## 3 Results  
### Comparison of the ARE Reporter System in Murine Hepatocytes and OSII in BY4742 Yeast Cells  
The four test substances, allicin, phenylethyl isothiamate (PEITC), allyl isothianate (AITC) and sulforaphane (SFN), were able to induce the luciferase reporter in both systems at low \(\upmu\)M concentrations (Figure 1). In the ARE assay, which was measured after 6 h of co-cultivation, the concentration-dependent increase of the luminescence signal compared to non-treated controls ranged from \(\sim\)2-fold (allicin) to \(\sim\)4-fold (PEITC, AITC, SFN). Allicin showed significant ARE activation at 10 \(\upmu\)M, PEITC at 2.5-5 \(\upmu\)M, AITC at 5-10 \(\upmu\)M, and SFN at 2.5-7.5 \(\upmu\)M, respectively (Figure 1A,C,E,G). In comparison, after 1 h of co-cultivation with the four test substances, the OSII reporter in yeast cells showed a 15-fold (sulforaphane) to \(>\)50-fold induction (allicin) (Figure 1 B,D,F,H). The ARE reporter was more sensitive to sulforaphane (2.5 vs. 10 \(\upmu\)M; Figure 1G,H) but the OSII reporter system was more sensitive to allicin (5 vs. 10 \(\upmu\)M; Figure 1A,B) and PEITC (0.5 vs. 2.5 \(\upmu\)M; Figure 1C,D). Figure 1: Responses of the antioxidant response element (ARE) and the OS-induced promoter of the YKL071w gene (OSI1) reporters to allicin, phenylethyl isothiamate (PEITC), allyl isothiamate (AITC), sulforaphene (SFN). (**A**,**C**,**E**,**G**) show the ARE assay with murine hepatocytes transfected with the SIN-lenti-ARE gene construct as reporter. Measurements were taken 6 h after treatment (\(n=8\)). (**B**,**D**,**F**,**H**) show the OSI1 assay with yeast cells transformed with the OSI1 promoter–luciferase gene construct as reporter (\(n=3\)). Measurements are shown after 1 h of co-cultivation with the respective test substance. Attention is drawn to the different \(y\)-axis scales of the ARE and OSI1 tests. Error bars show standard deviation. A normal distribution was assumed, and a one-way ANOVA was performed with [PERSON]’s multiple comparisons test for significance (\({}^{**}\)_p_ \(<\) 0.01). RLU is the abbreviation for Relative Luminescence Units. Routinely, in the ARE assay with murine hepatocytes, an \"end point\" measurement is made, in this case, after 6 h of co-cultivation of the cells with the test substance. However, for the OSI1 reporter, no pre-cultivation of the yeast cells with the test substance is necessary, as the measurements in the 96-well plates are dynamic from time zero and show the increase in luminescence over time (Figure 2), i.e., a kinetic is established rather than an end-point measurement. Thus, it can be seen that within the first 30 min of exposure, all the test substances activated the OSI1 reporter in the low \(\upmu\)M range. The OSI1 reporter was particularly responsive to PEITC (0.5 \(\upmu\)M) and allicin (1 \(\upmu\)M), while AITC and SFN showed clear responses in the 5-10 \(\upmu\)M range (Figure 2A-D). The data from the measurements at the 1 h time point were incorporated into Figure 1B,D,F,G to facilitate a more direct comparison with the ARE reporter system. With the exception of the thiosulfinate allicin, all of the substances tested in the previous section were isothi cyanates. We, therefore, screened the non-isothiocyanate \(\upalpha\)-\(\upbeta\)-unsaturated electrophilic substances monomethyl and dimethyl fumarate for activity with the \(OSII\):luciferase reporter. The response was measured over 6 h, and the dose-dependent kinetics of the \(OSII\):luciferase response is clearly visible (Figure 3). Interestingly, the OSI1 test system responded more rapidly to allicin than to mono- and dimethyl fumarate, and this emphasizes the advantage of the \(OSII\):luciferase reporter giving a kinetic response, compared to the ARE reporter which only gives a single time point reading. Kavalactones are weak antioxidants present in the rhizome and roots of the kava plant (_Piper methyticum_ [PERSON]. [PERSON]), which were demonstrated to activate Keap1-Nrf2/ARE by a non-oxidative route [41].