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Research Article
Nuclear Receptor Research
Vol. 5 (2018), Article ID 101321, 12 pages
doi:10.11131/2018/101321

Biphasic hCAR Inhibition-Activation by Two Aminoazo Liver Carcinogens

Kenneth T. Bogen

Exponent Inc., Health Sciences, Oakland, CA 94612, USA

Received 29 September 2017; Accepted 21 February 2018

Editor: William Baldwin

Copyright © 2018 Kenneth T. Bogen. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Detailed dose-response data recently archived by the National Center for Biotechnology Information (NCBI) identified 853 human CAR (hCAR) agonists by quantitative high-throughput screening (qHTS) assays applied to >9,000 chemicals tested at ≥14 concentrations using n = 3–48 replicates. By re-examining NCBI data on 746 agonists with replicate data sets each satisfying additional quality criteria, ∼95% had average values of agonist-specific Hill-model slopes estimated by NCBI that exceed 1 (i.e., exhibited an overall sublinear low-dose dose-response), and two unambiguously biphasic hCAR inhibitor-agonists were identified, 4-aminoazobenzene (n = 37) and ortho-aminoazotoluene (n = 3), both of which also cause rodent liver tumors. Although evidently rare among hCAR agonists, such biphasic responses add to evidence that nuclear receptors can exhibit complex patterns of low-dose response, consistent with previous observations and theoretical predictions for endpoints governed by ultrasensitive molecular switches. The pronounced biphasic hCAR response 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 receptor-mediated biphasic cellular responses to any single-chemical exposure reported to date.

1. Introduction

The constitutive androstane receptor (CAR, NR1I3) is a moderately promiscuous nuclear receptor and xenosensor expressed primarily in hepatocytes. Normally phosphorylated and complexed with heat shock protein 90 (HSP90) and cytosolic CAR retention protein (CCRP) in cytosol, CAR can become activated, e.g., in mice by binding to a ligand, such as 1,4-bis[2-(3,5- dichloropyridyloxy)]benzene (TCPOBOP), or in mice or humans by being dephosphorylated via phenobarbital-mediated recruitment of protein phosphatase 2 (PP2A), after which CAR translocates to the nucleus where it heterodimerizes with nuclear receptor RXR and then interacts with promoter complexes of target genes that regulate many physiological processes including lipid metabolism, glucose metabolism, hormonal regulation, cell growth, wound healing, and apoptosis [1,2,3,4,5,6,7]. CAR is thought to promote liver tumors in some rodents by stimulating downstream (e.g., CYP2b, Wisp, FoxM1, cMyc) receptors, multidrug transporters and resistance genes, and related epigenetic modifications (e.g., regions of altered DNA methylation) and microRNA dysregulation (e.g., miR-182 and miR-802 upregulation and miR-122 downregulation) that facilitate hepatocellular proliferation and associated shifts in energy and growth-directed metabolism [2,5,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22], particularly when coupled with β-catenin activation [23].

Mouse CAR (mCAR) is activated by nongenotoxic mouse liver tumor promoters such as phenobarbital and phenytoin via indirect activation, and via direct CAR or CAR-coactivator binding by TCPOBOP, di(2-ethylhexyl)phthalate (DEHP), and statins [3,5]. Activation of human CAR (hCAR), e.g., by phenobarbital, chlordane, or 6-(4-chlorophenyl)imidazo-[2,1-b,1,3]-thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime (CITCO), triggers patterns of downstream effects that overlap in many ways but differ in some respects (e.g., by excluding cell proliferation-specific and enhancing apoptotic gene activity), from those elicited by mCAR [2,3,24,25,26,27,28]. CAR is also activated by phthalate reproductive toxicants [29,30,31]. In contrast, CAR antagonists or inverse agonists reported to attenuate and/or inhibit basal levels of CAR activity include: the mCAR inhibitors endogenous androstanol and the pheromone androstenol; the peripheral benzodiazepine receptor ligand PK11195, which activates human CAR (hCAR) in human cell lines; the potent agonist of liver X receptor (LXR) and the human pregnane X receptor (hPXR) TO901317, which inhibits human, mouse, and rat CAR; the soybean and legume phytoestrogen coumestrol, which inhibits hCAR; the antifungal agent ketoconazole, which inhibits hCAR and mCAR; and the hCAR-inhibiting type II diabetes drug metformin [32].

Mechanistic and statistical models describe possible J- or U-shaped, biphasic patterns of dose-response, such as those governed by ultrasensitive molecular switches involving nuclear receptors [33,34,35,36,37,38]. 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.

2. Materials and Methods

2.1. Data on hCAR activation in human HepG2 cells

hCAR Activation in Human HepG2 Cells. Detailed, qHTS concentration-response data for in vitro hCAR activation in a line of human liver HepG2 cells containing a double-stable CYP2B6-driven luciferase reporter were recently archived by NCBI [49]. This HepG2-CYP2B6-hCAR cell line was constructed as previously described in detail [50,51] to express both the full-length hCAR protein, and a luciferase signal driven by the promoter of the prototypical hCAR-target CYP2B6, by co-transfecting the pEF6/V5-hCAR expression plasmid and a pGL4.17[luc2/Neo]-CYP2B6-2.2kb construct (containing phenobarbital- responsive and xenobiotic-responsive enhancer modules, PBREM and XREM) into HepG2 cells, followed by continual culture in selective media and then by selection of a single colony verified to contain both plasmids. The fact that constitutive activation in immortalized cells is a hallmark of CAR makes the identification of CAR activators extremely challenging—a drawback addressed in applications of HepG2-CYP2B6-hCAR cells to quantify CAR activation by using PK11195 (a commonly used ligand of the peripheral benzodiazepine receptor) as a potent and selective deactivator of hCAR; PK11195 competes directly with CITCO (by disrupting recruitment of co-activators such as steroid receptor coactivator-1 and glucocorticoid receptor interacting protein-1 to hCAR) and thus can effectively lower the high basal hCAR activity observed in HepG2-CYP2B6-hCAR cells [50]. Thus, as previously described [27,50,51,52,53], results for each tested chemical list reporter-signal intensities measured after 24 hours of exposure to each of 13 or 14 log-spaced chemical concentrations delivered in a dimethyl sulfoxide (DMSO) vehicle to which was added hCAR antagonist PK11195 (at 2.5 µM/well) to repress otherwise relatively high hCAR basal activity in HepG2 cells, using 3–48 replicates per concentration depending on the chemical tested. Raw well-specific plate reads were each normalized to a percent-activity scale in which 100% represents the maximal activity measured using the positive-control hCAR agonist CITCO, and 0% represents control activity measured in DMSO/PK11195-only wells placed in the first four columns of each 1,536-well plate. Each replicate set of agonist-specific NCBI hCAR activation data includes, among other reported information: (i) an estimated corresponding slope (n Hill ) from a Hill model fit to that data set, (ii) a corresponding value of R2 (the fraction of total response variance explained by the NCBI-fitted Hill model), and (iii) a model-fit Descriptor. The latter Descriptor was “Single” if a tested chemical was characterized as an “Activator” (i.e., agonist) based on substantially elevated activity observed only at a single test concentration.

