## 4 Discussion  
In this study, different substances were tested that are either known to be thiol-reactive (as is the case for thiosulfinates and isothiates) or known to induce the Nrf2 system without oxidizing or modifying the relevant cysteine residues. An overview of the chemical structures is given in Figure 4.  
Figure 3: Dynamic response of yeast cells containing the OSI1:luciferase reporter to (**A**) monomethylyl fumarate (MMF) (**B**) dimethyl fumarate (DMF). MMF and DMF were dissolved in DMSO. In the assay was 5% (_v/v_). \(n=3\). RLU is the abbreviation for Relative Luminescence Units. Figure 4: Overview of the chemical structure of the tested compounds. The compounds can be roughly divided into five chemical groups. Allicin belongs to the thiosulfinates, which are typical for representatives of the genus _Alliium_. The isothicyanates AITC, PETIC, and sulforaphane belong to the isothicyanates and are mainly found in the Brasiscaceae family. Methyl fumarates are esters of fumaratic acid and are synthetic in nature. All these compounds have a thiol reactivity in common. The kavalactones (methylsticn, yangonin, kavail) and the diterpene lactone andrographolide are derived from the plant _Piper methstticum_ G. Forst and activate the Keep/Nrf2 system in a non-oxidative way. Keap1-Nrf2/ARE-based assays represent an elegant and relatively easy-to-use approach to examine the oxidative potential of electrophilic substances on mammalian cells [16]. These assays are usually conducted with immortalized human or murine cell lines, thereby allowing high-throughput screening. However, these are cancer cell lines originating mostly from tumor biopsies. In the last few years, research has shown that, aside from its well-known antioxidant functions, Nrf2 has roles in the development and the progression of cancer. Furthermore, it has been shown that Nrf2 is also involved in many oxidative stress-independent intracellular processes, such as the cell cycle [40], metabolism [43; 44], and differentiation [45]. Tumor cells have been shown to highjack the Nrf2/ARE system to adjust cellular processes to their specific requirements [46]. Moreover, many human solid cancers, as well as commonly used cell lines, show gain-of-function mutations of Nrf2 or loss-of-function mutations of Keep1 [47; 48]. All these aspects make the use of cancer cell lines for Keep1-Nrf2/ARE-based assays at least questionable. For these reasons, we used freshly isolated primary cells in our study to better represent physiological conditions. In this case, the ARE-based assay requires the keeping and sacrifice of animals to obtain test cells, which are then transfected with ARE reporter construct before an assay. In the standard ARE protocol, from the onset of an assay with test substances, a six-hour incubation period is used and gives a point reading as output. Furthermore, the ARE procedure requires a prior assessment of the test substances for their effects on cell viability (e.g., CTB\({}^{\otimes}\)) to decide on a suitable working concentration. In contrast, an overnight culture of yeast cells, already stably transformed with the OSI1 reporter construct, provides the starting inoculum for the log-phase cells used for the OSI1 reporter assay. The OSI1 reporter assay gives a continual output after the addition of test substances over any chosen concentration- and time-range, allowing for kinetic observations. Furthermore, the plate reader allows parallel measurements of luminescence and cell growth kinetics. Concentrations of test substance which reduce yeast cell growth show a reduced luminescence response compared to lower concentrations of the test substance. Thus, dose-dependency can be seen up to concentrations that begin to inhibit growth. Furthermore, because cell culture itself is stressful, there is some induction of the ARE protection system leading to relatively high background luminescence in the controls.