Sex-Dependent Occlusive Cardiovascular Disease Effects of Short-Term Thirdhand Smoke Exposure

Abstract

Introduction

Thirdhand smoke (THS) is associated with many public health and disease concerns, such as respiratory illness, cancer, lipidemia, and cardiovascular disease (CVD). We have previously shown that a moderate to long-term exposure to THS increases the risk of thrombosis. However, whether short-term exposure to THS would produce any effects remains to be discovered. Therefore, this study investigated the impact of 1-month THS exposure on platelet function, in vivo and in vitro, and on cytokine response, in a sex-dependent manner.

Aims and Methods

Secondhand smoke or clean air (CA) exposed upholstery materials for 1 week were kept in cages housed with 5–6 mice, and the procedure was repeated for 4 weeks. These THS-exposed mice were evaluated for thrombogenesis and platelet function assays. In addition, cytokines expression was evaluated from pooled serum.

Results

Compared to the CA group, THS exposure significantly shortened the tail bleeding time and carotid artery thrombus formation. Moreover, the female mice appeared more sensitive to THS exposure than males. Furthermore, platelet aggregation, dense granule secretion, and P-selectin activation markers were significantly elevated due to THS exposure. In addition, high-throughput screening showed at least 30 cytokines differentially modulated by THS in females relative to 26 in male mice.

Conclusions

Collectively, these results demonstrate that 1 month of THS exposure represents a high health risk, in part, by triggering a prothrombotic phenotype that appears to be more significant in females, who are at a much higher risk for occlusive CVD. Additionally, changes in cytokine levels mediate some of the THS-induced occlusive effects.

Implications

This study revealed that THS exposure for 1 month is detrimental to the cardiovascular health of both sexes; however, females could be more aggressively affected than males. In addition, interleukins and chemokines could be critical factors for initiating prothrombotic activity due to THS exposure.

Introduction

Thirdhand smoke (THS) is the residual tobacco smoke that deposits on surfaces and is mainly composed of volatile or semi-volatile organic compounds that adsorb on surfaces, besides particulate matter.¹ Some of the surface-deposited organic residual layers may undergo further oxidation due to common acids like nitrous acid, producing carcinogens.² Moreover, some of the deposited smoke residues, with or without oxidation, depart from the surface and remain airborne or recycled, resulting in a primary source of THS exposure by inhalation or dermal contact.³ In addition, the unremitted part on the surface remains the cause of contact exposure, especially in infants.^4,5 The likelihood of nonsmokers getting exposed to THS could be higher than firsthand or secondhand smokers because they are unaware of its presence, besides being invisible and lacking the “typical” smoke smell.⁶ For example, dynamic real-time measurement of THS in the form of nicotinic-derived organic compounds in nonsmoking indoor modern movie theaters revealed the transport of hazardous THS pollutants by humans who smoked before entering the theater.⁷

The THS chemistry shows elevated levels of carcinogens at the place or space where firsthand smoke events occurred, mainly tobacco-specific nitrosamines (TSNA).^8,9 The human body can be exposed to these carcinogens via inhalation, dermal uptake, ingestion, and epidermal chemistry, in which adsorbed nicotine on the skin reacts with nitrous acid.¹⁰ To this end, exposure to THS has been shown or could be the cause of several diseases,¹¹ albeit a few clinical studies did determine the impact of THS on human health. For example, a recent study shows that the residents of houses of smokers at a younger age have a greater cancer risk.¹² Another study revealed epigenetic changes associated with THS exposure, including in many cellular pathways, such as cell death and oxidative stress.¹³

Concerning cardiovascular disease (CVD), we and others have shown that THS does increase the risk of CVD.^14–17 Furthermore, regarding occlusive CVD, we observed several in vitro and in vivo prothrombotic phenotypes associated with THS exposure.^14,17,18 Indeed, THS exposure increased platelet hyperactivity (eg, aggregation) and thrombosis risk,¹⁴ which was found to be the case with six or even three months of THS exposure.^14,18 Notably, a prothrombotic state due to THS exposure may result in ischemic stroke, myocardial infarction, or venous thromboembolism,¹⁹ all associated with human morbidity and mortality.