2.2. hCAR activation data analysis and modeling

Each set of replicate NCBI activation data for each of its 853 identified hCAR agonists was first screened first by applying all of three inclusion criteria (agonist classification = Activator, Hill model fit R2> 0.9, and Descriptor ≠ Single), and then by applying a fourth criterion that at least three agonist-specific replicates remained after applying the first three criteria (i.e., n≥ 3) for each agonist. Replicate data sets for a total of 746 agonists satisfied these selection criteria, or ∼87.5% of the 853 hCAR agonists identified by NCBI. For all 746 of these agonists, NCBI-estimated n Hill values were characterized as a cumulative likelihood distribution to assess the overall magnitude of apparent nonlinearity in activation response. Log(concentration)-activation patterns for each of the 746 agonists were then plotted and visually screened for apparent patterns of biphasic inhibition-activation response. Apparent biphasic candidates were fit to a five-parameter mixed-lognormal response model by inverse-variance-weighted nonlinear least-squares regression, implemented using Mathematica 11.1Ⓡ software [54]. Goodness of fit was then characterized by R2 and a p-value (p fit ) from a corresponding chi-square goodness-of-fit test with degrees of freedom (df) equal to the number of data points fit minus the number of fitted parameters. Negativity of initial slope-parameter estimates was in each case assessed by corresponding t-test [54].

3. Results

Of the 853 hCAR agonists identified by NCBI [49], 746 were determined to meet the additional criteria for unambiguous dose-response characterization described in Materials and Methods. The cumulative distribution of n Hill slope values estimated by NCBI [49] for this set of 746 agonists was determined to be approximately lognormal, with a geometric mean (GM) and geometric standard deviation (GSD) of approximately 1.8 and 1.4, respectively (Figure 1). These n Hill estimates have an arithmetic mean (±1 standard deviation) value of 1.86 ± 0.61, and a large majority (∼95%) of them exceed 1, indicating a general sublinear pattern of activation dose-response. A detailed examination of agonist-specific dose-response patterns for these 746 agonists indicated two clearly biphasic patterns, those for 4-aminoazobenzene (para-aminoazobenzene, Aniline Yellow, C.I. Solvent Yellow, CAS RN 60-09-3, PubChem Chemical Identifier [CID] 6051) (Figure 1) and for ortho-aminoazotoluene (o-aminoazotoluene, Solvent Yellow 3, CAS RN 97-56-3, CID 7340) (Figure 2). Fits obtained to hCAR-activation data sets for these two agonists are discussed below.

F1
Figure 1: Cumulative distribution (solid curve) of NCBI-estimated Hill slope coefficient (n Hill ) values for those 746 hCAR activators, among 853 designated by NCBI [49] as hCAR activators in HepG2 cells based on that analysis of in vitro luciferase-reporter assay results for >9,000 tested chemicals, which met additional activation criteria described in Materials and Methods. The parameters (±1 standard error) of the corresponding estimated lognormal distribution (dashed curve) are GM = 1.79 ± 0.00095 and GSD = 1.37 ± 0.0013, where GM and GSD denote geometric mean and geometric standard deviation, respectively.

The following 5-parameter biphasic inhibition-activation model was fit to hCAR activation data in relation to 4-aminoazobenzene concentration C, as shown in Figure 1:

% Activity = 𝑎 𝑏 Φ [ ln ( 𝐶 / GM ) / ln ( GSD ) ] + 𝑐 Φ [ ln ( 𝐶 / 100 ) / ln ( GSD ) ] (1)

in which Φ denotes the cumulative standard normal distribution function. This data set includes n = 37 replicates reported at each of 13 concentrations. Parameter estimates ±1 standard error (SE) obtained for this fit and associated fit statistics are: a = – (0.723 ± 0.142)%, b = (2.93 ± 0.376)%, GM = (1.67 ± 0.0594)µM, GSD = 4.09 ± 0.266, and c = (77.6 ± 3.25)% (p fit = 0.45, R2 = 0.996). The estimated Y-intercept (a), inhibition slope (b), and activation slope (c) each differ significantly from zero (p = 0.00094, 0.000052, and <10−7, respectively), as do estimates for parameters GM (p = 0.023) and GSD (p <10−6), by 2-tail t-tests. A corresponding 4-parameter activation-only model, in which the rightmost term of Equation 1 is omitted and its “– b” replaced by “+ b”, explains nearly as much % Activity variability exhibited in this data set (R2 = 0.990) but nevertheless is clearly inconsistent with the mean response pattern due to its relatively small error at each concentration (p fit = ∼0).

The following 4-parameter biphasic inhibition-activation model was fit to hCAR activation data in relation to o-aminoazotoluene concentration C, as shown in Figure 2:

% Activity = 𝑏 Φ [ ln ( 𝐶 / GM 0 ) / ln ( 3 / 2 ) ] + 𝑐 Φ [ ln ( 𝐶 / GM ) / ln ( 2 ) ] + 5 𝑐 Φ [ ln ( 𝐶 / [ 25 GM ] ) / ln ( 2 ) ] (2)

This data set includes n = 3 replicates reported at each concentration. Parameter estimates ±1 SE obtained for this fit and associated fit statistics are: b = (4.54 ± 0.621)%, GM0 = (0.108 ± 0.0191)µM, c = (23.26 ± 1.26)%, and GM = 3.02 ± 0.265 (p fit = 0.49, R2 = 0.997). The estimated inhibition slope (b) differs significantly from zero (p = 0.000045), as do estimates for parameters GM0 (p = 0.00031), c (p < 10−7), and GM (p = ∼10−6), by 2-tail t-tests. A corresponding 3-parameter activation-only model (like that described above but with no Y-intercept term), explains nearly as much % Activity variability exhibited in this data set (R2 = 0.981), but nevertheless is clearly inconsistent with the mean response pattern due to its relatively small error at each concentration (p fit = ∼0).

