## 4 Discussion
Among the minor components of our CSC samples, \(\alpha\)-myrcene is a monoterpene commonly occurring in nature, especially in hops and cannabis [65], and formed during burning of the tobacco [66]; it is endowed with antioxidant and anti-inflammatory properties, and some studies reported lacking genotoxic effects and genoprotection against damage by tert-butyl hydroperoxide [67; 68; 69]. In one study, the genotoxic effects of myrcene in HepG2/C3A cells after metabolic activation were reported too [70]. Moreover, urs-12-en-28-ol is a pentacyclic triterpene, occurring in different plants and found to possess anti-inflammatory properties [71]. The other identified compounds (i.e., triacetin, 7-methyl-6-tridecene, (Z)-2,6,10-trimethyl-1,5,9-undecatriene and 6,11-dimethyl-2,6,10-dodecatrien-1-ol) are commonly reported as smoke additives or as metabolites produced during tobacco pyrolysis [62]. On the basis of the available evidence and considering the identified compounds, nicotine seems to potentially contribute to the genotoxicity and oxidative stress by CSC, although the involvement of other unidentified toxicants cannot be excluded. It is important to underline that CSC also contains some other compounds, such as myrcene and urs-12-en-28-ol, endowed with genoprotective and anti-inflammatory properties, which, however, fail to counteract the CSC damage in MDA-MB-468 cells. In regard to the specific mechanisms accounting for the CSC injury in triple-negative breast cancer MDA-MB-468 cells, we found that cell death mainly resulted from mitochondrial damage, as highlighted by the MTT assay, and from cell membrane disruption, which led to LDH enzyme leakage: this last effect usually correlates with necrotic cell death [47]. As expected, the cytotoxic effects of CSC were associated with an increased oxidative stress, glutathione depletion and enhanced genome instability, as shown by the \(\gamma\)H2 AX levels. In particular, genotoxicity can be due to the increased oxidative stress, although other direct genotoxic mechanisms, ascribable to the toxicants contained in the smoke mixture, cannot be excluded. According to our results, DNA damage by cigarette smoke has been reported in different in vitro models, such as human cervical cancer cells and embryonic stem cells [72; 73; 74]; [PERSON] et al. [72] showed that smoke condensate induced \(\gamma\)H2 AX in A549 human pulmonary adenocarcinoma cells and in normal human bronchial epithelial (NHBE) cells. Along with the genotoxic effects, our results highlighted that CSC markedly increased the cell accumulation in the G1 phase, despite lowering the S and G2/M phases with respect to the control. In line with our hypothesis, [PERSON] et al. [75] reported that cigarette smoke extract (CSE) increased the number of cells in the G1 phase and lowered the G2 one in a rat alveolar epithelial cell line; moreover, the G2 phase was also reduced by cigarette smoke in oral cancer cells [76]. These findings suggest an improved proliferative profile of CSC-treated MDA-MB-468 cells which can be reflected in the enhanced recovery abilities. In addition, the lowering of the S and G2/M phases suggests that CSC can impair the cell cycle checkpoints, thus allowing the cells to escape any control and proliferate in the presence of unrepaired DNA damages. Therefore, the induction of DNA damage by CSC along with the alteration in the cell cycle phases, probably consequent upon defective checkpoints, can be considered as events triggering of hyperproliferation and invasion of the MDA-MB-468 cells, and then chemoresistance. When CSC was assessed in combination with \(\beta\)-caryophyllene, cytotoxicity rose, probably as a consequence of the genotoxic damage and oxidative stress; however, this effect seems to be mainly ascribable to a mitochondrial toxicity rather than necrosis, since the LDH release was not altered with respect to CSC.