The adverse health effects of THS were also studied under short-term exposure settings. Hence, a 4-week THS exposure study protocol showed an increased risk of lung cancer because of significantly altered gene expression, namely activation of the P53 gene and endoplasmic reticulum stress genes.¹² Also, exposure to THS in utero and early life in male mice impacted immune cells and plasma cytokines, causing a significant response in chemokines and interleukins.¹⁶ However, the consequences of short-term, 4-week THS exposure on platelet activation have yet to be studied in adult C57BL/6 mice, as is the case in the cytokine response, in the context of sex. Therefore, this study will determine these issues- as per the protocol or model shown in Figure 1A on adult male and female mice. Of note, our previous studies of THS exposure for 3 months and those under in utero settings showed that female mice are more prone to thrombosis than males.

Figure 1.

THS exposure results in the delivery of nicotine to the mice: (A). Illustration of the setup for THS exposure. (B) ELISA results showing the cotinine levels in C57BL/6 male and female mice exposed to THS (or Clean Air) for 4 weeks (1 month), each bar represents mean ± STDEV, Mann–Whitney t-test is nonsignificant (ns) p-value > .05.

Materials and Methods

Reagents and Materials

Antibody: BioLegend Cat# 148305, RRID:AB_2565274, whereas thrombin Cat# P/N 384 and ADP Cat# P/N 386 were purchased from Chrono-log Corp, USA. Cotinine ELISA: CALBIOTECH Cat# C0096D. Upholstery fabric, Catalog No. K9975 DRESDEN purchased from Kovifabics.com, Carpet Catalog No. 16691934 purchased from Bedbathbeyond.com. Catalog # ARY028 Mouse XL Cytokine Array, R&D Systems.

Animals

All animal work protocols were approved by Texas A&M University Institutional Animal Care and Use Committee (TAMU-IACUC). All experiments were performed in a pathogen-free facility at TAMU in compliance with the institutional guidelines. C57BL/6 mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA).

Mice Exposure to THS.

The procedures and protocols for exposure to THS were similar to our previous studies.^14,17,18 Two sets of household materials (carpet, curtains, and upholstery) were utilized in our study. The first set was exposed to secondhand smoke from 40 cigarettes per day, with the exposure lasting for 1 week, before the materials were placed in cages housing 4–5 mice that are 5–6 weeks old to start the THS exposure. At the same time, the second set of materials were exposed in a similar manner for 1 week. The second set was then placed in the cages, and the first set was removed and re-exposed for another week. Once the exposure ended, we placed the first set in the cages and the second set was re-exposed. Once this exposure was complete, the second set was placed in the cages, and the first set was removed. This “alternate” exposure ensured that mice were initially exposed to fresh 1-week exposed materials, whereas the exposures that took place in the third and fourth week involved recycled fabric used in first and second week, respectively. Taken together, the overall 4-week exposure in and of itself included fresh and 2 weeks of exposed aged materials. Of note, we utilized 5–6 week-old mice-in part- to be consistent, as well as be able to make comparisons with our previous 3- and 6-month exposure studies.

Tail Bleeding Time Assay

A tail bleeding assay was performed by following our previous work.^14,20,21 Briefly, isoflurane was used to anesthetize the C57Bl/6 mice, which were kept warm on an isothermal blanket (37°C). After anesthesia, a scalpel was used to transect the tail, approximately 5 mm from the tip, and immediately the tail was submerged in a 0.9% 37°C saline. The bleeding time was determined by measuring the time for the bleeding to stop. To avoid excessive bleeding, the cutoff bleeding time was set for 10 minutes for statistical analysis; therefore, if bleeding lasted longer than 10 minutes, the experiment was stopped.