F2
Figure 2: Model fit (curve) to data (open points) on hCAR activation in luciferase-reporter human HepG2 cells cultured for 24 hours at the indicated 4-aminoazobenzene concentration, which were reported normalized to a scale of 0% for DMSO control wells (plotted at 0 µM) and 100% (the maximum CITCO-induced activity). Error bars = ±1 standard deviation of the mean (SDM), involving 37 replicates/concentration. Dotted line = background in DMSO-exposed cells.
F3
Figure 3: Model fit (curve) to data (open points) on hCAR activation in luciferase-reporter human HepG2 cells cultured for 24 hours at the indicated o-aminoazotoluene concentration (see Figure 2 legend). Error bars = ±1 SDM, involving 3 replicates/concentration. Dotted line = background in DMSO-exposed cells.

4. Discussion

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 receptor-mediated 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. Under this interpretation, such integration mediates a cellular “choice” between two alternative (basal or negatively activated, vs. activated) modes of downstream signaling that (perhaps more so in some tissues than others) determine or influence cellular metabolic and/or proliferative status. Such chemical-specific balancing by hCAR is clearly indicated by potent chemical-specific anti-activation of this receptor [50], and by the application of the potent and selective hCAR deactivator PK11195 to reduce the high basal hCAR activity observed in HepG2-CYP2B6-hCAR cells used in the activation assay described in Methods. Evidently, chemicals such as AAB and OAT have a relatively rare capacity to both antagonize and activate hCAR substantially, and to do the former at sufficiently lower concentrations than the latter to yield a demonstrably biphasic pattern of overall activation. The signal-balancing basis of biphasic hCAR activation hypothesized here for hCAR appears to differ markedly from that of more subtle biphasic activation patterns observed for Nrf2 and HSP70 receptors functioning as ultrasensitive molecular switches that trigger suites of cytoprotective gene expression in response to specific (e.g., oxidative or heat) stress conditions, but which at sub-threshold trigger levels appear to dampen switch-activation likelihood, perhaps to suppress energy-draining “false alarms” under conditions of transient or marginal stress [44,45,46,47,48].

Both aminoazo compounds identified here to have a biphasic hCAR response have long been known to induce rodent liver tumors [55,56,57,58,59] (see Appendix). As noted in the Introduction, constitutive androstane receptor (CAR) activation by rodent liver tumor promoters such as phenobarbital act at least in part by stimulating downstream receptors and related epigenetic modifications that trigger hepatocellular proliferation and supporting shifts in energy and growth-directed metabolism. In contrast, CAR antagonist/inhibitors inhibit basal CAR activity levels. Among the 746 hCAR activator data sets discussed, possible nonlinear, nonmonotonic patterns of hCAR activation were previously suggested for the mouse liver carcinogens toxaphene (CAS RN 8001-35-2, CID 5284469 or 102000395), aldrin (CAS RN 465-73-6, 124-96-9, 309-00-2; CID 2087, 24860538), and isodrin (CAS RN 465-73-6, 124-96-9, 309-00-2; CID 10066, 60196420) [[60], see Figures 1, S5 and S6 of that study]. In contrast, approximately linear and lognormal patterns of hCAR activation were observed for the structurally related mouse liver carcinogens chlordane (CAS RN 57-74-9, CID 5993) and dieldrin (CAS RN 60-57-1, 72-20-8, 128-10-9; CID 3038, 969491), respectively [[60], see Figures S7 and S8 of that study].

It remains to be determined whether and to what extent mouse or rat CAR activation exhibits a biphasic dose-response pattern in vitro and in vivo, and what role CAR plays in driving or modulating AAB and/or OAT tumorigenicity in those species, analogous to the role it has been demonstrated through studies comparing CAR-knockout and wild-type mice to play in tumors associated with the nongenotoxic rodent tumor promotors phenobarbital and toxaphene [60,61,62]. However, the aryl hydrocarbon receptor (AHR) may also play an important and possibly CAR-related role in liver tumors associated with AAB and OAT, because these chemicals are also AHR agonists [63,64,65], CAR is (including in hepatocytes) upregulated by AHR [66], CAR and AHR activation correlate with susceptibility to OAT-induced liver tumors [65], and (like CAR) AHR plays central, diverse, and signal-integrating roles in cellular (including hepatocellular) development, energy metabolism, inflammation, enzyme induction, endocrine, and other systems that can modulate tumor likelihood [67,68,69]. Reports of biphasic AHR activation currently appear to be limited to subtle indications observed for galangin and reversitrol [70,71], although more systematic investigation using qHTS methods might reveal additional and more pronounced examples.

The biphasic nature per se of hCAR activation by AAB and OAT, and the fact that these two chemicals can also elevate rodent liver tumor incidence, appear to be coincidental with no present evidence of any causal connection. However, if future studies indicate that AAB and OAT (and perhaps other similarly rare biphasic CAR activators) elevate liver tumors by CAR-dependent mechanisms, their biphasic activation patterns would clearly be important and complicating considerations that bear directly on potential cancer risks posed by related environmental exposures.

