Page 320 - 2022_01-Haematologica-web

Letters to the Editor
In utero thirdhand smoke exposure modulates platelet function in a sex-dependent manner
Thirdhand smoke (THS), the persistent residue of tobacco smoke that remains after a cigarette is extin- guished, materialized as a threat for human health over the last decade. These toxic residues end up depositing on surfaces and objects where tobacco has been used (e.g., homes) and persist for weeks/months after the last smoking.1 THS toxicants undergo chemical reactions and changes over time potentially making them more toxic.2 Given that the routes of exposure to THS involve skin absorption, inhalation and ingestion,3 it is thought to be more toxic by producing more toxicants in the blood of the exposed person.4 Indeed, there is a growing body of evidence documenting THS-induced health risks,5 including cardiovascular disease (CVD). For exam- ple, we previously showed that THS exposure modu- lates platelet function and enhances thrombogenesis in adult exposed mice.6 However, it has not yet been estab- lished whether prenatal/in utero THS exposure impacts platelet function and related disorders, which is para- mount since the developing embryo is especially sensi- tive to environmental toxicants, including cigarette smoke.7 Therefore, this study was designed to address this issue, utilizing the offspring of exposed females. In addition, we also examined whether sex differences exist in THS-induced effects.
We employed our innovative THS exposure approach which has been peer-reviewed4,6 and accepted as one that provides exposure conditions that mimic those in multiple real-life human situations.4 According to our experimental design, the female breeders were exposed to THS smoke or clean air starting 1 week before mating and throughout the whole pregnancy period by placing them in cages that are furnished with either THS or clean-air exposed materials. After delivery (post-natal day 4), the offspring were moved to clean air cages and housed until 8-10 weeks of age, before experimentation. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee.
We first sought to investigate the in vivo effect of in
utero THS exposure on hemostasis and thrombosis. Thus, the tail bleeding time assay revealed that the THS- exposed males and females exhibit substantially short- ened bleeding time compared to clean air exposed con- trols (Figure 1A). In fact, the average bleeding time in males was 395 ± 65.14 seconds (sec) in clean air group versus 68.80 ± 14.87 sec in the THS group; whereas in females it was 449.40 ± 45.07 seconds and 44.50 ± 12.56 sec for clean air and THS groups, respectively. As the time needed for cessation of bleeding was significantly reduced in the in utero THS–exposed mice, we therefore hypothesized that these mice are more vulnerable to thrombosis. This was tested by employing the FeCl3 carotid artery injury–induced thrombosis model. As depicted in Figure 1B, in utero THS mice of both sexes displayed a significant reduction in the occlusion time, with the average in males being 1,080 ± 60.00 sec in clean air group versus 210.80 ± 79.37 sec in THS group; whereas the females recorded 1,150 ± 114.30 sec and 281 ± 116.50 sec for the control and experimental groups, respectively. Taken together, these results show that in utero THS exposure enhances hemostasis and ren- ders mice at higher risk of developing thrombosis. However, when males are compared with females, the results did not show any sex-based differences for either hemostasis or thrombus formation.
Notably, the platelet and other blood cells count was measured in both in utero THS- and clean air-exposed mice, as changes in platelet number may contribute to the hemostasis and thrombosis phenotype observed. The in utero THS exposure did not affect the platelet count or other hematological parameters (Table 1).
In light of the bleeding time and thrombosis data, another set of experiments evaluated the manifestation of the potential prothrombotic phonotype at the level of platelet physiology by studying platelet functional parameters in vitro. Hence, we first determined the effect of in utero THS exposure on agonist-induced platelet aggregation, which was found (Figure 2A) to be substan- tially increased, in response to either thrombin (0.1 U/mL) or ADP (1 μmol/L) in male and female mice exposed to THS in utero. However, when comparing platelet aggregation of both sexes, our analysis did not
AB
Figure 1. In utero thirdhand smoke exposure shortens the bleeding time in the tail bleeding time assay, and the time to occlusion in the ferric chloride in vivo thrombosis model both in males and females. (A) Tail bleeding time assay in the in utero thirdhand smoke (THS)- and clean air (CA)–exposed mice compared in males and females. Each point represents the tail bleeding time of a single animal. (B) Ferric chloride–induced thrombosis model (time to occlusion) in the in utero THS- and clean air–exposed compared in males and female mice. Each point represents the occlusion time of a single animal. *Male and female data compared using two-way ANOVA while THS vs. CA comparison within the same sex was done using Student’s t-test.
312
haematologica | 2022; 107(1)