Effect of Prenatal Waterpipe Tobacco Smoke Exposure on Cardiac Biomarkers in Adult Offspring Rats

Abstract

Introduction:

The prevalence of waterpipe tobacco smoke (WTS) consumption is increased among pregnant woman. Prenatal cigarette smoke exposure increased the risk of developing cardiovascular diseases in offspring. The current study examined the effect of prenatal WTS exposure on inflammatory profile, oxidative stress, and cardiac biomarkers in adult offspring rats.

Methods:

Female rats received WTS (2 hours per day) or fresh air 1 day prior to mating and throughout the pregnancy period. The body and heart masses were measured in male offspring rats. The level of oxidative stress biomarkers, nitrate, inflammatory mediators (interleukin 6 [IL-6], tumor necrosis factor alpha [TNF-α]), and gene expression of protein kinase C epsilon, angiotensin 2 receptor one, and transforming growth factor beta1 were measured in cardiac tissue homogenates of 13-week-old male offspring rats.

Results:

Prenatal WTS exposure reduced body weight and increased heart to body weight ratio (P < .05). Prenatal WTS exposure did not affect oxidative stress biomarkers (superoxide dismutase, glutathione peroxidase, and thiobarbituric acid reactive substances) but significantly increased catalase activity and nitrate level (P < .05) in cardiac tissue of adult male offspring rats. In addition, prenatal exposure to WTS did not affect cardiac level of TNF-α and IL-6 as well as the gene expression of different cardiac modulators in adult male offspring rats (P > .05).

Conclusion:

Prenatal WTS exposure has detrimental consequences on adult offspring rats by increasing the ratio of heart to body mass, increasing the catalase activity and nitrate level in cardiac tissue of adult male offspring rats.

Keywords

waterpipe smoking offspring rats prenatal oxidative stress gene expression cardiac biomarkers

Introduction

There is more than 1 billion smokers worldwide¹ and 1 death every 8 seconds due to tobacco-related diseases.² Tobacco smoking increases the risk of all cardiovascular diseases such as hypertension, heart failure, myocardial infarction, and stroke.³ The popularity of smoking has increased among females whereby they now account for 200 million of the world’s 1 billion smokers.⁴ The impact of cigarette smoking is extended to the offspring. For example, maternal smoking during pregnancy is associated with increased blood pressure and body mass index in adult offspring.⁵ In addition, it reduces high-density lipoprotein cholesterol concentrations,⁶ increases carotid wall thickening of human heart,⁷ and induces structural atherogenic changes in coronary artery.⁸

Waterpipe tobacco smoke (WTS) has become a popular method of tobacco consumption worldwide,^9,10 especially among youths. The prevalence of WTS among pregnant women is increased in recent years and reached about 7% to 9% in Jordan and Lebanon.^11,12 Waterpipe tobacco smoke contains several toxic chemicals similar to those found in other forms of tobacco.¹³ However, numerous studies revealed that WTS contains higher amount of nicotine and carbon monoxide (CO) compared to smoking 1 cigarette.^14,15 In addition, WTS contains high levels of heavy metals (arsenic, chromium, lead), nitric oxide (NO), benzene, volatile aldehydes, polycyclic aromatic hydrocarbons, tar, and tobacco-specific nitrosamines.^14,15 The frequent, long and deep smoking sessions of WTS resulted in higher concentrations of all aforementioned toxicants compared to other forms of tobacco smoke.¹³

Several studies examined the effect on WTS on the cardiovascular system. A single session of WTS increased systolic and diastolic blood pressures, heart rate,¹⁶ and cardiac output in human subjects.¹⁷ Almedawar and colleagues reported a strong association between WTS exposure and coronary artery diseases (Almedawar, Walsh et al. 2016).¹⁸ Several mechanisms were suggested to explain the role of WTS on the development of cardiovascular diseases such as inducing reactive oxygen species (ROS), impairing the cellular redox balance, and triggering inflammation and injury in human primary endothelial cells.¹⁹

Prenatal exposure to WTS reduced birth weight, growth rate, and survival in offspring rats.²⁰ A human study revealed that prenatal WTS exposure reduced offspring birth weight and Apgar score as well as increased pulmonary complications at birth.²¹ Furthermore, animal studies showed that prenatal exposure to WTS increased the susceptibility to develop airway inflammation in murine model of asthma²² and memory impairment²³ in offspring animals. However, the effect of prenatal WTS on offspring’s cardiac biomarkers is still unknown. Therefore, the main objective of the current study was to examine the effect of prenatal WTS on inflammatory profile, oxidative stress, and cardiac biomarkers in adult offspring rats.

