Low-Dose Dose–Response for In Vitro Nrf2-ARE Activation in Human HepG2 Cells

Abstract

Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-like factor 2-related factor 2 (Nrf2), and the antioxidant response element (ARE) are interacting components of a master regulatory signaling pathway that coordinates redox homeostasis, cytoprotective responses, and shifts in stem cell state. This study reexamined detailed dose–response (DR) data reported for in vitro Nrf2-ARE activation in human hepatoblastoma HepG2 cell lines containing either a ARE-bla or ARE-luc reporter at 12 different concentrations of each of 15 chemicals. The normalized study data were combined among chemicals exhibiting a positive response, yielding n = 531 (179) DR data for 9 (7) chemicals using the ARE-bla (ARE-luc) assay. Three-parameter linear/kth-power regression fits obtained to each combined set of ARE-bla- or ARE-luc-assay response data provided good fits (R 2 = .99 or .91, respectively, P fit > .99) that each incorporate a highly significant negative initial linear slope (P = 4 × 10−5 or .00025) and an overall J-shaped DR pattern. Results from this reanalysis of high-resolution ARE response data support the hypothesis that nonlinear ARE-mediated adaptive cellular responses to oxidative stress are governed by an ultrasensitive molecular switch.

Linear‐No‐Threshold Default Assumptions are Unwarranted for Cytotoxic Endpoints Independently Triggered by Ultrasensitive Molecular Switches

“…). Similar results were obtained for combined ARE‐ luc assay data from the same study . As noted above, the observed J‐shaped response for chemical Nrf2‐ARE activation is consistent with predictions made for certain types of ultrasensitive molecular switches …”

“…These data are plotted in Fig. together with a three‐parameter linear/ k th‐degree polynomial I fit to the data by inverse‐variance‐weighted nonlinear regression assuming zero net change in activity (expressed in relation to activity measured in vehicle‐exposed cells and maximum activity measured in positive‐control cells exposed to β‐naphthoflavone) at a 0.1‐nM concentration of each chemical . The combined fit is consistent with the data ( R 2 = 0.987) and exhibits a highly significantly negative initial linear slope ( p = 0.000040, by two‐tail t ‐test) and overall J‐shaped dose–response pattern (Fig.…”

“…–5 of that study, kindly made available to me by Drs. Menghang Xia and Ruili Huang of the National Institutes of Health Chemical Genomics Center, I combined ARE‐ bla response data from that study over all chemicals that clearly exhibited a positive response at one or more concentrations, yielding a total of n = 531 combined data points for nine chemicals . These data are plotted in Fig.…”

Biphasic hCAR Inhibition-Activation by Two Aminoazo Liver Carcinogens

“…The two hCAR activators, 4-aminoazobenzene (AAB) and o-aminoazotoluene (OAT), were identified in this study to exhibit significantly biphasic patterns of hCAR inhibition-activation. The biphasic pattern observed for 4-aminoazobenzene is particularly noteworthy insofar as it was identified with statistical power that exceeds that of most if not all other biphasic receptormediated responses to any single-chemical exposure documented to date [39][40][41][42][43][44][45]. Although specific mechanisms underlying biphasic hCAR activation patterns identified here for two hCAR activators remain to be elucidated, their relatively low frequency (2, or ∼0.25%) among the 746 activation patterns examined in this study suggests that these biphasic patterns reflect an apparent CAR function to balance competing signals, that typically are integrated as different signaling chemicals bind competitively and/or otherwise interact at key regulatory domains within this receptor.…”

“…Such biphasic, apparently receptor-mediated dose-response patterns have been reported and reviewed [39][40][41][42][43]. Highly significantly biphasic dose-response patterns were shown recently to describe detailed sets of activation data for two highly conserved ultrasensitive molecular switches [44,45], nuclear factor erythroid 2-like factor 2 transcription factor (Nrf2), and heat shock protein 70 (HSP70), each of which also interact with CAR [46][47][48]. The present study examined detailed activation data for 853 hCAR agonists identified from a total of >9,000 chemicals screened using a quantitative high-throughput screening (qHTS) luciferase reporter assay-recently archived by the National Center for Biotechnology Information (NCBI) [49]to determine if responses exhibited by those identified CAR agonists include any clearly biphasic (inhibition-activation) patterns.…”