Fig. 9.

Induction of genes downstream of the antioxidant-response transcription factor, nrf2, do not protect against VPA effects in the OT. (A) Treatment of embryos from 6-18 hpf with 5 µM D3T induces expression of some genes (Gclc and Gstp1 that play a direct role in reduced glutathione (GSH) synthesis and usage) downstream of nrf2 transcription factor gene in total embryos. (B) However, pretreatment for 12 h with 5 µM D3T before incubation with 250 µM VPA did not significantly change the redox potential when compared to VPA only treated embryos at any of the tested time points (C). Data points (A-C) represent n=3 pools of 30 embryos. *P<0.05. Data are shown as mean±s.e.m. (D) Pretreatment for 12 h with 1μM (I″-V″) or 5 μM (I′′′-V′′′) D3T before incubation with 250 μM VPA provides no protection in the OT compared to embryos treated with VPA alone (I′-V′) despite upregulation of nrf2 target genes. (E) Average lobe length is significantly decreased for the 1 µM D3T treatment group, compared to controls, when measured at 120 hpf. (F) For the average lobe width, all treatment groups were significantly decreased compared to controls when measured at 72 hpf and 120 hpf. When measured at 96 hpf the 5 µM D3T treatment groups was significantly decreased compared to controls. (G) Average lobe depth was significantly decreased compared to the controls for the 1 µM D3T treatment group when measured at 96 hpf. At 48 hpf, length, width and depth are difficult to measure since during this time neuroepithelium is transitioning into neuroblasts). In E-G a two-way ANOVA with Šidák's multiple comparisons test was used for statistical analysis *P<0.05, **P<0.01, ***P<0.001. Data are shown as mean±s.e.m.