Appendix

Concerning AAB tumorigenicity, dietary AAB administration did not result in liver tumors as 0.056% in the diet administered to albino rats for 8 months followed by two months of basal diet [72], or as 0.106% in the diet (5.34 mg/kg) administered to Sprague-Dawley rats for 9 months [73]. However, hepatic tumors were induced in 40% of Wistar rats fed 0.2–1% AAB the diet for up to 28 months [58]. By one year after neonatal ICR/JCL mice were exposed to AAB, they developed neoplastic lesions of liver, lung and lymphoreticular tissues [74]. In male 12-day-old C57BL/6 X C3H/ HeF1 (B6C3F1) mice administered single i.p. doses of 0.017–0.15 µmol AAB per g body weight (equivalent to a maximum transient concentration range of approximately 17–150 µM, assuming a 5-g mouse pup body weight with a 0.2-L/kg volume of distribution), hepatoma multiplicity was approximately linearly related to dose, with an average of 11 hepatomas/mouse observed at 10 months in the high-dose group; in contrast, female B6C3F1 mice were resistant to tumor induction under these conditions, and similar administration of AAB to male F344 rats with or without co-administration of 0.1% of phenobarbital in drinking water for 1–24 months did not induce a significant number of hepatic tumors [75]. In this study, 24 hours after 12-day-old male B6C3F1 mice and F344 rats were injected i.p. with 0.3 µmol AAB per g body weight, ∼40-fold more hepatic N-(deoxyguanosin-8-yl)-AAB-DNA adducts were detected in these mice than in similarly exposed rats [75]. The International Agency for Research on Cancer (IARC) currently classifies AAB as a possible (class 2B) human carcinogen [76].

Concerning OAT tumorigenicity, by 15 months after newborn (∼17-g) male and female A/Jax mice were given a single subcutaneous injection of 0, 0.4, or 0.7 mg OAT, ∼42–50% of males had one or more liver tumors, whereas females had about a third as many and unexposed mice had none; the OAT-exposed mice also showed a ∼4-fold elevation in pulmonary tumors [59]. Although OAT induces liver tumors potently in several mouse strains (CBA, SWR, DBA/2, A/He, and DD), much less or no observed hepatotumorigenic potency was observed in other strains (AKR and CC57Br) and in rats [77,78]. OAT was first shown to induce CAR and CAR-dependent liver cell proliferation in mice [65,79]. Onset of liver cell proliferation in mice hepatectomized and then treated with OAT was more delayed (60–80% inhibited) in strains more susceptible to OAT-induced liver tumors compared to <15% inhibition of cell proliferation exhibited in less-susceptible strains using the same protocol and OAT treatment [79]. IARC currently classifies OAT as a possible (class 2B) human carcinogen [76].

AAB and OAT each exhibit in vitro mutagenic activity in the Ames test using Salmonella typhimurium strain TA 98 activated by liver enzymes, where such activity was suppressed by adding pentachlorophenol (PCP) and induced by adding 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the activating enzymes [80]. However, tumor multiplicity exhibited in tumor-susceptible mouse strains neonatally exposed to OAT was increased with PCP pretreatment but was reduced with TCCD metabolic-activation pretreatment; in contrast, PCP pretreatment was observed to inhibit AAB-induced carcinogenic activity in three strains of mice [81].

Competing Interests

The author prepared the paper during the normal course of his employment by Exponent (Health Sciences), which is a consulting firm that, among other services, provides advice on toxicological and risk analysis issues to private and public clients. Formulation of scientific questions addressed, review of the literature, synthesis and integration of scientific information, and conclusions drawn in the paper are the exclusive professional product of the author and are not necessarily those of Exponent or any of its clients. Only Exponent reviewed the submitted paper and funded its preparation.

Acknowledgments

Comments by anonymous reviewers improved this manuscript are gratefully acknowledged.