Carotid Artery Injury–Induced Thrombosis Model

The carotid artery thrombus formation experiments were conducted as described previously.^18,22 Briefly, avertin (2.5%) at a dose of 250 mg/kg of body weight was used to anesthetize the C57Bl/6 mice, with a heating pad used to maintain 37°C temperature. A skin incision of 5 mm deep is drawn below the jaw until the sternum, and the carotid artery was identified by removing the fascia. With the help of tweezers, the common carotid was kept separated from vessels and nerves. A Transonic Micro-Flowprobe (Transonic Systems Inc, Ithaca, NY, USA) was used to measure baseline blood flow. Presoaked filter discs in 7.5% FeCl3 were placed on top of the artery for 3 minutes, and after, filter discs were removed and washed with saline. The doppler flow probe was re-placed to monitor blood flow until stable occlusion of blood was formed. On completion of the procedure, mice were euthanized.

Platelet Count

An automated HemaVet950FS Blood Analyzer (RRID:SCR_020016) was used to count platelets.

Platelet-Rich Plasma and Washed Platelets Preparation

Platelet-rich plasma (PRP) was obtained from blood drawn from mice, as described previously.^20–22 Briefly, mice were anesthetized, and blood was drawn directly from the heart and collected in sodium citrate tubes (Fisher Scientific, Hampton, NH. USA). Tubes filled with blood were kept at room temperature, before centrifugation at 237 × g for 15 minutes. PRP was collected by gently transferring into clean collection tubes, and platelet count was obtained by using our automated hematology analyzer. Furthermore, washed platelets were obtained from PRP by centrifuging at 483 × g for 10 minutes, and the pellet was resuspended in HEPS-buffered saline containing 1 mM EGTA (pH 6.5) and platelet-activation inhibitors 10 ng/mL of PGI₂, and 0.37 U/mL of apyrase.

Platelet Aggregation and Dense Granule Release

Platelet aggregation and dense granule secretion in response to the agonist thrombin (0.1 U/mL), were measured using a model 700 aggregometer (Chrono-Log Corporation, Havertown, PA) RRID:SCR_023468 at 37°C, with the platelet count in PRP diluted to 7 × 10⁷/mL in a final volume of 250 μL. For the measurement of dense granule release, PRP was incubated with 12.5 μL of luciferase substrate/luciferase mixture, before the release of ATP was measured. Each aggregation and secretion assay experiment was performed at least three times, using blood pooled from 8 to 10 mice.

Flow Cytometry

Washed platelets were used for the flow cytometry-based analysis of P-Selectin expression with the agonist thrombin (0.1 U/mL). Furthermore, washed platelets were diluted to 100 000/ μL in calcium-free Tyrode’s buffer before adding the agonist, and 1 mM of calcium was added to platelets and incubated for 20 minutes at room temperature. The BD Accuri C6 plus flow cytometry (BD Biosciences; Franklin Lakes, NJ, USA) RRID:SCR_019591, was used to measure the binding of anti-P-selectin antibody conjugated with PE-fluorophore (1:1000 dilution).

Platelet Spreading Assay

A standard protocol of platelet spreading was followed with slight modifications.^20,23 Thus, 100 000 cells/µL of washed platelets were added to each glass coverslip precoated with fibrinogen. After platelet activation with thrombin (0.005 U/mL), adherent platelets were fixed, stained with phalloidin conjugated with TRITC fluorophore and imaged with an inverted fluorescent microscope. Spreading was assessed by counting activated platelets that have changed shape using ImageJ.

Cotinine Assay

Cotinine was measured using a Direct ELISA cotinine kit purchased from Calbiotech USA. Briefly, 10 µL of serum was obtained from the mice exposed to clean air (CA) or thirdhand smoke to measure the cotinine using ELISA by following the instructions of the manufacturer. A standard curve was generated to extrapolate the cotinine levels in serum.

