To the Editor,
1.
We commend Sato A and colleagues on their timely investigation into the effects of heated tobacco product (HTP) aerosol extract on senescence‐associated gene expression in human lung fibroblasts [1]. The study addresses a critical knowledge gap by comparing HTPs with conventional cigarettes, and the incorporation of both replicative senescence and doxorubicin‐induced senescence as positive controls adds considerable rigor [1]. The finding that repeated HTP exposure modulates CDKN2A and several matrix metalloproteinases (MMPs) is novel and thought‐provoking [1]. Nevertheless, we wish to raise a few points for consideration that may influence the interpretation of these results.
First, the selection of exposure doses warrants careful reflection. The authors set the HTP concentration 10‐fold higher than that of the reference cigarette (RC) based on the lactate dehydrogenase (LDH) release assay, which showed markedly lower cytotoxicity for HTPs [1]. While this is a logical starting point, it may inadvertently mask biologically relevant differences at equally non‐cytotoxic levels. Notably, the WST‐8 assay demonstrated that sub‐cytotoxic concentrations of both RC and HTP enhanced cellular metabolic activity (Figure 1C), indicating that subtle stress responses can occur far below the threshold for membrane damage [1]. Because the chemical profiles of the two aerosols differ substantially [2], choosing concentrations matched solely on cytotoxicity could obscure whether HTPs trigger pro‐senescence signals at potencies comparable to those of RC when neither product causes overt toxicity. We believe that a side‐by‐side comparison at multiple sub‐cytotoxic benchmarks would further strengthen the risk assessment.
Second, the patterns of aging hallmarks have raised questions about the phenotypic integrity we have observed. In the positive control models, the authors elegantly demonstrated that both replicative senescence and doxorubicin treatment significantly upregulate CDKN1A and IL6, two canonical hallmarks of senescence (Figure 2) [1]. This is consistent with prior work showing that CDKN1A is a reliable indicator of fibroblast senescence [3]. However, repeated exposure to either RC or HTP failed to alter the expression of CDKN1A or IL6 (Figure 3) [1]. Thus, the molecular signature induced by the tobacco products appears to be a partial, senescence‐like response rather than a fully established senescence program. We would therefore be cautious in equating these changes directly with cellular senescence, and feel that confirmatory studies at the protein level would be particularly valuable given the current exclusive reliance on mRNA data.
Third, the clinical interpretation of the MMP12 finding merits additional nuance. The authors appropriately note that MMP12 is strongly implicated in lung tissue destruction and emphysema [4], and they cite this association to suggest that repeated HTP exposure may contribute to long‐term fibrotic or emphysematous effects [1]. However, in the context of pulmonary emphysema, MMP12 is predominantly released by macrophages, not fibroblasts [4]. Although a murine model has shown that IQOS aerosol inhalation can produce apoptosis‐mediated emphysema [5], the present observation of MMP12 mRNA upregulation in fibroblasts cannot be directly translated into a risk for human emphysema without further validation of enzyme activity and tissue‐level remodeling. Clarifying how fibroblast‐derived MMP12 participates in matrix degradation, if at all, would greatly strengthen the connection between this in vitro result and clinical pathology.
In summary, the authors provide valuable evidence for the ongoing development of security assessment for HTPs. The questions we raise are intended to support constructive dialogue and help future experimental designs more clearly address these important issues.
Funding
The authors have nothing to report.
Disclosure
The authors have nothing to report.
Ethics Statement
The authors have nothing to report.
Data Availability Statement
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.
References
- 1. Sato A., Kasahara S., and Ishigami A., “Effects of Aerosol Extract From Heated Tobacco Products on Senescence‐Associated Gene Expression in Human Lung Fibroblasts,” Geriatrics & Gerontology International 26, no. 3 (2026): e70436, 10.1111/ggi.70436. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Hashizume T., Ishikawa S., Matsumura K., Ito S., and Fukushima T., “Chemical and in Vitro Toxicological Comparison of Emissions From a Heated Tobacco Product and the 1R6F Reference Cigarette,” Toxicology Reports 10 (2023): 281–292. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Fujita Y., Iketani M., Ito M., and Ohsawa I., “Temporal Changes in Mitochondrial Function and Reactive Oxygen Species Generation During the Development of Replicative Senescence in Human Fibroblasts,” Experimental Gerontology 165 (2022): 111866. [DOI] [PubMed] [Google Scholar]
- 4. Houghton A. M., “Matrix Metalloproteinases in Destructive Lung Disease,” Matrix Biology 44 (2015): 167–174. [DOI] [PubMed] [Google Scholar]
- 5. Arano‐Nitta N., Sato T., Komura M., et al., “Exposure to the Heated Tobacco Product IQOS Generates Apoptosis‐Mediated Pulmonary Emphysema in Murine Lungs,” American Journal of Physiology. Lung Cellular and Molecular Physiology 322 (2022): L699–L711. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.