References

  1. J. P. Hernandez, L. C. Mota, and W. S. Baldwin, “Activation of CAR and PXR by dietary, environmental and occupational chemicals alters drug metabolism, intermediary metabolism, and cell proliferation,” Current Pharmacogenomics and Personalized Medicine, vol. 7, no. 2, pp. 81–105, 2009. Publisher Full Text | Google Scholar
  2. H. Yang and H. Wang, “Signaling control of the constitutive androstane receptor (CAR),” Protein & Cell, vol. 5, no. 2, pp. 113–123, 2014. Publisher Full Text | Google Scholar
  3. K. Kobayashi, M. Hashimoto, P. Honkakoski, and M. Negishi, “Regulation of gene expression by CAR: an update,” Archives of Toxicology, vol. 89, no. 7, pp. 1045–1055, 2015. Publisher Full Text | Google Scholar
  4. R. Hao, S. Su, Y. Wan et al., “Bioinformatic analysis of microRNA networks following the activation of the constitutive androstane receptor (CAR) in mouse liver,” Biochimica et Biophysica Acta - Gene Regulatory Mechanisms, vol. 1859, no. 9, pp. 1228–1237, 2016. Publisher Full Text | Google Scholar
  5. Y. A. Kazantseva, Y. A. Pustylnyak, and V. O. Pustylnyak, “Role of nuclear constitutive androstane receptor in regulation of hepatocyte proliferation and hepatocarcinogenesis,” Biochemistry (Moscow), vol. 81, no. 4, pp. 338–347, 2016. Publisher Full Text | Google Scholar
  6. G. M. Hudson, K. L. Flannigan, S. L. Erickson et al., “Constitutive androstane receptor regulates the intestinal mucosal response to injury,” British Journal of Pharmacology, vol. 174, no. 12, pp. 1857–1871, 2017. Publisher Full Text | Google Scholar
  7. I. R. Miousse, L. A. Murphy, H. Lin et al., “Dose-response analysis of epigenetic, metabolic, and apical endpoints after short-term exposure to experimental hepatotoxicants,” Food and Chemical Toxicology, vol. 109, pp. 690–702, 2017. Publisher Full Text | Google Scholar
  8. Y. Yamamoto, R. Moore, T. L. Goldsworthy, M. Negishi, and R. R. Maronpot, “The orphan nuclear receptor constitutive active/androstane receptor is essential for liver tumor promotion by phenobarbital in mice,” Cancer Research, vol. 64, no. 20, pp. 7197–7200, 2004. Publisher Full Text | Google Scholar
  9. R. H. Costa, V. V. Kalinchenko, Y. Tan, and I. Wang, “The CAR nuclear receptor and hepatocyte proliferation,” Hepatology, vol. 42, no. 5, pp. 1004–1008, 2005. Publisher Full Text | Google Scholar
  10. W. E. Blanco-Bose, M. J. Murphy, A. Ehninger et al., “c-Myc and its target foxM1 are critical downstream effectors of constitutive androstane receptor (CAR) mediated direct liver hyperplasia,” Hepatology, vol. 48, no. 4, pp. 1302–1311, 2008. Publisher Full Text | Google Scholar
  11. J. M. Phillips and J. I. Goodman, “Multiple genes exhibit phenobarbital-induced constitutive active/androstane receptor-mediated DNA methylation changes during liver tumorigenesis and in liver tumors,” Toxicological Sciences, vol. 108, no. 2, pp. 273–289, 2009. Publisher Full Text | Google Scholar
  12. H. Lempiäinen, A. Müller, S. Brasa et al., “Phenobarbital mediates an epigenetic switch at the constitutive androstane receptor (CAR) target gene Cyp2b10 in the liver of B6C3F1 mice,” PLoS ONE, vol. 6, no. 3, Article ID e18216, 2011. Publisher Full Text | Google Scholar
  13. D. Takizawa, S. Kakizaki, N. Horiguchi, Y. Yamazaki, H. Tojima, and M. Mori, “Constitutive active/androstane receptor promotes hepatocarcinogenesis in a mouse model of non-alcoholic steatohepatitis,” Carcinogenesis, vol. 32, no. 4, pp. 576–583, 2011. Publisher Full Text | Google Scholar
  14. D. R. Geter, V. S. Bhat, B. Bhaskar Gollapudi, R. Sura, and S. D. Hester, “Dose-response modeling of early molecular and cellular key events in the CAR-mediated hepatocarcinogenesis pathway,” Toxicological Sciences, vol. 138, no. 2, pp. 425–445, 2014. Publisher Full Text | Google Scholar
  15. M. J. Lebaron, B. B. Gollapudi, C. Terry, R. Billington, and R. J. Rasoulpour, “Human relevance framework for rodent liver tumors induced by the insecticide sulfoxaflor,” Critical Reviews in Toxicology, vol. 44, no. 2, pp. 15–24, 2014. Publisher Full Text | Google Scholar
  16. R. Luisier, H. Lempiäinen, N. Scherbichler et al., “Phenobarbital induces cell cycle transcriptional responses in mouse liver humanized for constitutive androstane and pregnane X receptors,” Toxicological Sciences, vol. 139, no. 2, Article ID kfu038, pp. 501–511, 2014. Publisher Full Text | Google Scholar
  17. J. Gao, J. Yan, M. Xu, S. Ren, and W. Xie, “CAR suppresses hepatic gluconeogenesis by facilitating the ubiquitination and degradation of PGC1α,” Molecular Endocrinology, vol. 29, no. 11, pp. 1558–1570, 2015. Publisher Full Text | Google Scholar
  18. B. G. Lake, R. J. Price, and T. G. Osimitz, “Mode of action analysis for pesticide-induced rodent liver tumours involving activation of the constitutive androstane receptor: Relevance to human cancer risk,” Pest Management Science, vol. 71, no. 6, pp. 829–834, 2015. Publisher Full Text | Google Scholar
  19. J. Yan, B. Chen, J. Lu, and W. Xie, “Deciphering the roles of the constitutive androstane receptor in energy metabolism,” Acta Pharmacologica Sinica, vol. 36, no. 1, pp. 62–70, 2015. Publisher Full Text | Google Scholar
  20. K. Tamura, K. Inoue, M. Takahashi, S. Matsuo, Y. Kodama, and M. Yoshida, “A crucial role of constitutive androstane receptor (Car) in liver tumor development by imazalil in mice,” Journal of Toxicological Sciences, vol. 41, no. 6, pp. 801–811, 2016. Publisher Full Text | Google Scholar
  21. A. A. Yarushkin, Y. A. Kazantseva, E. A. Prokopyeva, D. N. Markova, Y. A. Pustylnyak, and V. O. Pustylnyak, “Constitutive androstane receptor activation evokes the expression of glycolytic genes,” Biochemical and Biophysical Research Communications, vol. 478, no. 3, pp. 1099–1105, 2016. Publisher Full Text | Google Scholar
  22. W. Huang, J. Zhang, M. Washington et al., “Xenobiotic stress induces hepatomegaly and liver tumors via the nuclear receptor constitutive androstane receptor,” Molecular Endocrinology, vol. 19, no. 6, pp. 1646–1653, 2005. Publisher Full Text | Google Scholar
  23. B. Dong, J.-S. Lee, Y.-Y. Park et al., “Activating CAR and β-catenin induces uncontrolled liver growth and tumorigenesis,” Nature Communications, vol. 6, article no. 6944, 2015. Publisher Full Text | Google Scholar
  24. J. Ross, S. M. Plummer, A. Rode et al., “Human constitutive androstane receptor (CAR) and pregnane X receptor (PXR) support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogens phenobarbital and chlordane in vivo,” Toxicological Sciences, vol. 116, no. 2, pp. 452–466, 2010. Publisher Full Text | Google Scholar
  25. F. Chen, S. M. Zamule, D. M. Coslo, T. Chen, and C. J. Omiecinski, “The human constitutive androstane receptor promotes the differentiation and maturation of hepatic-like cells,” Developmental Biology, vol. 384, no. 2, pp. 155–165, 2013. Publisher Full Text | Google Scholar
  26. D. Li, B. MacKowiak, T. G. Brayman et al., “Genome-wide analysis of human constitutive androstane receptor (CAR) transcriptome in wild-type and CAR-knockout HepaRG cells,” Biochemical Pharmacology, vol. 98, no. 1, pp. 190–202, 2015. Publisher Full Text | Google Scholar
