In this study, we presented a low-cost, simple, and reproducible method to survey the potential health risks of HTPs and assessed the impact of long-term HTP exposure in an APPKI mouse model. We validated HTP exposure based on serum cotinine concentrations. In addition, the results of body weight after long-term exposure and gene expression of inflammatory, oxidative stress, and immune cell trafficking markers in the whole lung validate the HTP exposure model [8]. Although histological analysis indicated minimal effects of HTPs on AD pathology, the gene expression profile of the cerebral cortex tissues suggested that the effects of HTPs on the CNS were primarily caused by changes in non-inflammatory pathways.
To determine the health risks of long-term exposure to HTPs in humans, in vivo experiments focusing on multiple aspects are essential [4, 19]. In order to translate the findings from in vivo experiments to humans, a reproducible and reliable experimental mouse model is required [22, 23]. In the present study, we presented a novel experimental method for long-term exposure to HTPs in a mouse model. The materials required for our protocol, including a plastic syringe, three-way stopcock, silicone tube, and acrylic chamber, are not specialized and are easy to purchase at a low cost. During the dose determination period in this study, the serum cotinine levels after exposure for 30 min to the HTP aerosols generated from 4 puffs of one HTP were the same as those in a previous study on cigarette smoking [15]. In addition, serum cotinine levels were measured after long-term exposure to HTP aerosols for 16 weeks in this model. Because nicotine rapidly reaches the CNS through the blood-brain barrier, the cotinine concentrations of cerebrospinal fluid are also elevated after tobacco exposure [24–26]. Based on our results, researchers might consider this model as a smoking exposure mouse model for evaluating the effects of HTPs on the CNS [5, 8, 15].
The key difference between our approach and the one described by Sawa et al. lies in the direct exposure of mice to aerosols, as opposed to the utilization of a sampling bag in their methodology [5]. It should be noted that, due to the direct delivery of HTP aerosols into the examination chamber, a portion of the aerosols may undergo liquid transformation due to dew condensation. Consequently, the mice could potentially ingest HTP-infused droplets through preening of their entire body [27]. Conversely, the method utilizing a sampling bag under warm conditions serves to prevent aerosol condensation and precludes ambient exposure to HTP aerosols [5]. While we inferred systemic exposure of the mice to HTPs based on serum cotinine concentration, body weight, and gene expression in the entire lung, our methodology could be further enhanced as a physiologically comprehensive experimental framework by incorporating measures to impede liquefaction of HTP aerosols within the experimental chamber.
Several studies have suggested a relationship between lung stressors such as air pollution, silicosis, or the pulmonary microbiome and the CNS [14, 28, 29]. The lung-brain axis is at least partially mediated by inflammation and/or oxidative stress [14, 28]. Although microglial activation is one of the triggers of AD, the present study revealed no statistically significant changes in microglial activation in pathological analysis and no inflammation or oxidative stress based on the relative gene expression of brain tissue [30]. Thus, researchers should focus on the less surveyed non-inflammatory pathways in addition to the inflammatory pathways to determine the effect of HTPs on the lung-brain axis in AD.
Gene expression profiles showed that a few genes categorized into inflammatory pathways were upregulated in the present study; however, genes participating in neurohypophyseal hormone, neuropeptide hormone, and galanin receptor activity were relatively upregulated in the HTP exposure group. Some reports that support the reduced harm of using HTPs were based on findings of reductions in inflammation or oxidative stress indicators [31, 32]. However, previous reports have shown that the health risk of HTPs for pulmonary emphysema was not caused by inflammatory pathways but rather via apoptosis-related pathways [8]. In the present study, similar to some previous studies on cigarette smoking, HTPs were found to affect the activity of neuropeptide hormones such as arginine vasopressin, oxytocin, and galanin: a hormone that was noted to modulate mesolimbic dopaminergic neurotransmission and is associated with nicotine addiction [33–37]. Although Aβ-dependent pathways play a key role in AD pathogenesis, Aβ-independent pathways are also known to be important [12, 13]. The association between the endocrine system and AD pathogenesis has recently garnered attention [38, 39]. The findings collectively suggest that the health risks of HTPs via Aβ-independent pathways, such as those in the endocrine system, in AD pathogenesis are worth noting. Further studies are needed to determine whether these pathways critically drive the toxic effects of HTPs on the CNS, as our GO analyses were performed using DEGs detected at the thresholds of |log2FC| > 1 and non-adjusted p value < 0.05.
This study has several limitations that should be considered. First, we determined the exposure conditions based on serum cotinine concentrations, as reported in a previous study on cigarette smoking [15]. Although serum cotinine concentration is a standard marker of tobacco smoke exposure, we did not assess the amounts of chemicals in aerosols generated by HTPs in our protocol [5, 40, 41]. Furthermore, an epidemiological study revealed that people using HTPs as alternatives to CCs may engage in dual use and thereby increase tobacco consumption [40, 42]. Thus, researchers should not directly apply the experimental conditions for CCs to HTPs. Second, the gene expression profile of the cerebral cortex did not show any DEGs with adjusted p values < 0.05, but 282 DEGs were identified with thresholds of |log2FC| > 1 and a non-adjusted p value of < 0.05. Therefore, the possibility of false positives in these results should be considered [43]. Evaluating the health risks of new emerging products, such as HTPs, requires a comprehensive survey because adverse outcomes may result from multiple mechanisms that can have multiple components [19]. Several reports have shown that the aerosol content of HTPs contains small amounts of residual harmful constituents, such as 1,3-butadiene, benzene, and formaldehyde, and comparable or higher amounts of other toxicants, such as nicotine, acetol, glycerol, and propylene glycol, compared to CCs [44, 45]. Further studies are required to validate how these mechanisms lead to adverse effects in humans. Third, we did not validate the DEGs individually using different methods such as RT-qPCR or western blot analysis. Bulk tissue RNA-seq yields an average gene transcript abundance that reflects convoluted signals from several sources of variation. As these genes cooperate interactively rather than function individually, further studies should be performed to analyze gene signature patterns, such as cell-type-specific or pathway-type specific gene expression levels, to help identify new targets for novel and more effective therapies [46]. Finally, the APPKI mouse model used in this study was designed to exhibit typical Aβ pathology and neuroinflammation in an age-dependent manner; hence, our control mice also exhibited strong Aβ pathology and neuroinflammation [20]. Although a few genes associated with inflammation were upregulated in our HTP exposure model, the effect of HTP exposure could be underestimated compared with that of genetic hyperinflammation.