# Biocompatibility of Membranes Based on a Mixture of Chitosan and _Lythri herba_ Aqueous Extract
## 2 Materials and Methods
### Biological Analysis  
#### 2.4.1 The Genotoxic Assay of the Aqueous Solution of Chitosan and _Lythri herba_ Extract  
To evaluate the genotoxicity, we used an SOS-Chromo assay kit developed by EBPI (Environmental Bio-Detection Products Inc., Mississauga, ON, Canada), solutions of chitosan, _Lythri herba_ plant extract (2 g/L, 1 g/L) and combinations of chitosan and extract of differing concentrations. It should be mentioned that only the high concentrations of the test solutions where an effect was assumed were used. The bacterium used in the SOS-Chromo test was a PQ37 mutant strain of _Escherichia coli_ developed as a genotoxicity test indicator organism and included a gene encoding the _\(\beta\)-galactosidase_ enzyme linked to an SOS promoter. When DNA damage occurred, the SOS system was activated, and _\(\beta\)-galactosidase_ was transcribed proportionally to the level of SOS induction. The production of _\(\beta\)-galactosidase_ was measured through its reaction with a blue chromogen as highlighted in the work protocol (SOS-ChromoTestTM kit, for rapid detection of genotoxicity or DNA damage, Product Number 5031).  
EBPI uses traditional metabolic bioactivation methods for indirect genotoxins by using S9 liver homogenate from Sprague Dawley rats induced by Aroclor 1254 in the presence of the necessary cofactors. Many carcinogens are known to require metabolic conversion to a reactive metabolite before interacting with DNA. Therefore, both direct and indirect genotoxins can be detected if S9 is included in the assay design.  
To carry out the quantitative evaluation, the following calculation formulas were used:  
* growth factor for bacteria by measuring alkaline phosphatase (G), \[\text{G}=\frac{A_{420S}-A_{420B}}{A_{420N}-A_{420B}}\] (2) A420S = sample absorbance, A420B = blank absorbance and A420N = negative control absorbance read at 420 nm.
* \(\beta\)-galactosidase activity (\(\beta\)-gal), \[\beta\text{-gal}=\frac{A_{600S}-A_{600B}}{A_{600N}-A_{600B}}\] (3) A600S = sample absorbance, A600B = blank absorbance and A600N = negative control absorbance read at 600 nm.
* Induction factor (IF),\[\text{IF}=\frac{\beta\text{-}gal}{G} \tag{4}\]  
\(\beta\text{-}gal=\beta\text{-}galactosidase\) activity, G = growth factor for bacteria by measuring alkaline phosphatase.  
* Survival rate (%), \[\text{Survival rate (\%)}=\frac{OD_{420}\text{ }sample}{OD_{420}\text{ }negative\text{ }control}\text{-}100\] (5)  
\(\text{OD}_{420,\text{ sample}}\) = sample absorbance, OD\({}_{420,\text{ control negative}}\) = negative control absorbance, read at 420 nm.  
The effect of _Lythri hera_ (Ly) concentrations tested between 19.23 \(\upmu\)g/mL and 153.85 \(\upmu\)g/mL were compared with those of 1% CS solution, as well as with genotoxic solutions of 2-AA (2-aminoanthracene) and 4-NQO (4-hydroxyquinoline 1-oxide). This last two compounds were recognized and validated as positive controls, mutagens (according to the kit procedure).  
The microplate with the samples to be analyzed was kept in the incubator at 37 \({}^{\circ}\)C for 2 h, during which the potentially genotoxic solutions interacted with the DNA of the SOS-Chromo assay bacteria and induced the de novo synthesis of \(\beta\text{-}galactosidase\). In the last step of the SOS-Chromo test, the relative amount of enzyme produced because of the induction of the SOS system is measured by adding a blue chromogenic substrate. The blue chromogen produces a blue color that allows for quantitative evaluation (spectrophotometry). Since the success of the \(\beta\text{-}galactosidase\) assay and production depend on bacterial viability during the assay, cell survival (ATP activity) was assessed using alkaline phosphatase. In this way, both the acute bacterial toxicity and the genotoxicity of the samples could be measured simultaneously. Absorbances at 600 nm to determine \(\beta\text{-}galactosidase\) production (genotoxicity) and at 420 nm to determine bacterial viability were read immediately.