Methodology

Animals

Twenty-four adult male and 24 female Wister rats (9-10 weeks old) were housed in accordance to Animal Care and Use Committee (IACUC) at Jordan University of Science and Technology (JUST). Rats were kept at 24°C ± 1°C with 12:12 hour light/dark cycle at room temperature with free access to water and food. Female rats were randomly assigned to WTS group and the control group (12 female rats in each group). Each male rat was placed with a female rat in the same cage for mating. All experimental procedures were approved by IACUC at JUST. The offspring rats were weaned on day 21 of lactation. Offspring male rats (1 male from each dam) were randomly assigned to grow until age of 13-week and the remaining rats were culled after weaning.

Mainstream Smoke

Twelve female rats from the WTS group were exposed to WTS utilizing whole-body exposure system for 2 hours per day with 1 hour break session. The WTS exposure started 1 day prior mating and continued daily till the delivery day (day 21).²³

Waterpipe exposure consists of 4 compartments: waterpipe machine, exposure chamber, pump, and CO analyzer. The waterpipe machine was loaded with 10 g of Two Apple flavor ma’assel in the head and covered with perforated foil to separate ma’assel from the quick-light charcoal that was used as a heat source. The vapor passed down to a glass bottom, filled with water, that cools the vapor. The waterpipe machine is connected to a diaphragm pump that draws the smoke from the machine and transports it to the animal chamber. The pump was programmed to give 171 puffs of 2.6 seconds duration with 17 seconds interval between each puff, with 530 mL puff volume.^24,25 The exposure chamber was connected to electrochemical sensor (Bacharach Monoxor II) to monitor the level of CO concentration in the chamber. The average level of CO was 882 ± 33 ppm (mean ± standard deviation [SD]) for all animals throughout the exposure sessions. The level of CO concentration was consistent with previous studies on rat model,^20,23,26 which is less than 0.1% of the total gases in the exposure chamber.²⁰

Measurement of Inflammatory Mediators and Oxidative Stress Biomarkers

After 13 weeks of delivery, male rats (1 male rat from each dam) were killed by decapitation without anesthesia because anesthesia affects the level of inflammatory mediators; interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α),²⁷ antioxidant enzymes,²⁸ and protein kinase C epsilon (PKC∊) expression.²⁹ Cardiac tissues were surgically excised, placed on a filter paper, weighed, adequately washed in phosphate buffered saline and frozen at −80°C until time of tissue homogenization as described previously.^22,30 Homogenized tissues were centrifuged to remove insoluble materials at 15 000 × g for 15 minutes, at 4°C.

The activity of antioxidant enzymes; superoxide dismutase (SOD) and glutathione peroxidase (GPx, cat# 19160; and CGP1 respectively, Sigma-Aldrich Corp, Michigan), catalase (cat# 707002; Cayman Chemical, Michigan), and the concertation of thiobarbituric acid reactive substances (TBARS; cat# 10009055l Cayman Chemical, Michigan) were measured in the homogenized cardiac tissues according to the manufacturers’ instructions.

The level of NO was measured utilizing total NO and nitrate/nitrite parameter assay kit (cat# KGE001, R&D Systems, Minnesota) according to the manufacturer’s instructions. The concentrations of IL6 and TNF-α (cat# R6000B RTA00, respectively, R&D Systems) were measured from the cardiac tissues following the manufacturers’ instructions. The measured levels were normalized to the protein concentration in each sample. The absorbance was read by Epoch Biotek microplate reader (BioTek, Winooski, Vermont).

Measurement of the Gene Expression of Angiotensin 2 Receptor One, Transforming Growth Factor beta1, and PKC∊

Cardiac tissues were mixed with 300 μL lysis buffer, then homogenized by ultrasonicator bath (Branson ultrasonic Corporation Danbury, CT). Total RNA was extracted from cardiac tissue using Quick-RNA MiniPrep Plus Kit (cat# R1057; Zymo research Irvine, California) according to manufacturer’s instructions. One μg of the total RNA was converted to complementary DNA (cDNA) using SuperScript IV VILO (cat# 11756050, Invitrogen, California). Master Mix and 5 μL of the diluted cDNA was used for the quantitative real-time polymerase chain reaction (PCR).