Cytokine Array

The cytokines array kits were purchased from R&D Systems (A proteome Profiler Mouse XL Cytokine Array, Cat# ARY028). Each kit is supplied with four arrays, that were utilized for four independent groups as follows: Males CA and THS-exposed groups, as well as females CA and exposed groups. Each array has the capacity to evaluate 111 cytokines, which includes groups of chemokines, growth factors, and interleukins. Each time, all four arrays of one kit were used together, and array images were captured together. Data were presented as ratio of the exposed group and CA group, of which the top 10% of cytokines that were modulated, either as upregulated > 1 or downregulated < 1 in both kits were plotted. For each single array, blood was collected from the exposed and CA mice, and allowed to coagulate to obtain serum. We pooled serum from five mice, with 40 μL from each mouse pooled to obtain 200 μL of serum. All steps of the protocol were strictly followed according to the manufacturer’s guidelines. Cytokines were measured in both sexes, and each sex had its control group.

Statistical Analysis

Experiments were performed at least three times, and in each group, the number of animals was 5–10 mice. The statistical analysis of data was performed using the GraphPad Prism (RRID:SCR_002798). For sex differences, two-way ANOVA was used to obtain multiple comparisons using Bonferroni or Tukey statistical hypothesis testing. In addition, to determine the significance of occlusion time in males or females, an unpaired t-test with Welch’s correction was employed. Finally, the spreading data were analyzed by Mann–Whitney test.

Results

Determining Cotinine Levels in Serum From Mice Exposed to THS for 1-Month

To determine if exposure to THS for 1 month is sufficient to cause changes in platelet function, in a sex-dependent manner, we first sought to measure cotinine levels in male and female mice to confirm nicotine delivery due to THS exposure. Our ELISA results show the serum cotinine levels in the exposed female and male mice were 66.75 ± 2.7 ng/ml and 72.2 ± 0.86 ng/mL, respectively, whereas it was undetectable in the CA exposed control mice (Figure 1B); no sex differences were observed in the level of cotinine (CA data not shown). Thus, these results do indicate that the 1-month of THS exposure appears to be a significant time for exposure of mice to “nicotine” or its metabolites. Moreover, these data support the notion that similar to our long-term studies, exposure to THS for as “short” as 1 month could potentially lead to changes in platelet function and increases the risk of thrombosis.

Effect of 1-Month THS Exposure on the Tail Bleeding Time and the Occlusion Time

Bleeding time, a standard platelet function test, has prognostic implications in the context of occlusive CVD. Therefore, we analyzed the tail bleeding time on male and female mice subjected to 1-month of THS exposure. Our results show a significant shortening of bleeding time in male and female mice compared to their respective clean-air exposure groups (Figure 2A). Specifically, our statistical analyses show that the average bleeding time in the CA control female or male mouse groups varied significantly from 272 ± 64 seconds to 175 ± 65. In contrast, in THS exposure groups, it drops to 30.57 ± 20.42 or 38.33 ± 22.13 in females and males, respectively (Figure 2A), which suggests a prothrombotic phenotype. We further analyzed the difference in the change in bleeding time among genders due to exposure to THS. We observed that female mice showed significantly much more shortening in bleeding time than males, representing a more robust phenotype of THS (Figure 2B). These results indicate that females might be more sensitive to THS exposure. Our results also confer that control female mice exhibit significantly longer bleeding time compared to control group male mice (Figure 2A). It is to be noted or well-known that females exhibit longer bleeding times compared to males.²⁴ We next decided to determine THS effects on thrombogenesis using the ferric chloride carotid artery-induced thrombosis¹⁹ model and compare both genders. We observed a statistically significant¹² drop in the occlusion time in both genders due to exposure to THS, as shown in Figure 2C. In addition, and regarding sex differences, the females in the control group showed longer occlusion time compared to the male control group (Figure 2C). Statistically, we also observed, as was the case with the bleeding time, that exposure to THS showed a more robust phenotype in females than in the male group, which was inferred from the mean differences in female-THS versus male-THS (Figure 2D).

Figure 2.

