Water quality assessment using the AREc32 reporter gene assay indicative of the oxidative stress response pathway

Abstract

The reporter gene assay AREc32 is based on the induction of the Nrf2 mediated oxidative stress response pathway in the human breast cancer cell line MCF7, where eight copies of the antioxidant response element (ARE) are linked to a reporter gene encoding for luciferase. The Nrf2-ARE pathway is responsive to many chemicals that cause oxidative stress, among them a large number of pesticides and skin irritants. We adopted and validated the AREc32 bioassay for water quality testing. tert-Butylhydroquinone served as the positive control, phenol as the negative control and other reactive chemicals were assessed for their specificity. An environmentally relevant reference chemical, benzo(a)pyrene was the most potent inducer of all tested chemicals. The concentration causing an induction ratio (IR) of 1.5 (EC(IR1.5)) was chosen as the effect benchmark value. The assay was applied to 21 water samples ranging from sewage to drinking water, including secondary treatment and various tertiary treatment options (ozonation, biologically activated carbon filtration, membrane filtration, reverse osmosis, advanced oxidation, chlorination, chloramination). The samples were enriched by solid phase extraction. In most samples the oxidative stress response was far more sensitive than cytotoxicity. The primary and secondary treated effluent exceeded the effect threshold IR 1.5 at a relative enrichment factor (REF) of 1, i.e., the native samples were active. All tertiary treated samples were less potent and their EC(IR1.5) lay between REF 1 and 10. The Nrf2 pathway was induced at a REF of approximately 10 for surface waters and drinking water, and above this enrichment cytotoxicity took over in most samples and quenched the induction. The blank (ultrapure water run through the sample enrichment process) was cytotoxic at an REF of 100, which is the limit of concentrations range that can be evaluated. Treatment typically decreased both the cytotoxicity and oxidative stress response apart from drinking water treatment where chlorination caused an increase in oxidative stress response, presumably due to the formation of disinfection by-products. This study demonstrates the relevance and applicability of the oxidative stress response pathway for water quality monitoring.

In vitro bioassays to evaluate complex chemical mixtures in recycled water

“…The tBHQ-EQ of two effluents were 13 and 23 μg/L for GV Sec and RR Eff, respectively, which were in a similar range of the value reported in Australia (tBHQ-EQ 41-52 μg/L) for similar treatment processes (Escher et al, 2012). The Nrf2-Keap-ARE pathway plays a central role in the protection of cells against oxidative and xenobiotic damage and a very wide range of chemicals have been reported to induce this pathway.…”

“…For Caco-2 and SK-N-SH cytotoxicity test, the carrier solvents (MeOH for Caco-2 and DMSO for SK-N-SH) were used as the positive control. The cell viability test of Nrf2-MDA-MB (Nrf2-MTS) was conducted alongside the assay, and thus the same reference compound as the assay was used which was similar to a previous process (Escher et al, 2012). …”

Benchmarking Organic Micropollutants in Wastewater, Recycled Water and Drinking Water with In Vitro Bioassays

“…38,39 These assays were found to be unsuitable for samples with high organic matter content. 38 In the present study no effects could be detected although there appeared to be a qualitative difference in growth inhibition between the GSH-and the GSH+ strains.…”

Coupling passive sampling with in vitro bioassays and chemical analysis to understand combined effects of bioaccumulative chemicals in blood of marine turtles

“…To screen the passive sampling extracts of turtle blood samples, we selected the CAFLUX assay for arylhydrocarbon receptor (AhR)-mediated activity (Nagy et al, 2002;Jin et al, 2013), and the AREc32 assay for Nrf2-mediated oxidative stress response (Wang et al, 2006;Escher et al, 2012).…”

“…For oxidative stress response without an apparent limit until cytotoxicity occurs, EC IR1.5 that causes 1.5-fold induction relative to control signals (right above the lowest detectable response) was used as a benchmark metric (Escher et al, 2012).…”