A total 20 μL reaction volume of the genetic expression of PKC∊, angiotensin-2 receptor type 1 (AT₁R), and transforming growth factor beta1 (TGFβ1) were analyzed by real-time PCR using specific primers (Rn00572010, Rn02758772, and Rn01785893, respectively, cat# 4331182, 4331182, and 4331182; Applied Biosystems, California) and Gene Expression Master Mix (cat# 4369016, Taqman, Applied Biosystems). Each sample was run in triplicate and normalized to actin (Rn00667869; Cat# 4331182, Applied Biosystems). The threshold cycles (Ct) was measured and relative gene expression was calculated using 2^−ΔΔCT method. The ΔCt for gene expression level was calculated relative to actin gene expression in WTS group and control. The 2^−ΔΔCT method was used to compare the expression among the 2 groups (WTS and control).

Statistics

Data were presented as mean ± SD. D’Agostino and Pearson omnibus and Shapiro-Wilk normality tests were used to check for the normality of data. Student t test or Mann Whitney test were used to analyze the difference between 2 groups. Statistical analysis was performed using GraphPad Prism 4 software. Value of P < .05 was considered statistically significant.

Results

The Effect of Prenatal WTS Exposure on Heart to Body Mass in Adult Male Offspring Rats

Prenatal exposure to WTS significantly decreased the average number of offspring rats per dam compared to control (7.50 ± 1.15 in WTS group vs 10.62 ± 0.72 in control, P = .03). Prenatal exposure to WTS did not affect the gender ratio at birth (0.56 ± 0.15 female: total born pups ratio in control group vs 0.40 ± 0.21 female: total born pups ratio in WTS). Offspring rats that were exposed to prenatal WTS showed significant reduction in body mass (344.2 ± 10.21 g in the control group vs 293.0 ± 19.27 g in WTS group, P = .002; Figure 1A) while the heart mass was not affected (1.003 ± 0.11 g in the control group vs 1.17 ± 0.19 g in WTS, P = .09; Figure 1B). The body and heart masses of offspring rats that were exposed to prenatal WTS were ∼15% less and ∼16% higher than control animals, respectively (Figure 1D). Rats born to WTS exposed dams showed significant increase in heart to body mass ratio (0.003 ± 0.003 in the control group vs 0.004 ± 0.0004 in WTS group, P = .002; Figure 1C). Moreover, the ratio of heart to body mass was ∼ 31% higher in offspring rats that were exposed to prenatal WTS than control offspring (Figure 1D).

Figure 1.

Effect of prenatal WTS exposure on male offspring weight and heart weight. Pregnant rats were exposed to either fresh air (control) or WTS for 2 hours per day. Male offspring rats’ (A) weight, (B) heart weight, (C) ratio of heart weight to body weight, and (D) percentage change of body and heart weights and ratio of WTS group were measured. *Indicates a significant difference from the control group. Values represent mean ± SD of data from 6 to 8 adult male offspring rats in each group. P < .05 was considered statistically significant. SD indicates standard deviation; WTS, waterpipe tobacco smoke.

Effect of Prenatal WTS Exposure on NO Level in Adult Male Offspring Rats

Prenatal WTS exposure increased the concentration of nitrate in the heart of adult male offspring rats compared to unexposed offspring rats (0.97 ± 0.41 μmol/mg protein in the control group vs 2.33 ± 0.96 μmol/mg protein in WTS group, P = .0004; Figure 2). Nitrate is an indirect indicator of the NO level.

Figure 2.

Effect of prenatal WTS exposure on nitrate concentration in cardiac tissue of adult male offspring rats. Pregnant rats were exposed to either fresh air (control) or WTS for 2 hours per day. The concentration of nitrate was measured in heart tissue homogenate of adult male offspring rats. *Indicates a significant difference from the control group. Values represent the mean ± SD of data from 12 adult male offspring rats in each group. P < .05 was considered statistically significant. SD indicates standard deviation; WTS, waterpipe tobacco smoke.

Effects of Prenatal WTS Exposure on Inflammatory Mediators in Adult Male Offspring Rats

Prenatal WTS exposure did not affect the concentrations of TNF-α (1.06 ± 0.33 unit\mg protein in the control group vs 0.89 ± 0.23 unit\mg protein in WTS group, P = .15) and IL-6 (27.27 ± 5.91 pg/mg protein in the control group vs 29.98 ± 7.25 pg\mg protein in WTS group, P = .47) in adult male offspring rats (Figure 3A and 3B).

