Canadad The Lawson Health Research Institute, The University of Western Ontario, Canadae Department of Obstetrics and Gynecology, McMaster University, Canada

☆ None of the authors has anything to declare regardin⁎ Corresponding author at: The Department of Phy

University of Western Ontario, London, Ontario N6A 5C1,E-mail address: Daniel.Hardy@schulich.uwo.ca (D.B. H

0041-008X/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.taap.2013.12.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 October 2013Revised 9 December 2013Accepted 12 December 2013Available online 22 December 2013

Keywords:Nicotine replacement therapyTriglyceridesFatty acid synthaseLiverLiver X receptorObesity

While nicotine replacement therapy is assumed to be a safer alternative to smoking during pregnancy, the long-term consequences for the offspring remain elusive. Animal studies now suggest thatmaternal nicotine exposureduring perinatal life leads to a wide range of adverse outcomes for the offspring including increased adiposity.The focus of this study was to investigate if nicotine exposure during pregnancy and lactation leads to alterationsin hepatic triglyceride synthesis. FemaleWistar ratswere randomly assigned to receive daily subcutaneous injec-tions of saline (vehicle) or nicotine bitartrate (1 mg/kg/day) for two weeks prior to mating until weaning. Atpostnatal day 180 (PND 180), nicotine exposed offspring exhibited significantly elevated levels of circulatingand hepatic triglycerides in themale offspring. Thiswas concomitant with increased expression of fatty acid syn-thase (FAS), the critical hepatic enzyme in de novo triglyceride synthesis. Given that FAS is regulated by the nu-clear receptor Liver X receptor (LXRα), we measured LXRα expression in both control and nicotine-exposedoffspring. Nicotine exposure during pregnancy and lactation led to an increase in hepatic LXRα protein expres-sion and enriched binding to the putative LXRE element on the FAS promoter in PND 180 male offspring. Thiswas also associatedwith significantly enhanced acetylation of histone H3 [K9,14] surrounding the FAS promoter,a hallmark of chromatin activation. Collectively, these findings suggest that nicotine exposure during pregnancyand lactation leads to an increase in circulating and hepatic triglycerides long-term via changes in the transcrip-tional and epigenetic regulation of the hepatic lipogenic pathway.

© 2013 Elsevier Inc. All rights reserved.

Introduction

It is well established that smoking during pregnancy is associatedwith numerous adverse obstetrical outcomes including an increasedrisk of spontaneous abortions (Ness et al., 1999), placental complica-tions (Ananth et al., 1999), impaired fetal growth (Andres and Day,2000; Meyer et al., 1976) and perinatal mortality Andres and Day,2000; Meyer et al., 1976). Although rates of smoking during pregnancyhave declined, approximately 9–20% of mothers world-wide continueto smoke during pregnancy (Al-Sahab et al., 2010; Dhalwani et al.,2013; Paterson et al., 2003; Rogers, 2009; Tong, et al., 2013). This trans-lates into approximately ~75,000 babies born each year in Canada alonewhowere exposed to first hand smoke in utero (Andres and Day, 2000;Paterson et al., 2003). Furthermore, almost half of the women who are

g potential conflicts of interest.siology & Pharmacology, TheCanada. Fax: +1 519 661 3827.ardy).

ghts reserved.

able to quit smoking during pregnancy relapse within four months ofdelivery (Tong et al., 2009). This is of great concern considering that arecent meta-analysis of thirty prospective studies found that babiesborn to womenwho smoked regularly during pregnancy have a 47% in-crease in the odds of becoming overweight (Weng et al., 2012). More-over, the association between smoking and a predisposition ofchildren being overweight was demonstrated to be largely unaffectedby the socioeconomic status of the mother, fetal growth and maternalweight (Oken et al., 2008). This suggests that it is the direct and long-term effect of intrauterine exposure to the chemicals in cigarettesmoke and not lifestyle factors associated with smoking that accountsfor the increased risk of obesity in the offspring of women who smokein pregnancy.

Nicotine replacement therapy (NRT) has been widely developed asan effective therapy for smoking cessation (Okuyemi et al., 2000;Oncken and Kranzler, 2003). NRT provides a substitute source of nico-tine that significantly reduces the symptoms of nicotine withdrawaland leads to pleasurable experiences such as mood modulation andstimulation (Benowitz, 2010; Benowitz and Jacob, 1990). Yet, often

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due to low adherence there is limited clinical data available on the effi-cacy and safety of NRT use in pregnancy (Coleman et al., 2012; Pollaket al., 2007; Wisborg, et al., 2000). However, animal studies now dem-onstrate that nicotine may be the single most important component ofcigarette smoke leading to long-term adverse metabolic outcomes(Bruin et al., 2007; Gao et al., 2005; Gao et al., 2008; Holloway et al.,2005; Newman, et al., 1999; Pausova et al., 2003; Williams andKanagasabai, 1984). Indeed, animals exposed during fetal and/or neona-tal life to nicotine have increased adiposity (Gao et al., 2008), abnormalglucose homeostasis (Bruin et al., 2007; Holloway et al., 2005; Sommet al., 2008) and elevated blood pressure (Fox et al., 2012; Gao et al.,2008). Although NRT drastically reduces the number of chemicals thatbothmother and fetus are exposed to, the long-term risk of nicotine ex-posure alone still remains elusive (Dempsey and Benowitz, 2001;Osadchy et al., 2009).

