## 3 Results  
### Volatile Chemical Profile of Cigarette Smoke Condensate (CSC)  
SPME-GC/MS analysis allowed for the revelation of the presence of eight volatile compounds in the CSC sample, as shown in Table 2; among them, neophytadiene (56.4%) was the most abundant, followed by nicotine (26.7%) and 7-methyl-6-tridecene (6.9%); other minor compounds, including \(\alpha\)-myrcene, triacetin, (Z)-2,6,10-trimethyl-1,5,9-undecatriene, 6,11-dimethyl-2,6,10-dodecatrien-1-ol and urs-12-en-28-ol, were found with relative percentages from 0.8% to 3.1%. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Gene** & **Brand** & & **Primer (\(\mathbf{5^{\prime}\to 3^{\prime}}\))** & **Annealing (\(\mathbf{{}^{\circ}}\)C)** & **Efficiency (\%)** & \(\mathbf{R^{2}}\) \\ \hline \multirow{2}{*}{BIRC-5} & \multirow{2}{*}{Bio-Rad} & Forward & \multirow{2}{*}{N/A (Cod. qHsaCED0001615)} & \multirow{2}{*}{60} & \multirow{2}{*}{97.0} & \multirow{2}{*}{0.999} \\ \cline{3-3} \cline{5-6}  & & Reverse & & & \\ \hline \multirow{2}{*}{MMP2} & \multirow{2}{*}{Bio-Rad} & Forward & \multirow{2}{*}{N/A (Cod. qHsaCED0042560)} & \multirow{2}{*}{60} & \multirow{2}{*}{99.0} & \multirow{2}{*}{0.999} \\ \cline{3-3} \cline{5-6}  & & Reverse & & & \\ \hline \multirow{2}{*}{TPX2} & \multirow{2}{*}{Bio-Rad} & Forward & \multirow{2}{*}{N/A (Cod. qHsaCID0016024)} & \multirow{2}{*}{60} & \multirow{2}{*}{98.0} & \multirow{2}{*}{0.997} \\ \cline{3-3} \cline{5-6}  & & Reverse & & & \\ \hline \multirow{2}{*}{IL-8} & \multirow{2}{*}{Bio-Rad} & Forward & \multirow{2}{*}{N/A (Cod. qHsaCED0023767)} & \multirow{2}{*}{60} & \multirow{2}{*}{97.0} & \multirow{2}{*}{0.999} \\ \cline{3-3} \cline{5-6}  & & Reverse & & & \\ \hline \multirow{2}{*}{GAPDH} & \multirow{2}{*}{Bio-Rad} & Forward & \multirow{2}{*}{N/A (Cod. qHsaCED0038674)} & \multirow{2}{*}{60} & \multirow{2}{*}{97.0} & \multirow{2}{*}{0.999} \\ \cline{3-3} \cline{5-6}  & & Reverse & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: List of oligonucleotides, general conditions, and validation parameters used to perform RT-qPCR. Effects of \(\beta\)-Caryophyllene and Cigarette Smoke Condensate (CSC) on Viability and Recovery of Triple Negative Breast Cancer MDA-MB-468 Cells  
Preliminary cytotoxicity assays were performed to evaluate the effects of \(\beta\)-caryophyllene and CSC in triple-negative breast cancer MDA-MB-468 cells after 24 h exposure and to select the nontoxic concentrations to be assessed for the chemopre studies. According to our previous study [30], \(\beta\)-caryophyllene was cytotoxic starting from the concentration of 20 \(\mu\)g/mL (about 60% inhibition of cell viability), with early toxicity signs at 15 \(\mu\)g/mL (about 30% inhibition of cell viability) (Figure 4A). Similarly, CSC produced concentration-dependent cytotoxic effects, with early signs (about 30% inhibition of cell viability) at the concentration of 100 \(\mu\)g/mL (Figure 4B). In terms of potency, the IC\({}_{50}\) values showed that \(\beta\)-caryophyllene was about seven-fold more toxic than CSC (Table 3); as expected, the positive control doxorubicin was almost four-fold more cytotoxic than \(\beta\)-caryophyllene under the same experimental conditions. \begin{table}
\begin{tabular}{c c c c} \hline
**No.** & **Component 1** & **LRI 2** & **LRI 3** & **Smoke (\%) 4** \\ \hline
1 & \(\alpha\)-Myrcene & 979 & 981 & 0.8 \\
2 & Triacetin & 1340 & 1344 & 1.2 \\
3 & Nicotine & 1366 & 1360 & 26.7 \\
4 & (Z)-2,6,10-Trimethyl-1,5,9-undecatriene & 1410 & * & 2.4 \\
5 & 7-Methyl-6-tridecene & 1455 & * & 6.9 \\
6 & 6,11-Dimethyl-2,6,10-dodecatrien-1-ol & 1665 & 1658 & 3.1 \\
7 & Neophytadiene & 1912 & 1915 & 56.4 \\
8 & Urs-12-en-28-ol & 3385 & 3376 & 2.6 \\  & SUM (\%) & & 99.9 \\ \hline \end{tabular}  
* The components are reported according to their elution order on a polar column; 2 Linear Retention index (LRI) measured on a polar column; 3 LRI from literature; 4 Percentage mean values of CSC components; *: LRI not available.  
\end{table}