Objective
The objective of the study was to determine whether perinatal nicotine exposure adversely affects cardiovascular health in adulthood.
Study Design
C57Bl/6J female mice were randomized to 200 μg/mL nicotine in 2% saccharin or 2% saccharin alone from 2 weeks before breeding until weaning. Offspring weight, vital signs, and carotid artery vascular reactivity were studied. A second cohort was subjected to shaker stress on day 4 of 7 days. Selected mediators of vascular tone were evaluated by molecular studies. Student t or Mann-Whitney U test was performed for statistical analysis (significance: P < .05).
Results
Nicotine-exposed compared with control female offspring had significantly elevated mean blood pressure under normal and stress conditions. Nicotine females lacked heart rate elevation after stress. Nicotine males had higher mean heart rate and a blunted contractile response to phenylephrine compared with controls, without an increase in blood pressure.
Conclusion
Perinatal nicotine exposure has an impact on the developmental programming of future cardiovascular health, with adverse effects more evident in female offspring.
According to the hypothesis of the developmental origins of health and disease, an adverse environment during key early developmental periods has the potential to shift the long-term health trajectory toward disease states. Evidence in support of this have proliferated since the popularization of this concept by Barker and Osmond, who identified an increased risk for cardiovascular disease among adults whom had been exposed to famine in utero.
Cardiovascular disease remains a leading cause of death in both men and women, and maternal smoking has been linked to hypertension in adults. Nicotine has the potential to affect developmental programming through various pathways, including perturbations of maternal vascular tone, blood supply, and oxygen delivery to the uterus, alteration of placental enzyme activity, or via a direct effect on the fetus in utero or on the neonate via breast milk or second-hand smoke. Nicotine has been shown to readily cross the placenta both in vitro and in vivo and can be found in amniotic fluid in concentrations up to 88% higher and in cord blood in concentrations up to 15% higher than in the maternal circulation.
Both smoking and smokeless tobacco use have been associated with adverse outcomes for mothers and their babies, including preterm labor, intrauterine growth restriction, increased rates of stillbirth, and sudden infant death syndrome. The addictive properties of nicotine make cessation exceedingly difficult for some women despite such serious short-term risks.
According to the Centers for Disease Control and Prevention (CDC) Pregnancy Risk Assessment Monitoring data from 2008 show an overall prevalence of smoking in the 3 months prior to pregnancy of averaged 28% (range, 10.4–39.4%) and during the last 3 months of pregnancy was reported by 12.8% (5.1–28.7%) of women, with the highest rates in women aged 20-24 years. Moreover, nicotine exposure may continue if nicotine replacement is used as part of smoking cessation programs. This high rate of nicotine exposure in utero underscores the clinical relevance of investigating the long-term effects of nicotine on the exposed fetus and neonate.
We have previously demonstrated alterations in developmental programming of cardiovascular function using animal models including: a nitric oxide synthase 3 (NOS3) knockout mouse model of intrauterine growth deficiency, a soluble fms-like tyrosine kinase-1 induced model of preeclampsia, and an ApoE knockout mouse model of atherosclerosis. Our objective with this study was to assess the effect of perinatal nicotine exposure on cardiovascular function in adulthood under normal and stress conditions.
Material and methods
Animals
The Institutional Animal Care and Use Committee (IACUC) at the University of Texas Medical Branch at Galveston approved the study protocol. C57Bl/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME). All dams were housed individually except during 12 hour breeding periods and after delivery, when offspring were co-housed with their natural mothers and littermates. After weaning, offspring were separated by litter and sex and co-housed in groups of 3-4. The animal facility was temperature and humidity controlled, with automatically controlled 12 hour light and dark cycles. Mice were allowed to consume regular chow and drinking solution ad libitum. Certified personnel and veterinary staff provided regular maintenance and animal care according to IACUC guidelines. The animals were killed by carbon dioxide inhalation according to the IACUC and American Veterinary Medical Association guidelines.
Breeding
Female C57Bl/6J mice were randomized to receive either 200 μg/mL nicotine (catalog no. N-0267, ± nicotine, purity >99%; Sigma-Aldrich, St. Louis, MO) in 2% saccharin (weight/volume) (catalog no. S1002, saccharin sodium salt hydrate, purity ≥98%; Sigma-Aldrich) or 2% saccharin solution alone as control as the sole source of water for 2 weeks prior to breeding.
Thirteen dams were exposed to nicotine solution and 9 to saccharin alone. Male C57Bl/6J mice were placed with individual females for 12 hour breeding periods, the only times at which deionized water was substituted for the assigned drinking solutions to prevent exposing males to nicotine. Drinking solutions were continued throughout pregnancy and weaning on postnatal day 21, at which time offspring were separated into groups by litter and sex. This created 4 offspring groups: nicotine-exposed females, nicotine-exposed males, control females, and control males.