Figure 3.

Effect of prenatal WTS exposure on inflammatory mediators in cardiac tissue of adult male offspring rats. Pregnant rats were exposed to either fresh air (control) or WTS for 2 hours per day. The levels of (A) TNF-α and (B) IL-6 were measured in heart tissue homogenate of adult male offspring rats. Values represent the mean ± SD of data from 12 adult male offspring rats in each group. P < .05 was considered statistically significant. IL-6 indicates interleukin; SD, standard deviation; TNF-α, tumor necrosis factor alpha; WTS, waterpipe tobacco smoke.

Effect of Prenatal WTS Exposure on Gene Expression in Adult Male Offspring Rats

Prenatal WTS exposure did not affect the level of PKC∊ gene expression (1.17 ± 1.13 folds change) in male offspring rats as compared to unexposed group (P = .64; Figure 3A). Further, prenatal WTS exposure did not change the expression level of TGF-β1 (1.19 ± 0.90 fold in WTS, P = .50) and AT₁R (1.16 ± 0.79 folds change in WTS group, P = .55) genes in male offspring rats compared to unexposed offspring rats (Figure 4B and 4C).

Figure 4.

Effect of prenatal WTS exposure on gene expression in cardiac tissue of adult male offspring rats. Pregnant rats were exposed to either fresh air (control) or WTS for 2 hours per day. The gene expression levels of (A) PKC∊, (B) TGF-β1, and (C) AT1R were measured in heart tissue homogenate of adult male offspring rats. Values represent the mean ± SD of data from 12 adult male offspring rats in each group. P < .05 was considered statistically significant. AT1R indicates angiotensin 2 receptor one; PKC∊, protein kinase C epsilon; SD, standard deviation; WTS, waterpipe tobacco smoke.

Effects of Prenatal WTS Exposure on Oxidative Stress Biomarkers in Adult Male Offspring Rats

Prenatal WTS exposure did not affect the activity of SOD enzyme (0.61 ± 0.28 unit/mg protein in the control group vs 0.55 ± 0.24 unit\mg protein in WTS group, P = .59) and GPx (11.61 ± 5.85 unit\mg protein in the control group vs 10.65 ± 4.08 unit\mg protein in WTS group, P = .64) in adult male offspring rats (Figure 4A and 4B). However, prenatal WTS exposure significantly increased the enzymatic activity of catalase in male offspring rats (305.4 ± 212.60 unit\mg protein in the control group vs 1864 ± 1506.00 unit\mg protein in WTS group, P = .001; Figure 4C). The concentration of TBARS was not altered in male offspring rats by prenatal WTS exposure (0.92 ± 0.21 μM\mg protein in the control group vs 1.21 ± 0.74 μM\mg protein in WTS group, P = .22; Figure 5D).

Figure 5.

Effect of prenatal WTS exposure on oxidative stress biomarkers in cardiac tissue of adult male offspring rats. Pregnant rats were exposed to either fresh air (control) or WTS for 2 hours per day. The activities of (A) SOD, (B) GPx, (C) catalase, and level of (D) TBARS were measured in heart tissue homogenate of adult male offspring rats. *Indicates a significant difference from the control group. Values represent the mean ± SD of data from 12 adult male offspring rats in each group. P < .05 was considered statistically significant. GPx indicates glutathione peroxidase; SD, standard deviation; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; WTS, waterpipe tobacco smoke.

Discussion

In this study, we have examined for the first time, the effects of prenatal exposure to WTS on cardiac gene expression, oxidative stress biomarkers, and inflammatory mediators in adult male offspring rats. Only male offspring rats were used in this study to prevent any confounding differences due to sex.³¹

