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. 2025 Oct 14;28(11):113764.
doi: 10.1016/j.isci.2025.113764. eCollection 2025 Nov 21.

Human and zebrafish mineralocorticoid receptors reporter cell assays to assess the activity of chemicals

Anna Toso  1   2 , Abdelhay Boulahtouf  1 , Marina Grimaldi  1 , Audrey Sansaloni  1 , Yoshinao Katsu  3   4 , Michael E Baker  5   6 , Aurélie Escande  7 , Clémentine Garoche  1 , Selim Aït-Aïssa  8 , Patrick Balaguer  1
Affiliations

Human and zebrafish mineralocorticoid receptors reporter cell assays to assess the activity of chemicals

Anna Toso et al. iScience. .

Abstract

The action of environmental chemicals (ECs) on the mineralocorticoid receptor (MR) has been suggested to impair physiological processes regulated by this nuclear receptor. However, it remains understudied both as a target of ECs and with respect to potential species-specific differences. In this regard, we have developed reporter cell lines to identify the response to different steroids, ECs, and urban wastewater (WW) sample extracts of human MR (hMR) and zebrafish MR (zfMR). Most of the steroids had a higher efficacy on zfMR than hMR, while the ECs were antagonists to both hMR and zfMR, with a lower potency on the latter. Interestingly, WW sample extracts revealed the presence of MR activity with a greater activity on zfMR compared to hMR, suggesting the presence of steroids in WW. These screening tools have proven to be powerful tools for characterizing the interaction of chemicals with MRs and revealing their presence in environmental samples.

Keywords: Biochemistry; Biological sciences; Cell biology; Environmental monitoring; Natural sciences.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Aldosterone, dexamethasone, bimedrazole, and cortisol do not modulate luciferase expression in the UG5LN cell line The cells were treated with steroids at 10−8 M and 10−5 M except for bimedrazol, which was used at 10−8 M and 10−6 M. Results are expressed as the percentage of the maximum luciferase activity in presence of DMSO. Data are presented as means ± SD values. n (replicates per experiment) = 4 per group.
Figure 2
Figure 2
Cortisol and dexamethasone had an higherpotency and efficacy on zfMR than on hMR Dose-response curves of aldosterone, dexamethasone, cortisol, and bimedrazole in UG5LN-GAL4-hMR (A) and UG5LN-GAL4-zfMR cells (B). Results are expressed as the percentage of the maximum luciferase activity induced by 10−8 M aldosterone. The curves are presented as a non-linear regression; log (ligand) versus response. EC50 values (10−9 M) are shown in Tables 3 and 4. Data are presented as means ± SD values. n (replicates per experiment) = 4 per group.
Figure 3
Figure 3
Dose-response curves of dexamethasone in the absence or presence of aldosterone 10−9 M in HG5LN-GAL4-hMR cells Results are expressed as the percentage of the maximum luciferase activity induced by 10−8 M aldosterone. The curves are presented as a non-linear regression; log (ligand) versus response. EC50 and IC50 values (10−9 M) are shown in Table 3. Data are presented as means ± SD values. n (replicates per experiment) = 4 per group.
Figure 4
Figure 4
Medrol, drospirenone, progesterone, 11-deoxycortisol, spironolactone and drospirenone had an higher efficacy on zfMR than on hMR Dose-response curves of medrol (A), drospirenone (B), progesterone (C), 11-deoxycortisol (D), spironolactone (E), and drospirenone (F) in UG5LN-GAL4-hMR and UG5LN-GAL4-zfMR cells. Chemicals were tested alone (agonism test) or in the presence of aldosterone 1 nM in UG5LN-GAL4-hMR cells and 0.5 10−9 M in UG5LN-GAL4-zfMR cells (antagonism test). Results are expressed as the percentage of the maximum luciferase activity induced by 10−8 M aldosterone. The curves are presented as a non-linear regression; log (ligand) versus response. EC50 values (10−9 M) and IC50 values (10−9 M) are shown in Tables 3 and 4. Data are presented as means ± SD values. n (replicates per experiment) = 4 per group.
Figure 5
Figure 5
Canrenone, dihydrotestosterone, pregnenolone,promegestone, mifepristone dihydroprogesterone, finerenone and eperone fully antagonized hMR and zfMR (A–H) Dose-response curves of canrenone (A), dihydrotestosterone (B), pregnenolone (C), promegestone (D) mifepristone (E), dihydroprogesterone (F), finerenone (G), and eplerenone (H) in UG5LN-GAL4-hMR and UG5LN-GAL4-zfMR cells. Chemicals were tested in the presence of aldosterone 10−9 M in UG5LN-GAL4-hMR cells and 0.5 10−9 M in UG5LN-GAL4-zfMR cells (antagonism test). Results are expressed as the percentage of the maximum luciferase activity induced by 10−8 M aldosterone. The curves are presented as a non-linear regression; log (ligand) versus response. IC50 values (10−9 M) are shown in Tables 3 and 4. Data are presented as means ± SD values. n (replicates per experiment) = 4 per group.
Figure 6
Figure 6
The substitution of the threonine 870 by a leucine increased the efficacy of steroids on hMR Dose-response curves of (A) 17α-hydroxyprogesterone, medrol, progesterone, drospirenone, and spironolactone and (B) finerenone and eplerenone in UG5LN-GAL4-hMR (T870L) cells. 17α-hydroxyprogesterone, medrol, progesterone, drospirenone, and spironolactone were tested alone (agonism test). (B) Finerenone and eplerenone were tested in the presence of aldosterone 0.5 10−9 M (antagonism test). Results are expressed as the percentage of the maximum luciferase activity induced by 10−8 M aldosterone. The curves are presented as a non-linear regression; log (ligand) versus response. EC50 values (10−9 M) and IC50 values (10−9 M) are shown in Tables 5 and 6. Data are presented as means ± SD values. n (replicates per experiment) = 4 per group.
Figure 7
Figure 7
HMR and zfMR are targets of environmental chemicals Dose-response curves of environmental chemicals in UG5LN-GAL4-hMR (A and B) and UG5LN-GAL4-zfMR (C and D) cells. Chemicals were tested in the presence of aldosterone 10−9 M in UG5LN-GAL4-hMR cells and 0.5 10−9 M in UG5LN-GAL4-zfMR cells (antagonism test). Results are expressed as the percentage of the maximum luciferase activity induced by 10−8 M aldosterone. The curves are presented as a non-linear regression; log (ligand) versus response. IC50 values (10−6 M) are shown in Table 7. Data are presented as means ± SD values. n (replicates per experiment) = 4 per group.
Figure 8
Figure 8
hMR and zfMR activities of waste water samples (A–C) Mineralocorticoid activities of waste water influent and effluent of two urban sites, A and B, were tested in the UG5LN GAL4-hMR and UG5LN-GAL4-zfMR cells. Firstly, influent and effluent of the two sites were tested at the maximal concentration of 3 REF for influents and 6 REF for effluents (A). Secondly, serial dilutions of waste water influents A (B) and B (C) were tested in UG5LN-GAL4-hMR, UG5LN-GAL4-zfMR, and UG5LN-GAL4-hMR (T870L) cells. Sample concentration data are expressed in REF. Results are expressed as the percentage of the maximum luciferase activity induced by 10−8 M aldosterone. The curves are presented as a non-linear regression; log (REF) versus response. Data are presented as means ± SD values. n (replicates per experiment) = 4 per group.
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