Numerous clinical studies have found that adults exposed tosmoking in utero have increased plasma triglycerides, a characteristicoften linked with obesity and an independent risk factor significantlyassociated with cardiovascular (CV) disease (Bansal et al., 2007;Bosello and Zamboni, 2000; Cupul-Uicab et al., 2012; Nordestgaardet al., 2007; Power et al., 2010; Riediger and Clara, 2011). Given thatnearly one-third of Canadian children and youth (5– to 17-year old)are either overweight or obese (Roberts et al., 2012), and the risk thatelevated triglycerides pose (Bansal et al., 2007; Nordestgaard et al.,2007), it is clear that strategies are warranted for the prevention or re-duction of hypertriglyceridemia in these children. We have previouslyreported that in rats, fetal and neonatal exposure to nicotine increasescirculating triglyceride levels in adult male offspring (Holloway et al.,2005). There are three main sources of free fatty acids that contributeto increased triglycerides; dietary, circulating, and de novo synthesis(Jensen-Urstad and Semenkovich, 2012). In brief, de novo lipogenesisin the liver begins with the carboxylation of acetyl-coA to malonyl-coA through the actions of acetyl-coA carboxylase (ACCα) (Kim,1997). Fatty acid synthase (FAS) then converts malonyl-coA to itsmajor product, palmitic acid (Jensen-Urstad and Semenkovich, 2012).Stearoyl-CoA-1 (SCD-1) catalyzes the conversion of this saturatedfatty acid into a monounsaturated fatty acid, which subsequently un-dergoes desaturation and elongation reactions (Miyazaki and Ntambi,2003). Finally, acyl CoA:diacylglycerol acyltransferase (DGAT) catalyzesthe final step of triglyceride synthesis (Cases et al., 1998; Smith et al.,2000). These synthetic enzymes play critical roles in the de novo synthe-sis pathway as animal models of FAS, SCD-1 and ACCα ablation all leadto the disruption of triglyceride homeostasis (Chakravarthy et al., 2005;Chu et al., 2006; Mao et al., 2006).

The liver X receptor (LXR) is a key nuclear receptor involved in thetranscriptional regulation of de novo triglyceride synthesis (Hortonet al., 2003; Schultz et al., 2000). In mice, the oral administration of anLXRα agonist, T0901317 led to elevated plasma triglyceride levels con-comitant with increased gene activity of key synthetic enzymes ACCα,FAS and SCD-1 (Schultz et al., 2000). Similarly, the sterol regulatoryelement-binding protein-1c (SREBP-1c) is able to regulate these fattyacid synthetic genes. However, studies involving LXRα/β−/−mice treat-ed with T0901317 did not lead to an increase in triglyceride levels(Horton et al., 2003). These studies suggest that LXRs play a criticalrole in de novo hepatic lipogenesis (Schultz et al., 2000).

Given its role in regulating fatty acid homeostasis (Schultz et al.,2000), cholesterol homeostasis (Repa et al., 2000) and gluconeogenesis(Mitro et al., 2007), LXRα has become an attractive candidate inelucidating the molecular mechanisms underlying metabolic derange-ments following early life insults. Several studies from our laboratoryhave implicated that alterations in LXR activity during adverse perinataldevelopment led to symptoms ofmetabolic syndrome including hyper-cholesterolemia and impaired glucose tolerance/homeostasis in adult-hood (Osumek et al., 2013; Sohi et al., 2011; Vo et al., 2013). Howeverto date, the effects of in utero nicotine exposure on LXRα activity andLXR-mediated hepatic lipogenesis in postnatal life are unknown. This

study was designed to test the hypothesis that hypertriglyceridemia inoffspring exposed to nicotine during fetal and neonatal life (Hollowayet al., 2005) involves transcriptional and epigenetic regulation of LXR-target genes involved in hepatic de novo triglyceride synthesis.

Materials and methods

Animals and dietary regime. All animal experiments were approvedby the Animal Research Ethics Board at McMaster University, in accor-dancewith the guidelines of the Canadian Council for Animal Care. Nul-liparous female Wistar rats (200–250 g, Harlan, Indianapolis, IN, USA)were randomly assigned to receive daily subcutaneous injections of sa-line (vehicle) or nicotine bitartrate (1 mg/kg per day, Sigma-Aldrich,St. Louis, MO, USA) for 2 weeks prior to mating, during pregnancyuntil weaning (postnatal day 21) as previously described (Bruin et al.,2008a; Holloway et al., 2006). This dose of nicotine has been previouslyshown to lead to cotinine levels in maternal serum that are similar to“moderate” female smokers (80–163 ng/ml) and in nicotine-exposedoffspring serum at birth, that are comparable to infants nursed bysmoking mothers (Eskenazi and Bergmann, 1995; Holloway et al.,2006). Dams were allowed to deliver normally and at postnatal day 1(PND 1) all litters were culled to eight. After weaning offspring werecaged as sibling pairs and at PND 21 a subset of male offspring werefasted overnight and sacrificed by CO2 inhalation for liver tissue collec-tion. A second subset of animals was allowed to develop naturally. AtPND 180 male rat offspring were fasted overnight and sacrificed byCO2 inhalation for body weight measurements and blood and liver tis-sue collection. All animals were weighed at necropsy. Liver sampleswere snapped frozen in liquid nitrogen and stored at −80 °C until fur-ther molecular analysis. Blood was collected, allowed to clot, spun andserum was stored at−80 °C for analysis.

Plasma and hepatic lipid measurements. Total cholesterol, triglycerideand glucosemeasurements from blood and hepatic tissue samples wereautomatically detected using Cobas® Mira S analyzer at the MetabolicPhenotype Laboratory at Robarts Research Institute (London, Ontario,Canada). For triglyceride measurements, triglycerides were hydrolyzedby lipoprotein lipase to glycerol and fatty acids. Glycerol was then phos-phorylated to glycerol-3-phosphate by ATP in a reaction catalyzedby glycerol kinase (GK). The oxidation of glycerol-3-phosphate wascatalyzed by glycerol phosphate oxidase (GPO) to form dihydroxyace-tone phosphate and hydrogen peroxide (H2O2). In the presence ofperoxidase, H2O2 alters the oxidative coupling of 4-chlorophenol and4-aminophenazone to form a red-colored quinoneimine dye, whichwasmeasured at 512 nm. The increase in absorbance is directly propor-tional to the concentration of triglycerides in the sample. For cholesterolmeasurements, cholesterol esterase cleaved cholesterol esters, whichthen were converted to choleste-4-en-3-one and H2O2 by cholesteroloxidase. Cholesterol levels were quantified using a colorimetric assaythat measured the breakdown of H2O2 via the Trinder reaction as previ-ously described (Sohi et al., 2011). Glucose measurements weredetermined using a glucose assay kit from Roche Diagnostics (Roche,Misissauga, Ontario, Canada) that was run on the analyzer.

