Comparative cytotoxic and transcriptional effects of methanol, ethanol, and DMSO in the CMT-U27 canine mammary carcinoma cell line.

Organic solvents such as dimethyl sulfoxide (DMSO), ethanol, and methanol are widely used as vehicles in in vitro oncology research, yet their inherent toxicities can confound experimental outcomes. This study systematically evaluates the comparative cytotoxicity and transcriptional signatures of cell death pathways induced by these solvents in the canine mammary carcinoma cell line (CMTU27). Cells were treated with concentrations from 0.25% to 5% for 24, 48, and 72 h. Viability was assessed using the XTT assay, and cell death modes were characterized using Hoechst 33342/Annexin V/PI triple-fluorescence staining. Transcriptional alterations of apoptotic (CASP3, BAX, PUMA, ATM, BID), anti-apoptotic (BCL2, MCL1), and DNA damage/cell cycle (P53, P21, NOXA) genes were quantified via RT-qPCR. DMSO exhibited significant time- and dose-dependent cytotoxicity. No 50% viability concentration was reached after 24 h, whereas linear interpolation yielded approximate concentrations of 4.34% and 3.67% corresponding to 50% viability after 48 and 72 h, respectively. Fluorescent imaging revealed staining patterns consistent with apoptosis and secondary necrosis. Transcriptionally, DMSO markedly upregulated CASP3, BAX, PUMA, and ATM and downregulated MCL1, while BID and BCL2 showed concentration-dependent but not consistently significant changes. Conversely, ethanol and methanol maintained viability above 80% up to 5% (IC50 not calculated). PCA modeling revealed distinct spatial dissociation: ethanol predominantly induced genotoxic stress, strongly correlating with the P53, P21, and NOXA vectors, whereas methanol clustered closest to the control, despite showing significant gene-specific transcriptional alterations. In conclusion, our findings suggest that DMSO concentrations should be kept below 1% in CMT-U27 assays to minimize off-target apoptotic effects. Although ethanol showed limited cytotoxicity, it was associated with DNA damage-related transcriptional responses. Methanol exhibited the most favorable cytotoxicity profile among the tested solvents, although significant gene-specific transcriptional responses were observed, suggesting that its suitability as a vehicle solvent for CMT-U27-based in vitro studies should be evaluated according to the experimental endpoint.