Maternal data
Maternal weight was recorded at baseline, midgestation, and weaning. Cotinine, the major metabolite of nicotine, has a serum half-life of approximately 16-18 hours, considerably longer than that of nicotine (1-2 hours) and was used as a measure of maternal nicotine exposure. Tail vein blood (100 μL) was collected from all dams prior to exposure to drinking solution, before breeding, and at weaning. Blood was centrifuged for 10 minutes at 3000 rpm and serum was collected and stored at −80°C until the time of testing. Serum cotinine levels were determined using a mouse-specific colorimetric enzyme-linked immunosorbent cotinine assay kit, sensitive to 1 ng/mL (Calbiotech, Spring Valley, CA) according to the manufacturer’s instructions and were read by an automated spectrophotometer (Fusion 5.0; Ortho Clinical Diagnostics, Rochester, NY). All samples were run in duplicate.
Offspring data and vital sign measurement
Litters were counted and weighed on postnatal day 1 and offspring weighed at the time the animals were killed. The first of 2 offspring cohorts was tested at 5 months of age. The groups were comprised as follows: nicotine-exposed females (n = 12) and nicotine-exposed males (n = 11) born from 3 dams; and control females (n = 8) and control males (n = 7) born to 4 dams.
To continuously monitor vital signs, offspring were anesthetized using a mixture of ketamine (Ketathesia; Butler Animal Health Supply, Dublin, OH) and xylazine (Lloyd Labs, Shenandoah, IA). A vertical midline incision was made in the neck, and the left carotid artery was identified and isolated. A small incision was made in the vessel, and a 0.4 mm catheter was introduced and the tip was advanced to the level of the aortic arch and then tunneled to a telemetric transducer encased in a plastic capsule (PAC-10 model; Data Sciences, St. Paul, MN) that was secured within a pouch of skin at the nape of the neck. The skin incisions were closed with 5-0 silk sutures, and mice were monitored and kept under a radiant warmer until fully recovered from anesthesia.
Blood pressure and other vital signs were continuously recorded in the unrestrained, conscious mice for 5 days after an initial 48 hour recovery period. Moving averages of systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate were obtained over 24 hour and 5 day periods. Mean arterial blood pressure was calculated as (SBP + 2 DBP)/3 from the recorded data and averaged over the same 24 hour and 5 day periods.
Offspring were killed after the completion of the telemetry studies. The aorta and kidneys were harvested, flash frozen in liquid nitrogen, and stored at −80°C for molecular studies. The noncannulated carotid arteries were used immediately after collection for the in vitro vascular reactivity experiments.
Shaker stress
The second offspring cohort was bred as described in the above-cited text and subjected to continuous telemetry vital sign monitoring over a 7 day period at 3 months of age. The cohort was comprised of nicotine-exposed females (n = 9) and nicotine-exposed males (n = 6), born to 4 dams, and control females (n = 6) and control males (n = 10), born to 3 dams.
Offspring were subjected to an acute stress by securing up to 4 cages onto a shaker platform, which was programmed to shake at 150 cycles/s and 2.86 cm/stroke for 14 separate 2 minute episodes. These stressor events were scheduled intermittently every 15-45 minutes over a 6 hour period on day 4 of recording. The moving averages of blood pressures and heart rates were calculated from 15 minutes before to 15 minutes after the times at which shaking was scheduled to occur for each of the 7 days, allowing an assessment of blood pressure and heart rate at baseline, on the day of stress, and for 2 days thereafter. This yielded 6.5 hours of vital sign monitoring over each 24 hour period per mouse. Offspring were killed and tissues collected and stored for future analysis.
Vascular reactivity studies
Each carotid artery was carefully dissected, divided into 2 mm segments and mounted in a 25 μm tungsten wire-myograph (model 410A; J. P. Trading I/S, Aarhus, Denmark). The segments were bathed in physiological salt solution, maintained at 37°C and pH approximately 7.4, and a mixture of 95% O 2 and 5% CO 2 was bubbled continuously through the solution. The contractile force was recorded using an isometric force transducer and analyzed using PowerLab acquisition and LabChart 7, version 7.1, playback software (ADInstruments, Inc USA, Colorado Springs, CO).