Quantitative real-time PCR analysis. Total RNA from male liver tissueat all ages (PND 1, PND 21, and PND 180) was extracted by the one-step method described by Chomczynski and Sacchi (Chomczynski andSacchi, 1987). RNA was treated with deoxyribonuclease to remove anycontaminating DNA. 4 μg of the total RNA was reverse transcribed tocDNA using random primers and Superscript II RNase H-reverse tran-scriptase (Invitrogen, Carlsbad, CA, USA). Primer sets directed for thegenes of interest (FAS, ACCα, SCD-1, and DGAT) were generated usingOligoPerfect™Designer (Invitrogen, Carlsbad, CA, USA) (Table 1).The Bio-Rad CFX384 Real Time System was employed to determinequantitative mRNA expression using the DNA binding dye SsoFast™EvaGreen® Supermix (Bio-Rad, Mississauga, Ontario, Canada). The

Table 1Real-time PCR primers.

Gene Primer (5′–3′) Reference no.

FAS FWD GGA CAT GGT CAC AGA CGA TGA C X62889.1REV CGT CGA ACT TGG ACA GAT CCT T

ACCα FWD TCC GTA TGT GAC CAA AGA CC NM_022193.1REV TAC GTT GTT CCC AAG GAC TG

SCD-1 FWD CTG TGT GGA GCC ACA GGA CTT AC NM_031841.1REV ATC CCG GGC CCA TTC ATA TAC

DGAT FWD TGC TCT TTT TCA CCC AGC TT AB062762.1REV CAC AGG CTT TCC TTC TTT GC

β-Actin FWD CAG CCT TCC TTC CTG GGT AT NM_0.31144.3REV AGG AGC CAG GGC AGT AAT TCT

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cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min,followed by 45 cycles of 95 °C for 15 s, and 60 °C for 1 min. The cyclethreshold was set at a level where the exponential increase in PCR am-plificationwas roughly corresponding between all samples. The relativefold changes were calculated using the comparative cycle times (Ct)methodwithβ-actin as the reference gene. All primer setswere demon-strated to have good linear correlation (slope ≈ −3.4) strongly sug-gesting equal priming efficiency (data not shown). ΔCt values for eachprimer set were calibrated to the experimental sampleswith the lowesttranscript abundance (highest Ct value). The relative abundance of eachprimer set comparedwith calibratorwasdetermined by the formula, 2ΔΔCt, in which ΔΔCt is the calibrated Ct value.

Protein extraction and Western immunoblotting analysis. Wistar ratliver tissue proteins at PND 1 and PND 180 were prepared using modi-fications of previously published methods (Vo et al., 2013). Tissue pro-tein was extracted from snap frozen liver samples using RIPA lysisbuffer solution (50 mM Tris–HCl, pH 7.4, NP-40 1%, Na-deoxycholate0.25%, 150 mM NaCl, 1 mM EDTA, 50 mM NAF, 1 mM NaV, 25 mM β-glycerophosphate), along with a protease inhibitor (Roche). The liversample was placed in 600 μl of RIPA lysis buffer and homogenizedwith the IKA T10 Basic S1 Dispersing Tool (IKAWorks Inc., Wilmington,NC). The homogenates were placed on ice for 5 min before rotation at4 °C for 10 min. The homogenates were centrifuged at 300 g for15 min at 4 °C. The supernatantwas transferred to fresh tubes and cen-trifuged at 20,000 g for 20 min at 4 °C. The supernatant was retained asthe protein preparation. Equal concentrations of total protein were nor-malized using a colorimetric BCA Protein Assay (Pierce Corp., Madison,WI, USA). Protein was fractionated in gradient polyacrylamide gels(Invitrogen, Carlsbad, CA, USA) and transferred onto polyvinylidenedifluoride membrane (Millipore, Etobicoke, Ontario, Canada). Blotswere probed with LXRα antibody (cat# sc-13068x, Santa Cruz Biotech-nology;1:500), FAS antibody (cat #sc-20140, Santa Cruz Biotechnology;1:1000) and ACCα (cat# sc-30212, Santa Cruz Biotechnology; 1:1000),and monoclonal horseradish peroxidase-conjugated β-actin (cat#A3854, Sigma-Aldrich, 1:50000) diluted in 5–7% milk-1×Tris-buffered saline-Tween 20 (0.01%) buffer and with horseradish peroxi-dase conjugated donkey anti-rabbit IgG (cat #711-035-152, JacksonImmunoResearch Laboratories, 1:10000) diluted in 5–7% milk-1×Tris-buffered saline-Tween 20 (0.01%) as the secondary antibody. Immuno-reactive bands were visualized using an enhanced chemiluminescencedetection system (Thermo Scientific, Waltham, MA).

