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Electronic supplementary material to:
Environmental levels of oestrogenic and antiandrogenic compounds feminise digit ratios in male rats and their unexposed male progeny
By Jacques Auger, Dominique Le Denmat, Raymond Berges, Ludivine Doridot, Benjamin Salmon, Marie Chantal Canivenc-Lavier, Florence Eustache
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Supplementary material, table 1. Left and right forepaw digit length ratios in adult control (unexposed) male and female Wistar rats (n=20 animals per group).
Male / Female / P*Left
2D : 3D / 0.864±0.020 / 0.877±0.022 / 0.023
2D : 4D / 0.897±0.026 / 0.927±0.025 / 0.002
2D : 5D / 1.214±0.060 / 1.235±0.078 / 0.35
3D : 4D / 1.038±0.018 / 1.056±0.022 / 0.005
3D : 5D / 1.404±0.063 / 1.408±0.094 / 0.98
4D : 5D / 1.354±0.062 / 1.333±0.084 / 0.39
Right
2D : 3D / 0.857±0.024 / 0.871±0.025 / 0.048
2D : 4D / 0.879±0.023 / 0.918±0.027 / 0.0001
2D : 5D / 1.170±0.071 / 1.169±0.049 / 0.74
3D : 4D / 1.027±0.015 / 1.054±0.025 / 0.0004
3D : 5D / 1.366±0.065 / 1.343±0.060 / 0.16
4D : 5D / 1.331±0.061 / 1.274±0.041 / 0.0015
* Mann-Whitney non-parametric comparisons
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Supplementary material, table 2. Right and left forepaw digit lengths (mm) in the F1 male generation exposed from conception to adulthood and F2 unexposed male generation sired by exposed fathers (F1) with control unexposed mothers. The last two columns report the result of a one-way ANOVA (F) and relative significance (p); Tukey post hoc, $ § p<0.01, * ‡ p<0.05 (° r p<0.10); (abbreviations and doses in exposed fathers: BPA: bisphenol A, 5 µg/kg/day; V: vinclozolin, 10 µg/kg/day, ‘low’ or 1000 µg/kg/day, ‘high’ and G: genistein, 1000 µg/kg/day)
ExposureNegative
Control / BPA / ‘low’V+G / BPA+ ‘low’V / BPA+G / BPA+ ‘low’V+G / ‘high’V+G
(Eustache et al.,
EHP 2009) / F / p
Left
2D / F1
F2 / 8.23±0.22
8.39±0.32 / 8.41±0.10
8.42±0.41 / 8.33±0.26
8.44±0.21 / 8.19±0.21
8.44±0.23 / 8.33±0.22
8.32±0.29 / 8.33±0.26
8.54±0.28 / 8.34±0.33
8.29±0.31 / 1.38
1.35 / 0.23
0.24
3D / F1
F2 / 9.38±0.22
9.67±0.31* / 9.29±0.23
9.26±0.45* / 9.41±0.23
9.47±0.26 / 9.33±0.23
9.54±0.28 / 9.35±0.27
9.37±0.34 / 9.53±0.22
9.54±0.42 / 9.47±0.26
9.55±0.37 / 1.63
2.38 / 0.15
0.03
4D / F1
F2 / 9.14±0.26
9.36±0.24 / 9.12±0.23
9.21±0.40 / 9.13±0.19
9.16±0.26 / 9.00±0.25
9.32±0.28 / 9.10±0.17
9.09±0.29 / 9.09±0.28
9.32±0.21 / 9.13±0.32
9.26±0.32 / 0.52
1.89 / 0.79
0.09
5D / F1
F2 / 7.02±0.31
7.26±0.41 / 7.08±0.18
7.25±0.41 / 7.03±0.25
7.19±0.34 / 7.01±0.26
7.07±0.29 / 7.04±0.18
7.02±0.24 / 7.04±0.19
7.28±0.25 / 7.02±0.23
7.24±0.31 / 0.12
1.65 / 0.99
0.14