After stabilization of vascular tone, the vessels were contracted twice with 60 mmol/L potassium chloride (KCl) over 30 minutes to enhance reproducibility of responses and then were washed with Krebs solution and allowed to return to baseline tone. Vessels were then precontracted with the α-1-adrenergic agonist phenylephrine (10 −7 and 10 −6 mmol/L), and then relaxation responses to cumulative concentrations of the endothelium-dependent vasorelaxant acetylcholine (10 −9 to 10 −5 mmol/L), the endothelium-independent vasorelaxant sodium-nitroprusside (10 −9 to 10 −5 mmol/L), and the β-adrenoreceptor agonist isoproterenol (10 −9 to 10 −5 mmol/L) were evaluated.
Vessels were then washed, allowed to equilibrate, and induced to contract using increasing concentrations of phenylephrine (PE; 10 −9 to 10 −5 mmol/L) alone and in the presence of the nonselective nitric oxide synthase inhibitor N -omega-nitro-L-arginine methyl ester (L-NAME; 10 −4 mmol/L), and with serotonin (10 −9 to 10 −5 mmol/L). After each agent was used, the vessels were washed and allowed to return to basal tone. The precontraction to PE was used as reference in calculation of the percent relaxation, and the second response to KCl was used as the baseline in calculation of contractile responses.
The drugs used in the in vitro experiments were purchased from Sigma Chemical Co (St. Louis, MO). Stock solutions of all of the drugs (10 −2 mol/L) were prepared in deionized water and stored at −20°C. The composition of physiological salt solution was NaCl, 119 mmol/L; KCl, 4.7 mmol/L; NaH 2 PO 4 , 1.2 mmol/L; NaHCO 3, 25 mmol/L; MgCl 2 , 1.2 mmol/L; CaCl 2 , 2.5 mmol/L; ethylenediaminetetraacetic acid, 0.026 mmol/L; and glucose, 11.5 mmol/L.
Molecular studies
To elucidate the mechanisms behind differences seen in blood pressure, molecular studies of selected mediators of hypertension, vascular function, and nicotinic receptors were evaluated in the nonshaker cohort. Aorta and kidney tissues were homogenized using a Bullet Blender (Next Advance, Inc, Averill Park, NY).
Ribonucleic acid (RNA) extraction was performed using the RNAqueous kit (Ambion, Austin, TX) according to the manufacturer’s instructions. Reverse transcription into complementary deoxyribonucleic acid (cDNA) was performed using the MultiScribe reverse transcriptase system (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The cDNA was amplified on an ABI 7900 HT Fast real-time polymerase chain reaction (PCR) system (Applied Biosystems) using mouse-specific Taqman primer/probe sets (all from Applied Biosystems) for the following: NOS3 (catalog no. Mm00435217_m1), the angiotensin II type-1a receptor (AGTR1a, catalog no. Mm00616371_m1), angiotensin II type-1b receptor (AGTR1b, catalog no. Mm02620758_s1), and the angiotensin II type 2 receptor (AGTR2, catalog no. Mm01341373_m1) and the nicotinic acetylcholine receptor α-7 subtype (CHRNA7, catalog no. Mm01312230_m1).
Amplification was performed according to the manufacturer’s protocol under the following conditions: 95°C (20 seconds) for 1 cycle followed by 40 cycles each of 95°C (3 seconds) and 60°C (30 seconds). All samples were run in triplicate. Target gene expression was related to the expression levels of 18s messenger RNA (mRNA) in each specific sample (catalog no. Hs99999901_s1). All PCR reactions for each sample were performed simultaneously on the same plate.
Western blot was performed to assess the angiotensin levels in the kidney. To maximize protein yield of total homogenate and minimize background, proteins were extracted from tissues using 1× radioimmunoprecipitation assay buffer (consisting of 50 mM Tris HCl, 150 nM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 1 nM EDTA, 0.25% sodium deoxycholate, 1 mM each of the protease inhibitor phenylmethylsulfonyl fluoride and protease inhibitor cocktail [catalog no. P8340; Sigma]).
Proteins were quantified using a bicinchoninic assay protein assay kit (Pierce, Rockford, IL). Equal protein samples were loaded on sodium dodecyl sulfate polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA) and run according to the manufacturer’s instructions. Polyvinylidene fluoride membranes (Invitrogen Corp, Carlsbad, CA) containing protein were incubated overnight with antibody to angiotensin at a dilution of 1:100 (N-10: sc-7419, Santa Cruz Biotechnology, Inc, Santa Cruz, CA). Chemiluminescence was detected using the Amersham ECL Plus detection reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Detected bands were then analyzed densitometrically and normalized to monoclonal anti-β-actin at a dilution of 1:15,000 (Sigma-Aldrich) using ImageJ 1.44 software (US National Institutes of Health, Bethesda, MD).