The popularity of waterpipe smoke is increased, especially among pregnant women.^11,32 Khabour and colleagues reported that prenatal exposure to WTS did not alter male to female ratio but it reduced offspring weight at week 13 compared to unexposed rats,²⁰ consistent with the current finding. However, the current study revealed that prenatal WTS exposure reduced the number of born offspring rats, inconsistent with previous findings.²⁰ This inconsistency could be due to the larger number of animals utilized in the current study. Moreover, the results showed that prenatal exposure to WTS induced a substantial increase (16%, close to significant threshold, P = .09) in the heart mass in male offspring rats compared to unexposed offspring. In addition, the ratio of heart to body masses was increased in male offspring rats (31%) that were exposed to prenatal WTS, consistent with the effect of direct exposure to cigarette smoke.³³ These results collectively suggest an impact of WTS on cardiac muscles. The significant change in heart to body mass ratio indicates that WTS induced cardiac hypertrophy to some extent. In fact, heart to body mass ratio is used as an index of cardiac hypertrophy,³⁴ a leading cause of cardiovascular morbidity and mortality.³⁵ However, other studies showed that heart mass to tibia length is a better predictor to cardiac hypertrophy.^36,37 Therefore, future experiments on larger number of animals are needed to examine histological sections of the heart, assess heart mass to tibia length, as well as measure other markers of cardiac hypertrophy are needed to withdraw definitive conclusions about the effect of prenatal WTS exposure on cardiac hypertrophy.

Several studies examined the consequences of prenatal smoking exposure on offspring’s cardiovascular function. It has been shown that prenatal cigarette smoke induced reprogramming of the mechanisms that control blood pressure in infants and this would increase the susceptibility to develop cardiovascular diseases at later stages.³⁸ Further, prenatal cigarette exposure resulted in increased carotid artery intima-media thickness in young adult offspring⁷ and hence their risk to develop stroke³⁹ and cardiovascular diseases⁴⁰ is increased.

Nitric oxide is a lipophilic molecule that protects against cardiovascular diseases.⁴¹ Nicotine, the major component in cigarette smoke, enhances the release of NO by activation of nicotinic acetylcholine receptors.⁴² The current study revealed that prenatal exposure to WTS increased the level of nitrates, as a marker of NO, in the cardiac tissue of male offspring rats. Consistent finding was observed with prenatal nicotine exposure in rats.⁴³ Measuring the level of NO is difficult due to its lipid solubility and short half-life. There are few sensitive, specific, and reliable detection techniques that are available to directly and quantitatively measure NO levels. Hence, most tests rely on indirect techniques such as measuring the activity of the synthesizing enzymes (NO synthase),⁴⁴ accumulation of the second messenger that is produced by activating soluble guanylyl cyclase (cGMP),⁴⁵ as well as levels of the stable metabolites (nitrite and nitrate).⁴⁶ Elevated levels of NO contributes to cell apoptosis and disturb cell survival.⁴⁷ In addition, high levels of NO and superoxide radicals form peroxynitrite⁴⁸ that triggers oxidative stress, cell necrosis, and apoptosis.⁴⁹ Further, elevated level of peroxynitrite is critical for the development of myocardial infarction and chronic heart failure.⁴⁹

Several inflammatory mediators, such as IL-6 and TNF-α, contribute to the development of atherosclerosis, ischemic heart disease, and myocardial infarction.⁵⁰ Elevated levels of IL-6 induced cardiac remodeling through increasing collagen production⁵¹ and facilitating fibroblast migration and proliferation.⁵² Increased levels of TNF-α enhanced ventricular remodeling.⁵³ Direct exposure to cigarette smoke increased the level of TNF-α and IL-6 in animal models.^33,54 The cardiac levels of IL-6 and TNF-α in adult male offspring rats were not altered by prenatal WTS exposure in the current study. Mohsenzadeh and colleagues found that prenatal nicotine exposure increased the serum level of IL-6 and TNF-α in offspring rats.⁵⁵ Further, direct WTS exposure increased the levels of IL-6 and TNF-α in cardiac tissue⁵⁴ and lungs²⁶ of mice. This inconsistency in results could be due to the differences between direct versus prenatal exposure to WTS.

There are several markers of heart remodeling; PKCε, TGF-β1, and AT₁R. The PKCε plays a protective role against ischemic reperfusion injury.⁵⁶ The TGF-β1 induces cardiac hypertrophy,⁵⁷ remodeling,^58,59 and fibrosis.⁶⁰ The current study did not find any significant effect of prenatal WTS exposure on PKCε, TGF-β1, and AT₁R gene expressions in cardiac tissue of offspring rats. However, Lawrence and colleagues found that the level of PKCε expression in offspring cardiac tissue was decreased by prenatal nicotine administration from gestational day 4 to 21.⁶¹ In addition, Chou and Chen revealed increased level of TGF-β1 expression in cardiac tissue of 21-day-old offspring rats that were exposed to nicotine from gestational day 7 to postnatal day 10.⁶² Xiao and colleagues showed increased expression level of AT₁R in the heart of 5-month-old male offspring rats that were exposed to nicotine from day 4 of gestation to day 10 after birth.⁶³ This inconsistent results could be explained by the difference in number of animals, the exposure duration, time of measuring the expression level, and nicotine concentration. However, the relation between messenger RNA and protein levels is complex and is not always linear due to post-transcriptional and post-translational processes as well as protein degradation.^64,65 Therefore, future studies should focus on examining other biomarkers of remodeling such as galectin-3, urocortin, and copeptin among others, at the gene expression and protein level, to withdraw definitive conclusions.