Tail bleeding time and arterial occlusion time are shortened in THS-exposed mice, in a sex-dependent manner: Male and female mice exposed to Clean Air or THS are assessed for hemostasis by tail bleeding time (A) and for thrombogenesis by the FeCl₃ carotid arterial thrombosis mouse model (C). Each group has 5 to 8 mice. The mean values are plotted as interleaved scatter plots with STDEV, a two-way ANOVA statistic showing significance as p-value *≤.05, **≤.01, ****≤.0001. Furthermore, we evaluated the sex differences in bleeding time or occlusion time among males and females of the exposed groups (B) and (D), respectively. Each scatter bar represents the mean with STDEV; statistical significance was observed with unpaired t-test with Mann–Whitney test p-value *≤.05 or ***≤.001; CA: Clean Air; THS: thirdhand smoke.

Effect of 1-Month THS Exposure on Platelet Aggregation, Dense Granule Secretion, p-Selectin/CD62-P Expression and Platelet Spreading

We first sought to determine if 1-month THS exposure would lead to enhanced platelet “sensitivity” to one of the major agonists, specifically thrombin, in terms of aggregation. Our results demonstrate a significantly increased platelet aggregation or activation in the 1-month THS exposure compared to CA, in both female and male mice, as shown in Figure 3, A and B respectively; the mean values with STDEV showing the percentage of maximal aggregation as shown in Figure 3E. Likewise, measuring platelet dense granule secretion is essential to assessing platelet function. Utilizing the ATP luminesce assay, it was found that THS exposure significantly increased platelet secretion among female and male mice compared to their respective CA-exposed groups (Figure 3, C and D); the mean values are plotted as bar graphs (Figure 3F). However, platelet aggregation or dense granule secretion did not show any gender difference.

Figure 3.

Evaluation of platelet activity due to THS exposure. Platelet-rich plasma (PRP) of female (A, C) or male (B, D) mice exposed to Clean Air or THS were analyzed for platelet aggregation, and dense granule secretion, respectively in a Chrono-log 700-X Lummi aggregometer, in the presence of the agonist thrombin (0.1 U/mL). Each experiment was repeated three times with blood pooled from 5 to 8 mice each time. The mean values of aggregation (E) and dense granule release (F) are plotted with STDEV. Similarly, pooled washed platelets from male or female mice exposed to Clean Air or THS were analyzed using flow cytometry to evaluate the CD62-P expression (G). Statistical analysis by two-way ANOVA, shown as p-value ≤ .05, significance*, ns: represents nonsignificant (p-value ≥ .05); CA: Clean Air; THS: thirdhand smoke; Thr: Thrombin. (H–K) Platelet spreading assay. Washed platelets (pooled from two mice) were treated with or without thrombin 0.005 U/mL, fixed and stained. Images of actin filaments were captured with Nikon inverted microscopic and processed using the Shape Index tool in ImageJ (H). Activated platelets were counted using ImageJ (n = 30 images) from each group and presented as mean values ± STDEV in females or males (I and J), respectively. The difference in fold change among males and females was also plotted (K). Each experiment was done in triplicates (****p value < .0001 or *** p value < .001).

CD62-P (P-Selectin) is a well-known marker for evaluating platelet activation. Thus, we measured CD62-P activation in both genders in response to thrombin. Our results show a significant elevation in the activation of platelets exposed to THS compared to control groups, as shown in Figure 3G. These results are indeed consistent with phenotype documented thus far and further confirm the enhanced platelet activation phenotype due to THS exposure.

Finally, spreading, which is a platelet functional response that is critical for thrombus formation, was also investigated. Our results show that the THS-exposed group has a significantly higher number of platelets that changed shape or are activated, relative to CA (Figure 3H–J), in response to the agonist thrombin. In addition, the comparison of fold change among males and females shows that females had significantly higher platelet reactivity (Figure 3K). These observations further document that the 1-month THS exposure is indeed sufficient to cause platelet hyperactivity in both sexes.