Statistical analysis
Male and female groups were analyzed separately. For vascular studies, the area under the curve (AUC), maximum effect (Emax), and the dose required to produce 50% of the maximum effect (log EC50) were calculated. Data are expressed as mean ± SEM for continuous data and median ± interquartile range for noncontinuous data. Student t or Mann-Whitney U tests were performed as appropriate using GraphPad Prism version 5.0c for Mac OSX (GraphPad Software, San Diego, CA, www.graphpad.com ; significance: P < .05).
Results
Maternal serum cotinine levels did not differ between groups at baseline and, as expected, were significantly higher in the dams randomized to nicotine at breeding and weaning ( Figure 1 ). Cotinine enzyme-linked immunoassay interassay variability was 14.4%, and intraassay variability was 4.1%. Maternal weights did not differ between groups at any time point, nor did litter counts or offspring weights on postnatal day 1, at 3 months (shaker cohort), or at 5 months (nonshaker cohort, Table 1 ).
| Characteristic | Control | Nicotine | P value |
|---|---|---|---|
| Maternal weight, g | |||
| Baseline | 20.47 ± 0.58 | 19.87 ± 0.57 | .40 |
| Midgestation | 32.46 ± 1.38 | 33.29 ± 2.02 | .75 |
| Weaning | 26.02 ± 0.72 | 25.82 ± 0.41 | 1.0 |
| Maternal cotinine, ng/mL | |||
| Baseline | 1.61 ± 0.52 | 1.54 ± 0.06 | .63 |
| Prior to breeding | 0.93 ± 0.27 | 57.42 ± 18.0 | .005 |
| Weaning | 1.24 ± 0.20 | 43.85 ± 10.21 | < .001 |
| Litter count, n | 8 (IQR 1.5) | 6 (IQR 3.75) | .37 |
| Pup birthweight, g | 1.40 ± 0.9 | 1.26 ± 0.16 | .35 |
| Male weight, g | |||
| 3 mo | 27.56 ± 0.77 | 27.96 ± 0.45 | .95 |
| 5 mo | 28.36 ± 0.62 | 27.11 ± 0.91 | .06 |
| Female weight, g | |||
| 3 mo | 22.93 ± 0.22 | 25.43 ± 1.4 | .10 |
| 5 mo | 23.24 ± 0.52 | 24.24 ± 0.21 | .14 |
Nonshaker cohort vital signs and vascular reactivity
Nicotine-exposed female offspring had persistently elevated systolic, diastolic, and mean blood pressures compared with controls when analyzed over 24 hour and 5 day periods ( Figure 2 , A and B). There was no difference in heart rate. Conversely, males exhibited no differences in blood pressures but had significantly lower mean heart rates in the nicotine group ( Figure 3 , A and B). No differences were noted in the in vitro contractility or relaxation responses of carotid artery segments among female groups to any of the agents tested ( Table 2 ). Males in the nicotine group exhibited a blunted contractile response to phenylephrine, which was mitigated in the presence of L-NAME ( Figure 4 , A and B). No differences were noted among males with regard to relaxation.
| Variable | AUC | Emax | Log EC50 |
|---|---|---|---|
| Female offspring | |||
| Control PE | 48.1 ± 8.7 | 29.0 ± 4.7 | −6.1 ± 0.4 |
| Nicotine PE | 51.4 ± 9.7 | 62.3 ± 29.2 | −6.5 ± 0.2 |
| Control PE plus L-NAME | 134.9 ± 79.1 | 72.72 ± 37.0 | −6.5 ± 0.2 |
| Nicotine PE plus L-NAME | 138.6 ± 29.5 | 113.0 ± 13.5 | −6.1 ± 0.5 |
| Control serotonin | 80.2 ± 36.8 | 41.5 ± 15.9 | −7.3 ± 0.1 |
| Nicotine serotonin | 118.0 ± 34.6 | 56.9 ± 14.6 | −7.3 ± 0.1 |
| Male offspring | |||
| Control PE a | 304.5 ± 22.8 a | 83.6 ± 6.5 | −6.9 ± 0.2 |
| Nicotine PE a | 99.4 ± 37.2 a | 54.6 ± 18.7 | −6.8 ± 0.2 |
| Control PE plus L-NAME | 150.8 ± 17.0 | 89.7 ± 3.2 | −6.8 ± 0.1 |
| Nicotine PE plus L-NAME | 202.8 ± 59.4 | 91.6 ± 33.4 | −6.6 ± 0.2 |
| Control serotonin | 144.5 ± 8.5 | 36.4 ± 20.2 | −7.28 ± 0.18 |
| Nicotine serotonin | 68.7 ± 26.0 | 91.62 ± 33.4 | −7.40 ± 0.26 |
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