Chromatin immunoprecipitation (ChIP). Chromatin was extractedfrom liver tissues excised at PND 180 frommale offspring as previouslydescribed (Sohi et al., 2011). In brief, a small piece of snap frozen liverwas homogenized and incubated in 0.5 mL of 1% formaldehyde for10 min at room temperature to cross-link proteins and DNA. Glycine(0.125 M, final concentration) was added to all samples to terminatecross-linking. Samples were microfuged at 950 g at room temperaturefor 5 min and supernatant was subsequently removed. The liver tissuewas then washed once with cold PBS before being placed in 500 μl of

SDS lysis buffer (Millipore, Etobicoke, Ontario, Canada) with proteaseinhibitor cocktail (Roche, Mississauga, Ontario, Canada). Each samplewas sonicated to produce sheared, soluble chromatin. The lysateswere diluted ten times with the addition of ChIP dilution buffer(Millipore, Etobicoke, Ontario, Canada) and aliquoted to 300 μlamounts. Each of the aliquotswas preclearedwith protein A/G Plus aga-rose beads (40 μl, Millipore, Etobicoke, Ontario, Canada) and rotated for2 h at 4 °C. In order to pellet the beads, samples were microfuged at20,000 g, and the supernatant containing the sheared chromatin wasplaced in new tubes. The aliquots were incubated with 3 μg of antibod-ies against LXRα (cat# sc-13068x, Santa Cruz Biotechnology, SantaCruz, California) and acetylated histone H3 (lysine 9,14, cat #05-399,Millipore, Etobicoke, Ontario, Canada) and rotated overnight at 4 °C.Two aliquots were reserved as ‘controls’ — one incubated without anti-body (‘input’) and another with non-immune IgG (Millipore, Etobicoke,Ontario, Canada). Protein A/G Plus agarose beads (60 μl) were addedto each tube, the mixtures rocked for 1 h at 4 °C and the immunecomplexes collected by centrifugation. The beads containing theimmunoprecipitated complexes were washed sequentially for 5 mininwash buffer I (20 mMTris–HCl, pH 8.1, 2 mMEDTA, 0.1% SDS, 1% Tri-ton X-100, 150 mM NaCl), wash buffer II (same as I, except containing500 mM NaCl), wash buffer III (10 mM Tris–HCl, pH 8.1, 1 mM EDTA,1% NP-40, 1% deoxycholate, 0.25 M LiCl), and in 2 × TE buffer. Thebeads were eluted with 250 μl elution buffer (1% SDS, 0.1 mMNaHCO3 + 20 μg salmon spermDNA (Sigma-Aldrich, Oakville, Ontario,Canada) at room temperature. The elution step was repeated onceand eluates were combined. Crosslinking of the immunoprecipitatedchromatin complexes and ‘input controls’ (10% of the total solublechromatin) was reversed by heating the samples at 65 °C for 4 h. Pro-teinase K (15 μg (Invitrogen, Carlsbad, CA, USA)) was added to eachsample in buffer (50 mM Tris–HCl, pH 8.5, 1% SDS, 10 mM EDTA) andincubated for 1 h at 45 °C. The DNA was purified by phenol-chloroform extraction and precipitated in EtOH overnight at −20 °C.The supernatant was removed and pellets were dried. Both samplesand ‘input’ controls’ were diluted in 10–100 μl TE buffer prior to PCRanalysis. Real-time PCR was employed using forward (5′-GCCACGATGACCGGTAGTAA-3′) and reverse (5′-GCGTTGCTAGGCAATAGGGT-3′)primers (PE Applied Biosystems, Boston, MA, USA) that amplify a−690 bp to −561 bp region encompassing the published FAS LXREsite (Joseph et al., 2002). Using serial dilutions of rat liver chromosomalDNA, the primers were demonstrated to have equal efficiency in prim-ing to their target sequences. ChIP signal was calculated by comparingthe relative abundance of the immunoprecipitated chromatin com-pared with input chromatin using the 2ΔΔCt formula.

Statistics. All statistical analyses were performed using (Graph PadPrism Software, SanDiego, California, USA). The results from triglyceride,cholesterol and glucose measurements are expressed as mean ± SEM.The results from quantitative RT-PCR, ChIP and immunoblot analysisare expressed as the mean of arbitrary values ± SEM. All results wereevaluated using an unpaired Student's t test, where a p-value of lessthan 0.05 was considered significant.

Results

Weight responses in PND 180 male offspring exposed to nicotine duringpregnancy and lactation

At PND 1, nicotine-exposed offspring weighed significantly less(6.21 ± 0.06 g) compared to control (6.64. ± 0.13 g) (p b 0.05)(Table 2). However by PND 180, an increase in body weight from563 ± 5 g to 605 ± 4 g (p b 0.05) was observed in nicotine-exposedmale offspring (Table 2). This increase in weight is noteworthy consid-ering that previous studies in this nicotine animal model have observedthat the growth trajectory of nicotine-exposed offspring was signifi-cantly enhanced compared to control, despite nicotine administration

Table 2Outcome measures.

Outcome measure Control Nicotine p-Value

PND 1 body weight (g) 6.64 ± 0.13 6.21 ± 0.06 0.016Litter size 12.6 ± 1.1 14.6 ± 0.9 0.197PND 21 body weight—males 57.2 ± 1.5 56.6 ± 2.8 0.851PND 21 liver weight—males 2.35 ± 0.09 2.35 ± 0.15 0.971PND 21 liver:body weight ratio—males 4.1 ± 0.1 4.1 ± 0.1 0.727PND 21 body weight—females 53.0 ± 1.6 54.7 ± 2.8 0.590PND 21 liver weight—females 2.26 ± 0.10 2.32 ± 0.13 0.725PND 21 liver:body weight ratio—females 4.2 ± 0.1 4.2 ± 0.1 0.97126 week body weight—males 563.6 ± 13.1 605.6 ± 9.0 0.01326 week liver weight—males 19.6 ± 0.8 21.0 ± 0.8 0.10926 week liver:body weight ratio—males 4.1 ± 0.1 4.1 ± 0.1 0.72726 week body weight—females 320.2 ± 15.9 320.9 ± 10.4 0.97026 week liver weight—females 11.2 ± 0.5 11.3 ± 0.5 0.90526 week liver:body weight ratio—females 3.5 ± 0.2 3.5 ± 0.1 0.969

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having no effect on gestational length, maternal food intake ormaternalweight gain (Bruin et al., 2008b; Holloway et al., 2005). Similar to previ-ous reports (Bruin et al., 2007; Holloway et al., 2005), exposure to nico-tine during gestation did not alter litter size (Table 2) while survivalrates in both groups was 100% by postnatal day 4 (data not shown).For both genders, body weight and liver weight were equal in both ex-perimental groups by postnatal day 21 (Table 2).

