# Has the Toxicity of Therapeutic Deep Eutectic Systems
## 2 Cell Lines
### Gene Expression  
Although the above findings classify THEDESs as generally safe, little is known about their interaction at the cellular and molecular level, and how they can affect DNA structure and gene expression [63].  
Considering the little evidence about genotoxicity, similar strategies to those carried out in the studies with NADESs could be followed. They must be focused on genes related to the mechanism of action of drugs whose solubility is intended to be increased.  
The evidence to date for the genotoxicity of DESs/NADESs is more extensive, having been tested in different cell lines and methods. Between them, gene expression or DNA damage are measured. [PERSON] et al. (2020) [64] carried out a comet assay, a single-cell gel electrophoresis for measuring DNA stand breaks, to assess the genotoxicity of tested NADESs applied for the extraction of two medicinal plants as follows: ChCl: glycerol/_Sideritis scardica_, ChCl: glycerol/_Plantago major_, Citric acid-1,2--propanediol 1:4 (CAPD)/_Sideritis scardica_ and CAPD/_Plantago major_. All tested NADESs were harmless for CCL1 cells, and the genotoxicity was concentration-dependent [64]. Ammonium-based DESs with glycerin, ethylene glycol, triethylene glycol, or urea were tested on MCF-7 cells to evaluate DNA integrity. The results obtained via DNA fragmentation analysis on agarose gel show that the cell deaths were not caused by DNA damage [42].  
Trans-resveratrol is a natural polyphenol widely used in cosme and the pharmaceutical industry, both in its topical and oral forms (e.g., antioxidants and hypolipidemic supplements). When exposed to UV light, it is isomerized to trans-1,4,6-trihydroxyphenanthrene (THP), with genotoxic effects. Proline/glycerol NADES showed a photo-protective effect under UV exposure, inhibiting the isomerization of resveratrol and THP formation [65]. Moreover, another study evaluated resveratrol, improving its bioavailability via lipid conjugation. Endothelial permeability protection was evaluated by MMP-9 inhibition, which plays an important role in the inflammation and healing processes. Resveratrol-linoleic acid dissolved in NADESs, composed by 1,2-propanediol:ChCl:water, showed the highest MMP-9 inhibitory effect in THP-1 tested cells [66].  
A study carried out by [PERSON] et al. (2019) [67] tested the protective effect to hyperosmotic stress produced by sorbitol addition of glycerol and xylitol in keratinocyte HaCaT cells. The inflammatory cytokines' expression was measured to evaluate stress cell levels. The results show that both polyols decreased IL-1\(\alpha\) expression, while only glycerol affects _IL-1\(\beta\)_ and _NFAT5_ expression, despite having similar chemical structures.  
[PERSON] et al. (2022) [68] tested a fermented NADES finger extract on HTC-116/R cells (colorectal cancer model). The NADES ginger extract has a higher phenolic content and antioxidant activity than organic solvent extract. The NADESs were composed of a mixture of betaine/lactate/water. The fermented NADES extract were shown to inhibit the expression of _NF-kB_ (via activity assay) and _CXC chemokine receptor 4_ (_CXCR4_) (using Real time PCR) genes, enhancing the therapeutic effects in oxipalatin-resistant CRC cells.  
Likewise, similar studies, such as [PERSON] et al. (2019) [69], were carried out in ionic liquids, demonstrating their influence on the expression of _p53_ and _Bel-2_ family genes, and showing NADESs' cytotoxic and apoptotic effect on hepatocellular carcinoma (HepG2) cells treated with f1-dodecyl-3-methylimidazolium chloride.  
To the best of our knowledge, the only study really considered as a THEDES formed by betaine-urea DESs containing imipenem was tested in monocyte cells, showing that this formulation increases therapeutic efficacy. The immunogenic response of the THP-1 cells was measured by the proinflammatory cytokines gene expressions (_IL-1_, _IL-6_, and _TNF-\(\alpha\)_). This THEDES showed no immunogenic effect, suggesting its potential as a formulation [56].