To combine the effects of NP with expression data, several possible housekeeping genes used for quantification were investigated. The measured concentrations of the housekeeping genes rpl13 (mitochondrial), efla and в-actin based on the amount of RNA used for reverse transcription is shown in Figure 2. No significant differences were found. However, the expression of в-actin gene appeared smaller in the p262-NP/ p22-NP/ p252-NP group compared to the other groups (p33-NP/ p353-NP/ p363-NP, t-NP and DMSO alone). Zebrafish embryos treated with NP methylated on the 2’ position (p22-NP, p252-NP, p262-NP) compared with zebrafish embryos treated with NP methylated on the 3’ position (p33-NP, p353-NP, p363-NP) showed a mean difference of 0.47 (relative expression). This difference failed to be statistical significant. Assigning the data of the NP known for antiestrogenic effects (p22-NP and p262-NP,) to one group and data for tNP known to exert estrogenic effects (p33-NP, p252-NP, p353-NP and p363-NP) to the other group reveals that the expression of fi-actin gene in the antiestrogenic group is significantly different from the DMSO control while the comparison between the estrogenic group and DMSO control shows no significant differences (0,07±0.04 and 0,54±0.17 respectively).

However, since no marked differences were observed using the other housekeeping genes all further data were related to the mean of the expression observed with rpl13a gene and ef1a gene. This combination shows a homogeneous distribution over all groups (Figure 2d) and was used to normalize the expression data.
Fig2Nonylphenol Induces Expression_decrypted
Figure 2 : Expression levels of the housekeeping genes rpl13a (a), efla (b), p~actin (c) of zebra fish embryos treated with different synthetic isomers of nonylphenols (NPs) with a concentration of 10 nmol/L for 1 hr. Technical NP (t-NP) is a mixture of more than 20 NP isomers. Dimethyl sulfoxide (DMSO) was used as vehicle control. Fig 1d represents the mean of pr113a and efla mRNA levels. Values are means of 5 independent experiments with SEM

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