## 3 Results
### Volatile Chemical Profile of Cigarette Smoke Condensate (CSC)
Data are displayed as mean \(\pm\) SE of at least two experiments with at least three technical replicates (\(n=6\)). *** \(p<0.001\), significant lowering of cell viability the respect to the control (ANOVA + [PERSON]’s multiple comparison post-hoc test). \({}^{\circ}\)\(p<0.05\) and \({}^{\circ\circ\circ}\)\(p<0.001\), significant difference the respect to CSC (Student’s \(t\)-test). Effects of \(\beta\)-Caryophyllene and Cigarette Smoke Condensate (CSC) on H2 AX Histone Phosphorylation and Cell Cycle Modulation  
Under our experimental conditions, we also evaluated the genomic stability of triple-negative breast cancer MDA-MB-468 cells, which represents a key feature of the different cancer cells, by measuring the levels of phosphorylated H2 AX histone (\(\gamma\)H2 AX). Moreover, considering that genomic instability and altered cell cycle are often responsible for deregulated cancer cell proliferation and invasion, we also assessed the modulation of cell cycle phases by \(\beta\)-caryophyllene and CSC, as a possible mechanism accounting for the chemopre properties of \(\beta\)-caryophyllene towards cigarette smoke. According to the  
Figure 8: Bar graphs representing the levels of GSH (reduced glutathione) and GSSG (oxidized glutathione) induced by \(\beta\)-caryophyllene and cigarette smoke condensate (CSC) in MDA-MB-468 cancer cells after 24 h exposure as revealed by HPLC analysis. Data are displayed as mean \(\pm\) SE of at least three independent experiments with at least two technical replicates (\(n=6\)). *** \(p<0.001\), significant lowering of cell viability with respect to the control (ANOVA + [PERSON]'s multiple comparison post-hoc test). *** \(p<0.001\), significant difference with respect to CSC (Student’s \(t\)-test). Figure 7: Intracellular ROS levels induced by \(\beta\)-caryophyllene and cigarette smoke condensate (CSC) in MDA-MB-468 cancer cells after 24 h exposure. (**A**) Representative images of cells stained by DCF. Original magnification 10\(\times\). (**B**) Bar graph of DCF-fluorescence. Data are displayed as mean \(\pm\) SE of at least three independent experiments with at least three technical replicates (\(n=9\)). *** \(p<0.001\), significant lowering of cell viability with respect to the control (ANOVA + [PERSON]’s multiple comparison post-hoc test). *** \(p<0.001\), significant difference with respect to CSC (Student’s \(t\)-test). Scale bars = 20 μm. enhanced oxidative stress, the levels of \(\gamma\)H2 AX were found increased by CSC by at least 30%, without alterations due to the presence of \(\beta\)-caryophyllene (Figure 9); a similar trend was also found under pre-treatment and post-treatment exposures (Figure S2). At imaging analysis, the increase in the \(\gamma\)H2 AX levels were highlighted by the red fluorescence around the cells (Figure 9A). The lacking genoprotective effects of \(\beta\)-caryophyllene towards CSC in MDA-MB-468 cells could be ascribed to the inefficacy of DNA repair systems in restoring DNA integrity, leading to damage accumulation and cell-death activation. In line with this evidence, we also evaluated the role of \(\beta\)-caryophyllene in the control of cell cycle progression of the CSC-damaged MDA-MB-468 cells, which could promote the activation of cell death signaling in the presence of unsolved DNA damage. As displayed by both the histograms and graph bars (Figure 10A,B), \(\beta\)-caryophyllene produced almost a 40% reduction in the S cell cycle phase along with a slight but significant lowering of the G1 phase with respect to the control; moreover, it enhanced the G2/M phase by about 67% with respect to the control (Figure 10A,B). CSC stimulated the cell accumulation in the G1 phase, with about a 63% increase with respect to the control, while the S and G2/M phases were almost 14% and 60% lower than the control. This suggests higher proliferative abilities of the CSC-stimulated cells and a reduced control in the G2/M phase which allow damaged cells to escape.