Maternal nicotine exposure during pregnancy and lactation leads toincreased circulating and hepatic triglyceride levels exclusively in PND 180male offspring

Interestingly, exposure to maternal nicotine during gestation andlactation resulted in increased fasting serum triglycerides in male butnot female offspring exclusively at PND 180 (Figs. 1A–B). However, he-patic triglyceride levels were significantly elevated in both PND 180male and female nicotine-exposed offspring (Figs. 1C–D). Due to the

Fig. 1. The effect of nicotine exposure during pregnancy and lactation on circulating triglyceridepatic triglyceride concentrations (mg of lipid/g of tissue) measurements in (C) male and (D) fetermined using Student's unpaired t-test. *Statistically significant = (p b 0.05). n = 6–9/grou

lack of significant change in circulating triglyceride levels, PND 180 fe-male offspring were not further investigated in this study. Circulatinglevels of glucose and cholesterol were not significantly altered betweencontrol and nicotine-exposed fasted offspring of either sex at PND 180(data not shown).

Augmented triglyceride levels coincide with increases in the steady-statemRNA levels of hepatic fatty acid synthesis enzymes in the liver of PND180 male nicotine-exposed offspring

In order to elucidate the molecular mechanisms underlying the ele-vated hepatic and circulating triglyceride levels in nicotine-exposedmale adult rat offspring, we next examined the enzymes involved inthe fatty acid synthesis pathway leading to de novo triglyceride produc-tion in the liver. Quantitative real-time PCR revealed significant in-creases (p b 0.05) in hepatic fatty acid synthase (FAS) and acetyl CoAcarboxylase α (ACCα) steady-state mRNA in PND 180 male maternalnicotine-exposed (MNE) offspring, both enzymes involved in the initialsteps of fatty acid synthesis (Figs. 2A–B). No significant changes in thehepatic steady-state mRNA levels of stearoyl CoA 1 (SCD-1) and diglyc-eride acyltransferase (DGAT) were observed (Figs. 2C–D).

Increase in FAS mRNA expression is associated with a correspondingincrease in FAS protein levels exclusively in nicotine-exposedmale offspringat PND 180

Given the significant increases in the steady-state mRNA levels ofFAS and ACCα (Figs. 2A–B), we next performed western immunoblotanalysis to determine the protein levels of these lipogenic enzymes. Ex-posure to nicotine during pregnancy and lactation led to a significant in-crease in FAS protein levels in PND 180 offspring (Fig. 3A). Despitechanges in mRNA levels, there were no corresponding changes in pro-tein levels of ACCα (Fig. 3B) in PND 180 nicotine exposed offspring.

levels (mmol/L) in PND 180 (A) male and (B) female rat offspring and corresponding he-male offspring. Results are expressed as the mean ± SEM. The effects of nicotine were de-p per experimental group.

Fig. 2. The effect of nicotine exposure during pregnancy and lactation on hepatic steady-statemRNA levels of (A) FAS, (B) ACCα, (C) SCD-1 and (D) DGAT inmale rat offspring at PND 180.RNAwas extracted andmRNA levelswere assessedusing qRT-PCRusing primers specific for FAS, ACCα, SCD-1, andDGAT. Data is presented as arbitrary values and results are expressed asthe mean ± SEM. The effects of nicotine were determined using Student's unpaired t-test. *Statistically significant = (p b 0.05). n = 8–10 per experimental group.

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An increase in FAS expression is concomitant with an increase in hepaticLXRα protein levels and binding to the LXRE in the promoter of FAS in PND180 male rat offspring exposed to nicotine during pregnancy and lactation

To begin to decipher themechanisms for the increase in FAS expres-sion in these nicotine-exposed offspring, we next investigated whetherhepatic levels of LXRα were altered in PND 180 offspring exposed tonicotine, a key regulator of FAS and other various enzymes involved inthe fatty acid synthesis pathway (Joseph et al., 2002; Schultz et al.,2000). At PND 1 and PND 21, LXRα protein levels were not different be-tween control and nicotine-exposed offspring (Figs. 4A and B). In con-trast, nicotine in pregnancy and lactation led to a significant increase(p b 0.05) in hepatic LXRα protein levels in male offspring by PND180

Fig. 3. The effect of nicotine exposure during pregnancy and lactation on (A) FAS and (B) ACof FAS and ACCα were measured using Western blot analysis. The protein levels were quantiβ-actin. Data is presented as arbitrary values and results are expressed as the mean ± SEMsignificant = (p b 0.05). n = 5–7 per experimental group.

(Fig. 4C), suggesting that LXRα may be facilitating the long-term in-crease in FAS protein and mRNA expression observed in nicotine ex-posed offspring. To explore this further, we employed chromatinimmunoprecipitation (ChIP) to examine the in vivo binding of LXRα toits putative LXR binding element (LXRE) on the proximal rat promoter(−669 to −665) of FAS (Joseph et al., 2002). Primers were designedto surround the LXRE of the FAS gene andwere demonstrated to equallyamplify their target sequences over a range of chromatin concentrations(data not shown). In this small sample size (n = 5), while there was atrend for PND 180 male nicotine-exposed offspring to have increasedLXRα binding at the putative LXRE of the FAS promoter, thiswas not sig-nificant (Fig. 4D).

