Supplementary information

Characterising combined effects of anti-estrogenic chemicals on vitellogenin production in rainbow trout (Oncorhynchus mykiss) hepatocytes

Authors: Maria T. Hultman, Karina Petersen, Knut Erik Tollefsen

Supplementary Figure 1. Gene expression of putative transcripts included in the estrogen receptor signaling (ER) pathway in primary rainbow trout (Oncorhynchus mykiss) hepatocyte after exposure to 6.3E-10M 17β-estradiol (E2), 2.38E-8 M β-naphtoflavone (BNF), 1.1E−8M 4-hydroxytamoxifen (OHT) and mixture of these (BNF+OHT) for 48h. Data represent the mean of 3-4 individual cell isolations ± standard deviation. The statistical analysis was performed using a one-way ANOVA with a Tukey's post hoc test (p ≤ 0.05).

Supplementary Figure 2. Gene expression of cytochrome P450 1a (cyp1a) in primary rainbow trout (Oncorhynchus mykiss) hepatocyte after exposure to 6.3E-10 mol/L 17β-estradiol (E2), 2.38E-8 mol/L β-naphtoflavone (BNF), 1.1E−8 mol/L 4-hydroxytamoxifen (OHT) and mixture of these (BNF+OHT) for 48h in absence of 6.3E-10 mol/L E2. Data represent the mean of 3 individual cell isolations ± standard deviation. The statistical analysis (p ≤ 0.05) was performed using a one way-ANOVA with a Tukey's post hoc test (left graph) and a two-way ANOVA with a Bonferroni post hoc test (right graph). Letters denotes statistical significance in the one way-ANOVA, while * denotes statistical significant interaction in the two way-ANOVA.

Supplementary Figure 3. Gene expression of aryl hydrocarbon receptor nuclear translocator (arnt) in primary rainbow trout (Oncorhynchus mykiss) hepatocyte after exposure to 6.3E-10 mol/L 17β-estradiol (E2), 2.38E-8 mol/L β-naphtoflavone (BNF), 1.1E−8 mol/L 4-hydroxy-tamoxifen (OHT) and mixture of these (BNF+OHT) for 48h in absence (clear boxes) and co-exposure (filled boxes) with 6.3E-10 mol/L E2. Data represent the mean of 3 individual cell isolations ± standard deviation. The statistical analysis (p ≤ 0.05) was performed using a one-way ANOVA with a Tukey's post hoc test (left graph) and a two way-ANOVA with a Bonferroni post hoc test (right graph). Letters denotes statistical significance in the one way-ANOVA, while * denotes statistical significant interaction in the two way-ANOVA.

Experiment 2
Exposure / Concentration (mol/L) / Predicted effect (% of E2 control) / Observed effect (% of E2 at 6.3E-10M)± stdev / MDR
E2 / 6.3E-10 / 100
BNF / 2.38E-8 / 71 / 71 ± 5.4
OHT / 1.10E-8 / 39 / 51 ± 12
BNF + OHT / 2.38E-8 + 1.10E-8 / 38 (CA), 28 (IA) / 23 ± 4.7 / 2.0 (CA) 1.3 (IA)

Supplementary Table 1. Observed and predicted results normalised between DMSO control (0%) and positive control (E2: 6.3E-10 mol/L).

CA – Concentration addition; IA – Independent action; MDR - model deviation ratios