Cytokine Expression Level

Aside from the fact that cytokines are important in controlling growth and the immune system, they have also been shown to contribute to thrombogenesis. In addition, previous studies have shown that THS can cause changes in the expression level of many inflammatory cytokines (16). Therefore, we investigated whether exposure to THS for 1-month would modulate inflammatory cytokines; this was achieved by performing a high-throughput screening of 111 cytokines in the serum of both male and female mice, using two separate kits, to examine their differential expression, if any, in the context of sex. Our analysis of the cytokine arrays in the control and THS-exposed groups revealed robust changes in several cytokines in both female and male mice, as shown in Figure 4, A and B, respectively. Of note, for determining the differentially expressed cytokines, we selected the top 10% cytokines that consistently (in both kits) were either upregulated or downregulated, as shown in Figure 4, C and D. Our results show that the top 10% of cytokines modulated in females were different than in males (Figure 4, C and D, respectively). We believe this initial high-throughput cytokine data analysis could be essential for understanding the mechanistic impact of THS exposure, albeit still requires further classification and quantitative analysis.

Figure 4.

Cytokine screening of pooled serum from Clean Air (CA) and THS-exposed mice: Cytokines were measured from the serum of adult female (A) or male (B) mice that were exposed to THS or CA; each group represents serum pooled from five mice. The ratio of the THS to the CA exposed group data are plotted as XY bar graph representing the expression pattern of all 111 cytokines that the kit determines. The top 10% of modulated cytokines (from two separate array kits) that are either upregulated > 1 or downregulated < 1 in females (C) and males (D) are plotted in an XY bar graphs that represent relative expression.

Discussion

Since it was first incepted, it has become a well-established fact that THS residues persist for months, even after ventilation, that they reemit from the surface and can react with other elements in the surrounding environment to produce more toxic compounds.^1,8 While these THS residues are invisible, they were thought of as a threat to health and that they could potentially cause several diseases.^25,26 Hence, THS has become the subject of intense investigation, not only in the United States, but worldwide. It is important to note that in THS exposure, nicotine is likely not the only element to cause toxicity¹⁴; but rather, other toxicants- including nicotine derivatives- may be involved, due to the aging of THS smoke.⁸ To this end, a host of oxidized or reduced components emitted from the surroundings, such as formaldehyde, n-nitrosopyrrolidine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and other more reactive species have been reported.^1,14,18 Thus, toxicity induced by THS could be more severe (dangerous) than firsthand smoke. Indeed, aged THS was shown to be more toxic, with previous studies demonstrating that when the fresh sidestream smoke ages, it becomes 2–4 times more toxic.²⁷

Previous clinical and preclinical studies investigating the potential health hazards of THS exposure, documented that “victims” could mostly be nonsmokers, including children and pregnant women.^13,28 Consequently, the effects of THS on nonsmokers and children have been linked with respiratory disease, cancer, and CVD.²⁵ As for the THS effect on the cardiovascular system, we were the first to document that it increases the risk of thrombotic disease states.² Specifically, we have shown using a validated animal model that THS promotes prothrombotic activity in mice under moderate to long-term exposure conditions (3 or 6 months, respectively),^14,18 which was also observed when the exposure took place under in utero conditions.^3–6,17 Moreover, while some of the activation markers or measures of the prothrombotic platelet phenotype showed sex-dependent differences, they appeared to manifest in “washed platelets” but not PRP-based experiments²⁹; which warrants investigation in the future.

As part of our time-dependent analysis of the negative consequences of THS exposure, we conducted 1-month experiments in male and female mice separately while also assessing its impact on levels of inflammatory cytokine, which has not been done before. Interestingly, 1 month-THS exposure did show high levels of cotinine in the serum,^8,9 which provides evidence that even when the exposure is short, significant levels of tobacco makers can still accumulate or buildup. Therefore, one-month exposure to THS may be sufficient to trigger a prothrombotic phenotype in the exposed mice; since we previously showed that cotinine enhances platelet activation.²² Indeed, our findings showed a significant shortening of bleeding and occlusion time in both sexes. These results are consistent with our previous studies of 3- and 6-month THS exposure and support the notion that 1 month is sufficient to produce the aforementioned prothrombotic phenotype.^10,11 In our analysis, the bleeding and occlusion times shortened in all exposed mice, both females and males. However, the shortening was significantly more pronounced in female mice. In this connection, several studies on sex differences indicated that females have higher platelet reactivity, which may have implications from an antithrombotic treatment outcomes point of view.^15,30 Besides our in vivo work, we also demonstrated the hyperactive platelet phenotype in vitro, with platelet aggregation, dense granules release, and P-selectin exposure being enhanced; albeit no differences were observed among the male and female mice. Nonetheless, platelet spreading, which supports the notion that outside-in signaling is potentiated, was found to be enhanced to a much higher extent in females. While one cannot completely exclude the contribution of differences in weight to the sex differences, the fact that the levels of cotinine were not found to be different seems to suggest otherwise; aside from the fact that both males and females were subjected to identical exposure conditions.