Cα protein levels PND 180 male rat offspring. Protein was extracted and the expressionfied using densitometry and normalized to the protein levels of a housekeeping protein,. The effects of nicotine were determined using Student's unpaired t-test. *Statistically

Fig. 4. The effect of nicotine exposure during pregnancy and lactation on hepatic LXRα protein levels at (A) PND 1, (B) PND 21, (C) PND 180, alongwith (D) binding to the LXRE of the FASpromoter in PND 180 male offspring. A. Protein was extracted and the expression of LXRαwas analyzed usingWestern blot analysis. Data is presented as arbitrary values and results areexpressed as themean ± SEM. The effects of nicotine were determined using a Student's unpaired t-test. *Statistically significant = (p b 0.05). n = 7 per group. B. The in vivo binding ofLXRα to the hepatic LXRE of the FAS promoter in rat offspring at PND 180 was assessed by chromatin immunoprecipitation. Briefly, cross-linked chromatin immunoprecipicated using anantibody specific for LXRαwas isolated and the relative abundance of a region surrounding the LXRE (−669 to−655) of the FAS promoterwas quantifiedusing qRT-PCR. The relative levelof immunoprecipitated DNAwas normalized to total genomic DNA for each sample. Data is presented as arbitrary values and results are expressed as themean ± SEM. The effect of nic-otine was determined using Student's unpaired t-test. *Statistically significant = (p b 0.05). n = 5 per experimental group.

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Nicotine-induced transcriptional activation of hepatic FAS expression isassociated with an increase in the acetylation of histone H3 [K9,14]surrounding the LXRE of the FAS promoter region in PND 180male offspringexposed to nicotine during pregnancy and lactation

Since LXRα has been demonstrated to enhance the acetylation ofhistone H3 [K9,14] leading to increased hepatic FAS transcription(Yu et al., 2012), we next employed ChIP to investigate if chromatinremodeling could be an additional factor influencing the observedincrease in FAS steady-state mRNA and protein levels in PND 180male nicotine-exposed offspring. The acetylation of histone H3[K9,14] is well established to be associated with chromatin activa-tion (Lee et al., 1993). ChIP revealed that in the livers of PND 180male MNE offspring, there was significant enrichment in the acety-lation of histone H3 [K9,14] surrounding the putative LXRE (−669to −655) of the FAS promoter compared to control offspring(Fig. 5). This is concomitant with an increase in hepatic FAS mRNAand protein in these MNE offspring at PND 180 (Figs. 2A and 3A,respectively).

The steady-statemRNA levels of LXR-regulated fatty acid synthesis enzymeswere unaltered by nicotine-exposure during pregnancy and lactation in thelivers of PND 1 and PND 21 male rat offspring

In order to determine if nicotine exposure was directly affecting thelong-term changes observed in the hepatic fatty acid synthesis pathwayof nicotine-exposed male rat offspring, we examined whether parallelalterations were present from early life. At both postnatal day 1 (PND1) and 21 (PND 21, the longest window of direct nicotine exposure tooffspring), there were no changes in hepatic FAS or ACCα steady-statemRNA levels compared to control (Figs. 6A–D).

Discussion

In the liver, the fatty acid biosynthesis pathway facilitates thestorage of excess energy as cytosolic lipid droplets or circulatingtriglyceride-rich lipoproteins (Jensen-Urstad and Semenkovich, 2012).These triglycerides can later be oxidized to provide energy duringtimes of deficiency. However accumulation of excess intracellular

Fig. 5. The effect of nicotine exposure during pregnancy and lactation on hepatic acetylationof Histone H3 [K9,14] surrounding the LXRE of the FAS promoter in PND 180male offspringby chromatin immunoprecipitation. Briefly, cross-linked chromatin immunoprecipitatedusing an antibody specific for acetylated Histone H3 [K9,14] was isolated and the relativeabundance of a region surrounding the LXRE (−669 to −655) of the FAS promoter wasquantified using qRT-PCR. The relative level of immunoprecipitated DNA was normalizedto total genomic DNA for each sample. Data is presented as arbitrary values and resultsare expressed as the mean ± SEM. The effect of nicotine was determined using a Student'sunpaired t-test. *Statistically significant = (p b 0.05). n = 5 per experimental group.

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triglycerides, as it occurs during obesity (Bosello and Zamboni, 2000;Riediger and Clara, 2011), is characteristic of atherosclerosis and hepaticsteatosis aswell (Bansal et al., 2007; Donnelly et al., 2005; Nordestgaardet al., 2007). In this study we present evidence that maternal nicotineexposure during pregnancy and lactation led to augmented hepaticand circulating triglyceride levels in PND 180 male offspring. As men-tioned previously, there are three mains sources of free fatty acids thatcontribute to increased triglycerides; dietary, circulating, and de novosynthesis (Jensen-Urstad and Semenkovich, 2012). Our data demon-strates that the high triglycerides observed in offspring exposed to nic-otine during pregnancy and lactation are a result of increased hepaticFAS expression. FAS expression was enhanced in part due to increasedLXRα protein levels and binding to the LXRE of the FAS promoter inPND180 nicotine-exposedmale offspring. The increase in LXRα bindingat the FAS promoter was concomitant with significantly enhanced his-tone H3 acetylation [K9,14] surrounding the LXRE site, previously dem-onstrated to lead to the transcriptional activation of FAS (Yu et al.,2012). Given the well-established link between an adverse in utero en-vironment and the development of metabolic related disorders long-term (Fernandez-Twinn and Ozanne, 2006; Hales and Barker, 2001)and that smoking exposure leads to metabolic deficits in children(Behl et al., 2013; Ino, 2010; Oken et al., 2008; Weng et al., 2012), ourstudy provides further insight into the long-term consequences of nico-tine exposure in early life.

In our model, nicotine exposure during pregnancy and lactation ledto elevated plasma and hepatic triglycerides in adult male rat offspringsupporting two human studies that demonstrated that exposure to

tobacco in utero led to elevated triglyceride levels in adulthood(Cupul-Uicab et al., 2012; Power et al., 2010). Remarkably, there wereno differences in the levels of circulating triglycerides in nicotine ex-posed female offspring observed in this study, although the hepaticlevels of triglycerides were significantly increased. The disparity be-tween circulating and hepatic triglycerides in the female offspringmay be due to estradiol/estrogen receptor (ERα)-induced triglyceridelipolysis in other tissues (Benz et al., 2012; Wend et al., 2013). FemaleC57BL/6J mice on a high fat diet exhibit greater adipose tissue metabo-lism associated with increased expression of adipose triglyceride lipase(ATGL) compared to male counterparts (Benz et al., 2012). In addition,ERα-deficient femalemice exhibit less ATGL expression and adipose tis-sue metabolism in vivo and and in vitro (Benz et al., 2012; Wend et al.,2013). This may explain the decreased weight gain in our nicotine ex-posed females versus males at PND 180. Studies in pancreatic β-cellshave also demonstrated that the activation of the estrogen receptor byestradiol is involved in the down regulation of lipogenesis through thesuppression of regulatory targets including both LXRα and SREBP-1c(Tiano et al., 2011). Thus, estradiol may confer protective effects againstoverall lipogenic defects in nicotine-exposed female offspring althoughthis remains to be determined.