  27. C. Lynch, J. Zhao, R. Huang et al., “Quantitative high-throughput identification of drugs as modulators of human constitutive androstane receptor,” Scientific Reports, vol. 5, Article ID 10405, 2015. Publisher Full Text | Google Scholar
  28. T. Yamada, S. M. Cohen, and B. G. Lake, “The Mode of Action for Phenobarbital-Induced Rodent Liver Tumor Formation Is not Relevant for Humans: Recent Studies With Humanized Mice,” Toxicological sciences : an official journal of the Society of Toxicology, vol. 147, no. 2, pp. 298–299, 2015. Publisher Full Text | Google Scholar
  29. M. E. Wyde, S. E. Kirwan, F. Zhang et al., “Di-n-butyl phthalate activates constitutive androstane receptor and pregnane X receptor and enhances the expression of steroid-metabolizing enzymes in the liver of rat fetuses,” Toxicological Sciences, vol. 86, no. 2, pp. 281–290, 2005. Publisher Full Text | Google Scholar
  30. J. G. Dekeyser, E. M. Laurenzana, E. C. Peterson, T. Chen, and C. J. Omiecinski, “Selective phthalate activation of naturally occurring human constitutive androstane receptor splice variants and the pregnane X receptor,” Toxicological Sciences, vol. 120, no. 2, Article ID kfq394, pp. 381–391, 2011. Publisher Full Text | Google Scholar
  31. E. M. Laurenzana, D. M. Coslo, M. V. Vigilar, A. M. Roman, and C. J. Omiecinski, “Activation of the Constitutive Androstane Receptor by Monophthalates,” Chemical Research in Toxicology, vol. 29, no. 10, pp. 1651–1661, 2016. Publisher Full Text | Google Scholar
  32. M. T. Cherian, S. C. Chai, and T. Chen, “Small-molecule modulators of the constitutive androstane receptor,” Expert Opinion on Drug Metabolism & Toxicology, vol. 11, no. 7, pp. 1099–1114, 2015. Publisher Full Text | Google Scholar
  33. L. Li, M. E. Andersen, S. Heber, and Q. Zhang, “Non-monotonic dose-response relationship in steroid hormone receptor-mediated gene expression,” Molecular Endocrinology, vol. 38, no. 5-6, pp. 569–585, 2007. Publisher Full Text | Google Scholar
  34. Q. Zhang and M. E. Andersen, “Dose response relationship in anti-stress gene regulatory networks,” PLoS Computational Biology, vol. 3, no. 3, pp. 345–363, 2007.
  35. Q. Zhang, S. Bhattacharya, and M. E. Andersen, “Ultrasensitive response motifs: Basic amplifiers in molecular signalling networks,” Open Biology, vol. 3, Article ID 130031, 2013. Publisher Full Text | Google Scholar
  36. Q. Zhang, S. Bhattacharya, R. B. Conolly, H. J. Clewell, N. E. Kaminski, and M. E. Andersen, “Molecular signaling network motifs provide a mechanistic basis for cellular threshold responses,” Environmental Health Perspectives, vol. 122, no. 12, pp. 1261–1270, 2015. Publisher Full Text | Google Scholar
  37. Q. Zhang, S. Bhattacharya, J. Pi, R. A. Clewell, P. L. Carmichael, and M. E. Andersen, “Adaptive posttranslational control in cellular stress response pathways and its relationship to toxicity testing and safety assessment,” Toxicological Sciences, vol. 147, no. 2, Article ID kfv130, pp. 302–316, 2015. Publisher Full Text | Google Scholar
  38. C. Nweke and C. Ogbonna, “Statistical models for biphasic dose-response relationships (hormesis) in toxicological studies,” Ecotoxicology and Environmental Contamination, vol. 12, no. 1, pp. 39–55, 2017. Publisher Full Text | Google Scholar
  39. E. J. Calabrese, “Estrogen and related compounds: Biphasic dose responses,” Critical Reviews in Toxicology, vol. 31, no. 4-5, pp. 503–515, 2001. Publisher Full Text | Google Scholar
  40. E. J. Calabrese, “Adrenergic receptors: Biphasic dose responses,” Critical Reviews in Toxicology, vol. 31, no. 4-5, pp. 523–538, 2001. Publisher Full Text | Google Scholar
  41. E. J. Calabrese, “Opiates: Biphasic dose responses,” Critical Reviews in Toxicology, vol. 31, no. 4-5, pp. 585–604, 2001. Publisher Full Text | Google Scholar
  42. E. Calabrese, “Cancer biology and hormesis: Human tumor cell lines commonly display hormetic (biphasic) dose responses,” Critical Reviews in Toxicology, vol. 35, no. 6, pp. 463–582, 2005. Publisher Full Text | Google Scholar
  43. K. Javaherian, T.-Y. Lee, R. M. T. T. Sjin, G. E. Parris, and L. Hlatky, “Two endogenous antiangiogenic inhibitors, endostatin and angiostatin, demonstrate biphasic curves in their antitumor profiles,” Dose-Response, vol. 9, no. 3, pp. 369–376, 2011. Publisher Full Text | Google Scholar
  44. K. T. Bogen, “Low-dose dose–response for in vitro Nrf2-ARE activation in human HepG2 cells,” Dose-Response, vol. 15, no. 2, 2017. Publisher Full Text | Google Scholar
  45. K. T. Bogen, “Linear-No-Threshold Default Assumptions are Unwarranted for Cytotoxic Endpoints Independently Triggered by Ultrasensitive Molecular Switches,” Risk Analysis, vol. 37, no. 10, pp. 1808–1816, 2017. Publisher Full Text | Google Scholar
  46. Y. E. Timsit and M. Negishi, “Coordinated regulation of nuclear receptor CAR by CCRP/DNAJC7, HSP70 and the ubiquitin-proteasome system,” PLoS ONE, vol. 9, no. 5, Article ID e96092, 2014. Publisher Full Text | Google Scholar
  47. M. D. Merrell, J. P. Jackson, L. M. Augustine et al., “The Nrf2 activator oltipraz also activates the constitutive androstane receptor,” Drug Metabolism and Disposition, vol. 36, no. 8, pp. 1716–1721, 2008. Publisher Full Text | Google Scholar
  48. L. M. Aleksunes and C. D. Klaassen, “Coordinated regulation of hepatic phase I and II drug-metabolizing genes and transporters using AhR-, CAR-, PXR-, PPARα-, and Nrf2-null mice,” Drug Metabolism and Disposition, vol. 40, no. 7, pp. 1366–1379, 2012. Publisher Full Text | Google Scholar
  49. G. Kahl, The Dictionary of Genomics, Transcriptomics and Proteomics, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2015.
  50. C. Lynch, J. Zhao, R. Huang et al., “Quantitative High-Throughput Identification of Drugs as Modulators of Human Constitutive Androstane Receptor,” Scientific Reports, vol. 5, no. 1, 2015. Publisher Full Text | Google Scholar
  51. C. Lynch, J. Zhao, H. Wang, and M. Xia, “Quantitative High-Throughput Luciferase Screening in Identifying CAR Modulators,” in High-Throughput Screening Assays in Toxicology, vol. 1473 of Methods in Molecular Biology, pp. 33–42, Springer New York, New York, NY, 2016.
  52. R. Huang, M. Xia, M.-H. Cho et al., “Chemical genomics profiling of environmental chemical modulation of human nuclear receptors,” Environmental Health Perspectives, vol. 119, no. 8, pp. 1142–1148, 2011. Publisher Full Text | Google Scholar
  53. R. Huang, “A Quantitative High-Throughput Screening Data Analysis Pipeline for Activity Profiling,” in High-Throughput Screening Assays in Toxicology, vol. 1473 of Methods in Molecular Biology, pp. 111–122, Springer New York, New York, NY, 2016.
  54. Wolfram. Research, Wolfram Language and System Documentation Center, Wolfram Research, Inc, Champaign, IL, 2017, http://reference.wolfram.com/language/.
  55. A. H. M. Kirby, “Carcinogenic effect of aminoazobenzene [3],” Nature, vol. 154, no. 3917, pp. 668–669, 1944. Publisher Full Text | Google Scholar