Tobacco smoke triggers oxidative stress by producing ROS and attenuating the antioxidant systems.^66,67 Oxidative stress is involved in the pathogenesis of several cardiovascular diseases, such as cardiomyopathy, ischemic heart disease, and congestive heart failure.⁶⁸ Prenatal exposure to cigarette smoke significantly reduced the activity of antioxidant catalase and SOD in rat astrocytes.⁶⁹ Al-Sawalha and colleagues reported that prenatal exposure to WTS increased the activity of SOD, but not GPx and TBARS level, in mice lungs.²² However, prenatal WTS exposure did not affect the level and activity of oxidative stress biomarkers in offspring rats’ hippocampus.²³ The present study revealed that prenatal exposure to WTS did not affect the activities of SOD, GPx, as well as the level of TBARS, a marker of lipid peroxidation, in heart tissue homogenate in adult male offspring rats. On the other hand, prenatal WTS exposure increased the activity of catalase in male offspring rats by more than 6 fold as compared to unexposed offspring rats. This increase in catalase activity could represent a physiological adaptation and compensation to counteract the increase in oxidant load by WTS exposure, specifically hydrogen peroxide. Catalase, an antioxidant enzyme that is present in peroxisomes, converts hydrogen peroxide to water and oxygen.⁷⁰ Hydrogen peroxide induced oxidative stress and serves as a second messenger that is involved in several cellular responses such as proliferation and morphological changes.⁷¹ Further, hydrogen peroxide increased the synthesis of NO in mice cardiac myocyte.⁷² The increased levels of nitrate, an indicator of NO, in the current study might be due to the effect of elevated levels of hydrogen peroxide and this might explain the increased activity of catalase enzyme. Dieterich and colleagues reported an increased activity of catalase, but not SOD or GPx, in patients with end-stage heart failure.⁷³ Therefore, examining additional biomarkers of heart failure are needed. Future studies should also focus on evaluating the levels of ROS such as hydrogen peroxide, nitrotyrosine, and peroxynitrite among others.

The current study has several limitations. First, limited number of cardiac biomarkers to assess the inflammation, oxidative stress, as well as gene expression were examined. Second, the current study did not examine histological changes in the heart to evaluate inflammation, cellular hypertrophy or damage, endocardial thickness, and deposition of collagen among other changes. Third, the functional read outs of cardiovascular performance such as blood pressure and heart rate were not assessed. Fourth, the levels of nicotine and cotinine in offspring rats were not measured. Further studies are required to consider the aforementioned limitations to withdraw conclusions about the detrimental effect of prenatal WTS exposure of cardiac tissue of offspring.

In conclusion, the results showed that prenatal WTS exposure increased heart to body masses, the activity of catalase and concentration of nitrate, a marker of NO, in cardiac tissue of offspring rats. Further, prenatal WTS exposure did not affect the level of IL-6 and TNF-α inflammatory mediators, the activity of SOD and GPx antioxidant enzymes or the concentration of TBARS in cardiac tissue of offspring rats. Prenatal WTS exposure did not alter the gene expression of different cardiac modulators in male offspring rats. Therefore, careful measures should be adopted to enhance WTS cessation during pregnancy to limit the negative consequences on adult offspring.

Footnotes

Acknowledgments

The authors thank Jordan University of Science and Technology for funding this work. The authors thank Weam Alyacoub, BSc, and Yehya Almahmoud, BSc, for their technical assistance.

Author Contributions

N. A. Al-Sawalha contributed to conception, design, acquisition, analysis, and interpretation; and drafted the manuscript. M. S. AL-Filali contributed to acquisition. K. H. Alzoubi and O. F. Khabour contributed to interpretation. All authors critically revised the manuscript, gave final approval, and agree to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Deanship of Research in Jordan University of Science and Technology (grant number 85/2017).

ORCID iD

Nour A. Al-Sawalha

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