A significant increase in body weight was observed in nicotine ex-posed adult male offspring compared to control. These results comple-ment numerous epidemiological studies which have reported thatchildren born towomenwho smoked during pregnancy have an elevat-ed risk of obesity regardless of parental socioeconomic background, in-fant feeding patterns and gestational weight gain (Behl et al., 2013; Ino,2010; Oken et al., 2008; Weng et al., 2012). Our model has previouslyfound that nicotine exposure in perinatal life leads to elevated bodyand fat pad weight by 26 weeks, along with an increase in perivascularadipose tissue (Gao et al., 2005). Our current study suggests that the in-crease in body and fat padweight previously observed can be attributedin part to the higher levels of circulating and stored triglycerides. Clini-cally, an accumulation of excess intracellular triglycerides oftenprecedes the development of obesity (Bosello and Zamboni, 2000;Riediger and Clara, 2011).

The upregulation of de novo triglyceride synthesis can occur as a re-sult of elevated triglyceride synthesis and/or increased secretion by theliver (Yuan et al., 2007). Studies in both humans and rodents have dem-onstrated that alterations to the synthetic pathway can lead to impairedtriglyceride homeostasis (Attie et al., 2002; Jensen-Urstad andSemenkovich, 2012; Joseph et al., 2002; Schultz et al., 2000). In the pres-ent study, we observed that the steady-state mRNA levels of FAS andACCα, two enzymes involved in the triglyceride synthesis pathway(Abu-Elheiga et al., 2003; Abu-Elheiga et al., 2005; Chirala et al., 2003;Joseph et al., 2002; Joseph et al., 2002) were elevated in the liver ofmale nicotine-exposed offspring at PND 180. However, only FAS had acorresponding increase in protein expression, suggesting that alter-ations in FAS contribute to the increase in de novo triglyceride synthesisobserved in nicotine-exposed offspring. Interestingly, clinical studieshave demonstrated that obese patients with higher plasma triglyceridelevels also exhibited elevated hepatic lipogenesis and a greater contri-bution of endogenous triglycerides towards the circulating pool of tri-glycerides observed (Diraison et al., 2002). Similarly, rodent modelswith elevated plasma triglyceride levels are associated with increasedhepatic FAS expression (Barry and Bray, 1969; Morgan et al., 2008).Taken together, these studies suggest that perinatal nicotine exposuremay lead to long-term hypertriglyceridemia, in large part due to theFAS-dependent pathway of de novo triglyceride synthesis. In order to in-vestigate whether direct nicotine exposure led to the long-term alter-ations in the liver, PND 1 and 21 male rat livers were also analyzed. Itis noteworthy that the PND21 time pointwas examined as it representsthe longest window of direct nicotine exposure in the offspring. Sinceno significant changes were observed in hepatic LXRα protein levelsor the FAS and ACCα steady-state mRNA at either time point, this sug-gests that maternal nicotine exposure likely programs long-term

Fig. 6. The effect of nicotine exposure during pregnancy and lactation on hepatic steady-statemRNA levels of FAS and ACCα inmale rats at PND 1 and PND 21. Hepatic expression at post-natal day 1 of A. FASmRNA, B. ACCαmRNA,while hepatic expression at postnatal day 21 of C. FASmRNA,D. ACCαmRNAwasmeasured in vehicle and nicotine-exposed offspring (n = 5/group). RNA was extracted andmRNA levels were assessed using qRT-PCR using primers specific for FAS and ACCα. Data is presented as arbitrary values and results are expressed as themean ± SEM. The effects of nicotine were determined using Student's unpaired t-test. *Statistically significant = (p b 0.05). n = 4–5 per experimental group.

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hypertriglyceridemia through indirect mechanisms, such as catch-upgrowth, following nicotine exposure. We have recently demonstratedthat catch-up growth in low birth weight maternal protein restricted(MPR) pups is associated with increased hepatic phosphorylatedeIF2α, a hallmark of endoplasmic reticulum (ER) stress (Sohi et al.,2013). Moreover, ER stress has been associated with both augmentedLXRα and de novo lipogenesis (Basseri and Austin, 2008; Jwa et al.,2012; Lee et al., 2012). Although further support for this hypothesis isrequired, animal studies have demonstrated a link between direct nico-tine exposure and impaired fetal and neonatal growth (Huang et al.,2006; Wang et al., 2009).

The expression of FAS is under the direct regulatory control of vari-ous transcription factors including SREBP-1 and LXRα (Joseph et al.,2002; Kim et al., 1998; Magana and Osborne, 1996). Joseph et al. dem-onstrated that LXRα was able to induce FAS expression through bothSREBP-dependent pathway and SREBP-independent pathway, as evi-denced by distinct binding sites for each transcription factor on theFAS promoter (Joseph et al., 2002;Magana and Osborne, 1996). Howev-er treatmentwith an LXRα agonist inmice led to the transient elevationof LXRα expression concomitantwith a 15-fold induction of hepatic FASexpression and approximately 200% increase in plasma triglycerides(Joseph et al., 2002). In order to determine the regulatory relationshipbetween LXRα and SREBP-1 on FAS transcription, investigators treatedLXRα/β−/− mice with an LXRα agonist and observed no increase in tri-glyceride levels (Schultz et al., 2000). Since researchers observed a lackof regulation of SREBP-1 and FAS, it was proposed that LXRα directly ac-tivates SREBP-1 transcription, in addition to directly activating lipogenictarget genes including FAS (Schultz et al., 2000). The ablation of SREBP-1in genetically obese mice ameliorated fatty livers, however triglyceridelevels remained augmented compared to wild type mice (Yahagi et al.,2002). Thus, another regulatory pathway must be maintaining thehigh levels of triglycerides in these genetically obese mice. Therewas no significant difference in plasma triglyceride levels betweenobese and SREBP-1−/− obese mice, further suggesting that LXR may