  56. J. W. ORR, “The Production of Liver Tumours by Azo Compounds,” British Medical Bulletin, vol. 4, no. 5-6, pp. 385–388, 1946. Publisher Full Text | Google Scholar
  57. R. C. Garry and J. W. Cook, “Azo-dyes and Experimental Liver Tumours,” British Journal of Nutrition, vol. 1, no. 2-3, pp. 245–253, 1947. Publisher Full Text | Google Scholar
  58. A. H. Kirby and P. R. Peacock, “The induction of liver tumours by 4-aminoazobenzene and its N: N-dimethyl derivative in rats on a restricted diet,” The Journal of Pathology, vol. 59, no. 1-2, pp. 1–18, 1947. Publisher Full Text | Google Scholar
  59. Y. Nishizuka, K. Ito, and K. Nakakuki, “Liver tumor induction by a single injection of o-aminoazotoluene to newborn mice,” Jap J Cancer Res (GANN, vol. 56, no. 2, pp. 135–42, 10.20772/cancersci1959.56.2_135.
  60. B. D. Kerger, K. T. Bogen, A. E. Loccisano, and J. C. Lamb, “Proposed reference dose for toxaphene carcinogenicity based on constitutive androstane receptor-mediated mode of action,” Human and Ecological Risk Assessment: An International Journal, pp. 1–21, 2017. Publisher Full Text | Google Scholar
  61. C. R. Elcombe, R. C. Peffer, D. C. Wolf et al., “Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator,” Critical Reviews in Toxicology, vol. 44, no. 1, pp. 64–82, 2014. Publisher Full Text | Google Scholar
  62. Z. Wang, X. Li, Q. Wu, J. C. Lamb, and J. E. Klaunig, “Toxaphene-induced mouse liver tumorigenesis is mediated by the constitutive androstane receptor,” Journal of Applied Toxicology, vol. 37, no. 8, pp. 967–975, 2017. Publisher Full Text | Google Scholar
  63. T.-A. Kato, T. Matsuda, S. Matsui, T. Mizutani, and K.-I. Saeki, “Activation of the aryl hydrocarbon receptor by methyl yellow and related congeners: Structure-activity relationships in halogenated derivatives,” Biological & Pharmaceutical Bulletin, vol. 25, no. 4, pp. 466–471, 2002. Publisher Full Text | Google Scholar
  64. M. A. Smetanina, M. Y. Pakharukova, S. M. Kurinna et al., “Ortho-aminoazotoluene activates mouse constitutive androstane receptor (mCAR) and increases expression of mCAR target genes,” Toxicology and Applied Pharmacology, vol. 255, no. 1, pp. 76–85, 2011. Publisher Full Text | Google Scholar
  65. N. V. Baginskaya, E. V. Kashina, M. Y. Shamanina, S. I. Ilnitskaya, V. I. Kaledin, and V. A. Mordvinov, “Correlation of susceptibility to ortho-aminoazotoluene-induced hepatocarcinogenesis with Car and Ahr signaling pathway activation in mice,” Russian Journal of Genetics: Applied Research, vol. 6, no. 4, pp. 463–468, 2016. Publisher Full Text | Google Scholar
  66. R. D. Patel, B. D. Holhngshead, C. J. Omiecinski, and G. H. Perdew, “Aryl-hydrocarbon receptor activation regulates constitutive androstane receptor levels in murine and human liver,” Hepatology, vol. 46, no. 1, pp. 209–218, 2007. Publisher Full Text | Google Scholar
  67. T. V. Beischlag, J. L. Morales, B. D. Hollingshead, and G. H. Perdew, “The aryl hydrocarbon receptor complex and the control of gene expression,” Critical Reviews in Eukaryotic Gene Expression, vol. 18, no. 3, pp. 207–250, 2008. Publisher Full Text | Google Scholar
  68. K. Kawajiri and Y. Fujii-Kuriyama, “The aryl hydrocarbon receptor: A multifunctional chemical sensor for host defense and homeostatic maintenance,” Journal of Experimental Animal Science, vol. 66, no. 2, pp. 75–89, 2017. Publisher Full Text | Google Scholar
  69. P. Lu and W. Xie, “Xenobiotic receptors in the crosstalk between drug metabolism and energy metabolism,” in Drug Metabolism in Diseases, W. the crosstalk between drug metabolism and energy metabolism. Xie, Ed., pp. 258–78, Academic Press, London, UK, 2016.
  70. H. P. Ciolino and G. C. Yeh, “The flavonoid galangin is an inhibitor of CYP1A1 activity and an agonist/antagonist of the aryl hydrocarbon receptor,” British Journal of Cancer, vol. 79, no. 9-10, pp. 1340–1346, 1999. Publisher Full Text | Google Scholar
  71. S. U. N. Singh, R. F. Casper, P. C. Fritz et al., “Inhibition of dioxin effects on bone formation in vitro by a newly described aryl hydrocarbon receptor antagonist, resveratrol,” Journal of Endocrinology, vol. 167, no. 1, pp. 183–195, 2000. Publisher Full Text | Google Scholar
  72. J. A. Miller and C. A. Baumann, “The Carcinogenicity of Certain Azo Dyes Related to p-Dimethylaminoazobenzene,” Cancer Research, vol. 5, no. 4, pp. 227–234, 1945.
  73. JA. Miller and EC. Miller, “The carcinogenicity of certain derivatives of p-dimethylammoazobenzene in the rat,” J Exp Med, vol. 87, no. 2, pp. 13–156, 1948.
  74. K. Fujii, “Induction of tumors in transplacental or neonatal mice administered 3′-methyl-4-dimethylaminoazobenzene or 4-aminoazobenzene,” Cancer Letters, vol. 17, no. 3, pp. 321–325, 1983. Publisher Full Text | Google Scholar
  75. K. B. Delclos, W. G. Tarpley, E. C. Miller, and J. A. Miller, “4-Aminoazobenzene and N,N-Dimethyl-4-Aminoazobenzene as Equipotent Hepatic Carcinogens in Male C57BL/6 x C3H/He F1 Mice and Characterization of N-(Deoxyguanosin-8-yl)-4-Aminoazobenzene as the Major Persistent Hepatic DNA-Bound Dye in These Mice,” Cancer Research, vol. 44, no. 6, pp. 2540–2550, 1984.
  76. D. W. Bleyl, OveraIl Evaluations of Carcinogenicity: An Updating of IARC Monographs Volumes 1 to 42. Monographs on the Evaluation of the Carcinogenic Risks to Humans, Supplement 7. World Health Organization, International Agency for Research on Cancer (IARC), Geneva, Switzerland. Table 1, at. p. 56 ff.
  77. L. Y. Zacharova, L. F. Gulyaeva, V. V. Lyakhovich et al., “Cytochrome P4501A1 and 1A2 gene expression in the liver of 3-methylcholanthrene- and o-aminoazotuluene- treated mice: A comparison between PAH-responsive and PAH-nonresponsive strains,” Toxicological Sciences, vol. 73, no. 1, pp. 108–113, 2003. Publisher Full Text | Google Scholar
  78. V. I. Kaledin, S. I. Ilnitskaya, L. P. Ovchinnikova, N. A. Popova, L. A. Bogdanova, and T. S. Morozkova, “Mutagenic activation and carcinogenicity of aminoazo dyes ortho-aminoazotoluene and 3′-methyl-4-dimethylaminoazobenzene in experiments on suckling mice,” Biophysics (Russian Federation), vol. 59, no. 3, pp. 431–435, 2014. Publisher Full Text | Google Scholar
  79. M. Y. Pakharukova, M. A. Smetanina, V. I. Kaledin, V. F. Kobzev, I. V. Romanova, and T. I. Merkulova, “Activation of constitutive androstane receptor under the effect of hepatocarcinogenic aminoazo dyes in mouse and rat liver,” Bulletin of Experimental Biology and Medicine, vol. 144, no. 3, pp. 338–341, 2007. Publisher Full Text | Google Scholar
  80. O. A. Timofeeva, A. V. Eremeev, A. Goloshchapov et al., “Effects of o-aminoazotoluene on liver regeneration and p53 activation in mice susceptible and resistant to hepatocarcinogenesis,” Toxicology, vol. 254, no. 1-2, pp. 91–96, 2008. Publisher Full Text | Google Scholar
  81. L. P. Ovchinnikova, L. A. Bogdanova, and V. I. Kaledin, “Mutagenic Activation Reduces Carcinogenic Activity of Ortho-Aminoazotoluene for Mouse Liver,” Bulletin of Experimental Biology and Medicine, vol. 154, no. 5, pp. 664–668, 2013. Publisher Full Text | Google Scholar
Research Article
Nuclear Receptor Research
Vol. 5 (2018), Article ID 101321, 12 pages
doi:10.11131/2018/101321