Supplementary information

Supplementary table 2. A two-way ANOVA comparing gene expression in factorial treatments exposed to E2, BNF, OHT and BNF+OHT with and without presence of E2. The presented Fvaluerepresentsthe ratio of the experimental effect compared to the given 'error', while p ≤ 0.05 was determined as statistically significant. Significant values are given in bold.
F (p) / Vtg protein / er-α / vtg-1 / zrp / ahr2β / cyp1a / arnt
E2+ / BNF / 26.09 (0.002)* / 2.428 (0.1702) / 6.455 (0.044)* / 2.401 (0.1722) / 0.387 (0.5569) / 3.753 (0.1008) / 0.503 (0.5047)
OHT / 3.171 (0.1253) / 7.577 (0.0332)* / 3.002 (0.1339) / 1.347 (0.2899) / 0.270 (0.6218) / 0.054 (0.8236) / 0.010 (0.9229)
Mix / 0.099 (0.7627) / 0.425 (0.5388) / 0.045 (0.8391) / 0.6696 (0.4445) / 0.068 (0.8035) / 0.211 (0.6623) / 2.226 (0.1863)
E2- / BNF / NR / 1.158 (0.3232) / 1.865 (0.0.2210) / 1.033 (0.3438) / 0.410 (0.5455) / 2.798 (0.1454) / 4.781 (0.0714)
OHT / NR / 0.073 (0.7962) / 2.44 (0.1693) / 0.344 (0.5789) / 1.768 (0.2319) / 1.576 (0.2560) / 0.299 (0.6042)
Mix / NR / 0.776 (0.4124) / 0.747 (0.4207) / 2.40 (0.1723) / 13.06 (0.0112)* / 1.604 (0.2523) / 0.318 (0.5935)
BNF+ / E2 / NR / 17.51 (0.0058)* / 10.24 (0.0286)* / 16.13 (0.007)* / 0.005 (0.9459) / 1.458 (0.2727) / 0.322 (0.5909)
OHT / NR / 5.09 (0.0649) / 5.078 (0.0651) / 3.159 (0.1258) / 0.9441 (0.3687) / 0.679 (0.4414) / 0.041 (0.8460)
E2+OHT / NR / 5.187 (0.063) / 4.764 (0.0718) / 6.395 (0.0447)* / 2.645 (0.155) / 2.608 (0.1575) / 1.63 (0.2489)
BNF- / E2 / NR / 24.23 (0.0027)* / 11.69 (0.0142)* / 9.991 (0.0195)* / 0.878 (0.3849) / 0.913 (0.3761) / 9.949 (0.0197)*
OHT / 0.367 (0.5668) / 1.631 (0.2488) / 0.0001 (0.9917) / 0.002 (0.9699) / 0.010 (0.9255) / 0.754 (0.4185)
E2+BNF / NR / 2.307 (0.1796) / 2.047 (0.2025) / 1.868 (0.2207) / 0.0001 (0.9920) / 0.010 (0.9252) / 0.9662 (0.3636)
OHT+ / E2 / NR / 6.909 (0.0391)* / 3.664 (0.1041) / 1.091 (0.3365) / 2.632 (0.1559) / 0.003 (0.9591) / 1.551 (0.2594)
BNF / NR / 2.323 (0.1783) / 2.123 (0.1954) / 3.084 (0.1296) / 0.724 (0.4276) / 1.426 (0.2774) / 0.538 (0.4911)
E2+BNF / NR / 0.4139 (0.5438) / 1.602 (0.2526) / 0.1868 (0.6807) / 0.2022 (0.6687) / 0.3502 (0.5756) / 0.012 (0.9177)
OHT- / E2 / NR / 35.97 (0.001)* / 8.481 (0.0269)* / 5.582 (0.0561)* / 0.2351 (0.6450) / 1.832 (0.2247) / 0.554 (0.4848)
BNF / NR / 1.451 (0.2737) / 7.105 (0.0373)* / 0.334 (0.5844) / 0.004 (0.9537) / 4.246 (0.085) / 1.408 (0.2803)
E2+BNF / NR / 0.9758 (0.3614) / 6.734 (0.0409)* / 1.879 (0.2196) / 2.768 (0.1472) / 1.512 (0.2649) / 9.687 (0.0208)*
Abbreviations: E2 - 17β-estradiol; BNF – β-naphtoflavone; OHT – 4-Hydroxytamoxifen; Mix – BNF+OHT; ahr2β – aryl hydrocarbon receptor 2β; cyp1a – cytochrome P450 1a; er-α – estrogen receptor α; zrp – eggshell zona radiata protein; vtg-1 – vitellogenin 1; Vtg protein – vitellogenin protein; NR – No data reported.