contribute a larger role in augmenting hepatic de novo triglyceride syn-thesis in disease states such as obesity (Yahagi et al., 2002). In ourmodel, SREBP-1c protein expression was unchanged in PND 180 maleoffspring (data not shown) suggesting a prevailing regulatory role ofLXRα in the upregulation of synthetic genes in the liver in response toperinatal nicotine exposure during fetal and neonatal life. This furtherreinforces the important role of LXRα in perinatal life on long-termliver function given our previous studies which demonstrated thatLXR activity was altered in postnatal life due to a maternal low proteindiet ormaternal hypoxia, leading to hypercholesterolemia and impairedglucose tolerance/homeostasis, respectively, in adulthood (Osumeket al., 2013; Sohi et al., 2011; Vo et al., 2013).

To date, very little is known about the posttranslational histonemodifications that alter the activation of LXRα target genes. Our labora-tory has previously demonstrated that MPR through pregnancy andweaning leads to long-term hypercholesterolemia as a result of im-paired Cyp7a1 expression, an LXR target gene (Sohi et al., 2011). Thisdecreased Cyp7pa1 expression was influenced by diminished histoneH3 acetylation at the LXRE of the promoter of the Cyp7a1 gene, promot-ing a repressive chromatin environment in adulthood (Sohi et al., 2011).In addition, our laboratory has also shown that MPR during gestationonly led to decreased expression of hepatic LXRα long-term as a resultof decreased histone acetylation of histone H3 [K9,14] surrounding theproximal promoter of LXRα (Vo et al., 2013). In the present study, wehave demonstrated a significant increase in both hepatic LXRα proteinlevels and histone H3 [K9,14] acetylation surrounding the LXR respon-sive element of the hepatic FAS gene occurs in PND 180 male nicotine-exposed offspring. While not significant, there was also an increasingtrend of LXRα binding to the promoter of FAS in these same male nico-tine exposed offspring. Elegant in vitro studies by Yu et al. have demon-strated that human hepatic carcinoma cells (HepG2) treated with theLXR agonist T0901317 led to a rapid increase in the abundance of FAS(Yu et al., 2012). Moreover, this increase was associated with an in-crease in histone acetylation of H3 and H4 within 30 min of ligand

9N. Ma et al. / Toxicology and Applied Pharmacology 275 (2014) 1–11

addition, both associatedwith a permissive chromatin environment. In-terestingly, LXRα expressionwas necessary for these epigeneticmodifi-cations on FAS, corroborating the close connection between LXRα andFAS (Yu et al., 2012). Although a similar pattern of FAS induction wasobserved in our model of nicotine exposure, the underlying mecha-nisms initiating these posttranslational histone modifications to LXRare not well understood. Interestingly, studies using mouse primarycortical neurons have demonstrated that nicotine can directly lead toa more transcriptionally ‘permissive’ chromatin environment by alter-ing the expression of enzymes involved in histone H3 methylation andacetylation (Chase and Sharma, 2012).

While our results have found that nicotine exposure during preg-nancy and lactation leads to elevated levels of triglycerides in adultmale offspring, short-term dietary or pharmaceutical intervention inperinatal life might be useful in the prevention of these outcomes.Since the development of the liver occurs throughout neonatal andearly postnatal life (Greengard et al., 1972), it is plausible that targetingthis short period of development could help reverse or prevent adversehepatic outcomes in offspring. For example, short-term injections ofExendin-4™ in neonatal life has been demonstrated to prevent oxida-tive stress, impaired hepatic glucose production and hepatic insulin re-sistance normally exhibited in IUGR rat offspring (Raab et al., 2009).Given that folic acid supplementation has been demonstrated to reducelong-term hypertriglycidemia in IUGR piglets via repressive epigeneticmodifications silencing the promoter of FAS (Jing-Bo et al., 2012), itmight serve as an appropriate dietary intervention in MNE offspring.This is of great interest considering that smokingmothers and their off-spring have low serum folate levels (Cogswell et al., 2003; Jauniauxet al., 2007; Okumura and Tsukamoto, 2011; Stark et al., 2005; vanWersch et al., 2002). Therefore, it is conceivable that additional supple-mentation may reverse any aberrant epigenetic modifications underly-ing adult-onset elevations in triglyceride levels found in offspring.

In summary, our findings demonstrate that maternal nicotine expo-sure during pregnancy and lactation leads to increased circulating andhepatic triglyceride levels in postnatal life due in part to alterations inthe regulation of the hepatic triglyceride synthesis pathway. This data,if extrapolated to humans, provides some insight into the molecularmechanisms underlying the reported increased risk of obesity observedin children of smoking mothers.

Declaration of interest

There is no conflict of interest that could be perceived as prejudicingthe impartiality of the research reported.

Funding

This work was supported by the Canadian Institutes of Health Re-search (MOP 111001 to DBH and MOP 86474 to ACH).

Author contributions

NM designed and performed all themolecular analyses in this studyand was the primary author in writing this manuscript. MW assistedNM with some of the molecular analyses. CJN and ACH generated theanimal cohort and assisted with the design of experiments and manu-script writing. DBH assisted in the designing of the experiments, molec-ular analyses and in the preparation of this manuscript.

Acknowledgments

We thank Dr. Lin Zhao (Department of Obstetrics and Gynaecology,Western University) and Peter Vo for their technical assistance and thestaff of the Central Animal Facility (McMaster University) for assistancewith the animal work.

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