Biphasic hCAR Inhibition-Activation by Two Aminoazo Liver Carcinogens

Kenneth T. Bogen

Exponent Inc., Health Sciences, Oakland, CA 94612, USA

Received 29 September 2017; Accepted 21 February 2018

Editor: William Baldwin

Copyright © 2018 Kenneth T. Bogen. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Detailed dose-response data recently archived by the National Center for Biotechnology Information (NCBI) identified 853 human CAR (hCAR) agonists by quantitative high-throughput screening (qHTS) assays applied to >9,000 chemicals tested at ≥14 concentrations using n = 3–48 replicates. By re-examining NCBI data on 746 agonists with replicate data sets each satisfying additional quality criteria, ∼95% had average values of agonist-specific Hill-model slopes estimated by NCBI that exceed 1 (i.e., exhibited an overall sublinear low-dose dose-response), and two unambiguously biphasic hCAR inhibitor-agonists were identified, 4-aminoazobenzene (n = 37) and ortho-aminoazotoluene (n = 3), both of which also cause rodent liver tumors. Although evidently rare among hCAR agonists, such biphasic responses add to evidence that nuclear receptors can exhibit complex patterns of low-dose response, consistent with previous observations and theoretical predictions for endpoints governed by ultrasensitive molecular switches. The pronounced biphasic hCAR response 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 receptor-mediated biphasic cellular responses to any single-chemical exposure reported to date.

Research Article
Nuclear Receptor Research
Vol. 5 (2018), Article ID 101321, 12 pages
doi:10.11131/2018/101321

Biphasic hCAR Inhibition-Activation by Two Aminoazo Liver Carcinogens

Kenneth T. Bogen

Exponent Inc., Health Sciences, Oakland, CA 94612, USA

Received 29 September 2017; Accepted 21 February 2018

Editor: William Baldwin

Copyright © 2018 Kenneth T. Bogen. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to Cite this Article

Kenneth T. Bogen, “Biphasic hCAR Inhibition-Activation by Two Aminoazo Liver Carcinogens,” Nuclear Receptor Research, vol. 5, Article ID 101321, 12 pages, 2018. doi:10.11131/2018/101321