Right
2D / F1
F2 / 8.19±0.26
8.41±0.33 / 8.24±0.13
8.33±0.49 / 8.32±0.19
8.30±0.26 / 8.20±0.26
8.42±0.22 / 8.29±0.11
8.23±0.18 / 8.25±0.23
8.50±0.26 / 8.37±0.15
8.42±0.26 / 1.36
1.62 / 0.24
0.15
3D / F1
F2 / 9.15±0.34
9.62±0.32$ § / 9.24±0.32
9.36±0.40 / 9.31±0.21
9.22±0.33§ / 9.22±0.27
9.47±0.24 ° / 9.25±0.26
9.16±0.20 $ ° r / 9.41±0.25
9.40±0.37 / 9.28±0.20
9.48±0.31r / 1.33
4.03 / 0.25
0.001
4D / F1
F2 / 9.08±0.25*
9.35±0.28 $ / 8.86±0.23°
9.06±0.40 / 9.00±0.24
9.07±0.31 / 8.77±0.36 $*
9.06±0.26 / 8.83±0.29‡
8.80±0.25$ § / 8.93±0.16
9.20±0.39 § / 9.15±0.23 $ ‡ °
9.09±0.37 / 3.71
4.17 / 0.003
0.0008
5D / F1
F2 / 7.07±0.39
7.35±0.35° / 7.07±0.27
7.13±0.30 / 6.99±0.33
7.16±0.35 / 6.98±0.23
7.04±0.28 * ° / 7.08±0.20
7.07±0.22r / 7.09±0.29
7.37±0.27*r / 7.08±0.25
7.12±0.34 / 0.30
3.14 / 0.93
0.007
Supplementary material, table 3. Left and right forepaw digit length ratios in the F1 male generation exposed from conception to adulthood. The last two columns report the result of a one-way ANOVA (F) and relative significance (p); Tukey post hoc, £ $ @ ‡ Y § p<0.01, * † p<0.05 (° - p<0.10); (abbreviations and doses: BPA: bisphenol A, 5 µg/kg//day; V: vinclozolin, 10 µg/kg/day, ‘low’ or 1000 µg/kg/day, ‘high’ and G: genistein, 1000 µg/kg/day, see [15] in the reference list)
Left
2D : 3D / 0.878±0.023* / 0.906±0.021*† ° - / 0.886±0.024 / 0.878±0.018† / 0.892±0.027 / 0.880±0.019 - / 0.881±0.014 ° / 2.78 / 0.02
2D : 4D / 0.901±0.022 / 0.922±0.018 / 0.913±0.024 / 0.911±0.021 / 0.915±0.021 / 0.923±0.023 / 0.913±0.021 / 1.69 / 0.13
2D : 5D / 1.175±0.042 / 1.189±0.029 / 1.187±0.050 / 1.169±0.029 / 1.183±0.032 / 1.192±0.037 / 1.188±0.057 / 0.52 / 0.79
3D : 4D / 1.026±0.022 ° / 1.019±0.024 $ / 1.030±0.014 / 1.038±0.019 / 1.027±0.020 / 1.049±0.028 $ ° / 1.037±0.021 / 2.79 / 0.02
3D : 5D / 1.339±0.053 / 1.313±0.044 / 1.341±0.062 / 1.333±0.033 / 1.329±0.045 / 1.355±0.035 / 1.350±0.060 / 1.06 / 0.39
4D : 5D / 1.305±0.047 / 1.289±0.030 / 1.301±0.058 / 1.285±0.030 / 1.294±0.038 / 1.292±0.018 / 1.301±0.054 / 0.41 / 0.87
Right
2D : 3D / 0.896±0.035 / 0.893±0.023 / 0.894±0.012 / 0.890±0.027 / 0.897±0.019 / 0.876±0.022 ° / 0.902±0.024 ° / 1.55 / 0.17
2D : 4D / 0.902±0.024 $* ° / 0.931±0.022 ° / 0.925±0.024 / 0.936±0.029 * / 0.941±0.025$ / 0.924±0.023 / 0.915±0.025 / 3.64 / 0.003
2D : 5D / 1.161±0.038 / 1.168±0.052 / 1.193±0.060 / 1.175±0.031 / 1.172±0.023 / 1.183±0.038 / 1.168±0.052 / 0.89 / 0.51
3D : 4D / 1.007±0.027$ £ @ ‡ ° / 1.042±0.029 $ - / 1.035±0.024 ° / 1.052±0.033£ Y / 1.048±0.020‡ † / 1.054±0.021@ § / 1.015±0.021§ † Y - / 7.04 / 0.0001
3D : 5D / 1.297±0.062 / 1.308±0.069 / 1.335±0.058 / 1.321±0.043 / 1.306±0.021 / 1.330±0.055 / 1.313±0.054 / 0.87 / 0.52