As to the mechanism underlying THS toxicity in the context of occlusive CVD, it remains poorly defined. Due to the exposure and nature of the chemicals in THS, and given the potential contribution of “cytokines” to the THS phenotype,^31–34 especially since some cytokines were shown to regulate platelet function and contribute to inflammation associated thrombosis,^16,35 we sought to determine if THS modulates their levels. Our results show that 1 month of THS exposure did lead to alterations in chemokines, and interleukins relative to CA, in the male and female mice. To this end, previous studies also show that THS exposure impacts lung cells, and causes increased expression of cytokines, a suggesting proinflammatory environment.²⁶ Moreover, there are several elevated chemokines and interleukins that are linked to myocardial infarction and ischemic conditions.³⁶ Therefore, the stratification of modulated cytokines due to THS needs to be well contemplated to address the mechanism of cytotoxicity. Of note, our results show that IL-2 is downregulated in males, while in female mice it was also downregulated but was not amongst the top 10%, possibly due to functional impairment of T cells, thereby enhancing the risk for infectious diseases and cancer. Nonetheless, the modulated chemokines and proinflammatory proteins due to THS exposure need further characterization and analysis to better appreciate their overall impact on human health.

Conclusions

This study provides evidence that even when the exposure to THS is relatively short, namely 1 month, it is still dangerous to human health and can increase the risk of occlusive CVD, especially in vulnerable populations. The fact that a relatively short exposure to THS can result in such a “severe” phenotype is of importance and of significant public health relevance, and further underscores the dangers associated with THS exposure. In addition, our data suggest that females are likely at a much higher risk than males if exposed to THS—which may manifest in more severe disease states—and this is related to changes in chemokine and cytokine levels. The notion that cytokines may contribute to the THS effects in the context of platelets has not been shown before. Nonetheless, additional research is needed to further explore the detailed mechanism of THS toxicity to inform preventative and therapeutic strategies. Together, our findings should also inform policy to regulate THS exposure, guide tobacco cessation efforts, and may serve as the foundation of educational efforts to inform the public of the adverse health effects of THS.

Funding

Research reported in this publication was supported by the National Institute of Environmental Health Sciences, the National Heart, Lung, And Blood Institute and the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Awards Number R01HL145053, R21ES029345, R03ES030486, R56HL158730, R21HD105187, and R21ES034512. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Declaration of Interests

The authors have no conflicts of interests to declare.

Author Contributions

Shahnaz Qadri (Data curation [Lead], Formal analysis [Lead], Methodology [Lead], Writing—original draft [Lead], Writing—review & editing [Lead]), Ana Carolina R. G. Maia (Investigation [Supporting], Methodology [Supporting], Writing—review & editing [Supporting]), Hamdy E. A. Ali (Data curation [Supporting], Formal analysis [Supporting], Methodology [Supporting], Writing—review & editing [Supporting]), Ahmed Alarabi (Data curation [Supporting], Investigation [Supporting], Methodology [Supporting]), Fatima Alshbool (Conceptualization [Supporting], Formal analysis [Supporting], Funding acquisition [Supporting], Writing—original draft [Supporting], Writing—review & editing [Supporting]), and Fadi Khasawneh (Conceptualization [Lead], Formal analysis [Supporting], Funding acquisition [Lead], Writing—original draft [Supporting], Writing—review & editing [Supporting]).

Data Availability

The data underlying the results described in this manuscript will be made available upon a reasonable request to the corresponding author.