4D : 5D / 1.288±0.057° / 1.254±0.051 / 1.290±0.051* / 1.257±0.045 / 1.246±0.021°*- / 1.262±0.041- / 1.294±0.048 / 2.28 / 0.04
Supplementary material, table 4. Left and right forepaw digit length ratios in the F2 unexposed male generation sired by exposed fathers (F1) with control unexposed mothers. The last two columns report the result of a one-way ANOVA (F) and relative significance (p); Tukey post hoc, $ @ p<0.01, † p<0.05 (° - p<0.10); (abbreviations and doses in fathers: BPA: bisphenol A, 5 µg/kg//day; V: vinclozolin, 10 µg/kg/day, ‘low’ or 1000 µg/kg/day, ‘high’ and G: genistein, 1000 µg/kg//day, see [15] in the reference list)
Left
2D : 3D / 0.867±0.021$ † / 0.910±0.038$ @ / 0.891±0.023 / 0.885±0.026 / 0.888±0.027 / 0.897±0.030† / 0.869±0.028@ / 4.95 / 0.0002
2D : 4D / 0.896±0.024° / 0.915±0.032 / 0.922±0.025°‡ / 0.905±0.015 / 0.916±0.022 / 0.917±0.019 / 0.896±0.031‡ / 2.93 / 0.01
2D : 5D / 1.158±0.063 / 1.163±0.049 / 1.176±0.055 / 1.196±0.053° / 1.186±0.041 / 1.175±0.035 / 1.147±0.053° / 1.91 / 0.08
3D : 4D / 1.033±0.028 / 1.006±0.037° / 1.035±0.017° / 1.023±0.028 / 1.032±0.021 / 1.024±0.037 / 1.031±0.026 / 2.14 / 0.05
3D : 5D / 1.336±0.079† / 1.280±0.054$ °† / 1.320±0.056 / 1.351±0.052$ / 1.335±0.033° / 1.311±0.051 / 1.320±0.056 / 3.06 / 0.008
4D : 5D / 1.293±0.066 / 1.272±0.047° / 1.276±0.043 / 1.320±0.047° / 1.295±0.047 / 1.282±0.038 / 1.280±0.060 / 1.88 / 0.09
Right
2D : 3D / 0.874±0.027‡ / 0.889±0.039 / 0.902±0.037 / 0.890±0.018 / 0.899±0.022 / 0.906±0.036‡ / 0.889±0.014 / 2.09 / 0.06
2D : 4D / 0.899±0.031ú ° / 0.920±0.040 / 0.916±0.031 / 0.930±0.022° / 0.936±0.018ú / 0.925±0.033 / 0.927±0.029 / 2.40 / 0.03
2D : 5D / 1.146±0.073 / 1.170±0.074 / 1.162±0.072 / 1.198±0.032 / 1.165±0.029 / 1.155±0.044 / 1.184±0.044 / 1.62 / 0.15
3D : 4D / 1.029±0.026 / 1.034±0.025 / 1.020±0.039 ° / 1.045±0.021° / 1.042±0.030 / 1.022±0.040 / 1.044±0.025 / 2.39 / 0.03
3D : 5D / 1.311±0.074 / 1.315±0.065 / 1.289±0.067° / 1.347±0.042‡ ° / 1.297±0.044 / 1.277±0.061‡ ° / 1.333±0.049 / 3.09 / 0.008
4D : 5D / 1.275±0.071 / 1.272±0.066 / 1.268±0.061 / 1.289±0.046 / 1.245±0.042 / 1.249±0.048 / 1.277±0.039 / 1.44 / 0.20
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Supplementary material, table 5. Association between relative anogenital distance, measured at PND21, and right digit length 2D:4D ratio, measured in adulthood, for all exposure groups and controls from the F1 exposed and F2 unexposed male generations using Spearman's rank correlation coefficient ( r )
Relative anogenital distanceF1 / F2
All / Control / All / Control
Right 2D:4D / r = -0.11 p=0.31 / r = -0.05
p = 0.88 / r = -0.07
p = 0.44 / r = -0.15
p = 0.62