Figure 1S. Change in SOD Gene Expression in Hepatocytes Exposed to Treated and Untreated

Figure 1s. Change in SOD gene expression in hepatocytes exposed to treated and untreated wastewaters.

Gene expression was measured by qPCR. The filled and dotted lines represent the mean ± 1 standard deviation of the ethanol solvent control. Asterisks (*) indicate a significant (p<0.05) difference between the observed responses in the influent and effluent.

Figure 2s. Change in CYP1A1 gene expression in hepatocytes exposed to treated and untreated wastewaters.

Figure 3s. Change in CYP3A4 gene expression in hepatocytes exposed to treated and untreated wastewaters.

Figure 4s. Change in GST gene expression in hepatocytes exposed to treated and untreated wastewaters.

Figure 5s. Change in MDR gene expression in hepatocytes exposed to treated and un treated wastewaters.

Figure 6s. Change in VTG gene expression in hepatocytes exposed to treated and untreated wastewaters.

Gene expression was measured by qPCR. Asterisks (*) indicate a significant (p<0.05) difference between the observed responses in the influent and effluent.

Figure 7s. Change in ER gene expression in hepatocytes exposed to treated and untreated wastewaters.

Table 1s. List of target genes and reference genes

Gene name / Symbol / Accession Number / Forward primer / Reverse primer / Amplicon
Size (bp) / Reference
Target genes
Metallothionein / MT / M18104 / GCTCTAAAACTGGCTCTTGC / GTCTAGGCTCAAGATGGTAC / 236 / na
Superoxide dismutase
(Cu/Zn cytosolic) / SOD / AF469663 / TGGTCCTGTGAAGCTGATTG / TTGTCAGCTCCTGCAGTCAC / 201 / Fontagné et al. (2008)
Cytochrome P450 1A / CYP1A / U62796 / GATGTCAGTGGCAGCTTTGA / TCCTGGTCATCATGGCTGTA / 104 / Aluru and Vijayan (2006)
Cytochrome P450 3A / CYP3A / U96077.1 / TACATGCCATTTGGGGCGGGG / ACGGGCCTCCAGCCTCAGTTT / 195 / na
Glutathion-S-Transferase-P / GST / BQ036247 / ATTTTGGGACGGGCTGACA / CCTGGTGCTCTGCTCCAGTT / 81 / Salaberria et al. (2009)
P-glycoprotein (Abcb1) / GP1 / AY863424 / ACGTGCGCTCCCTGAACGTG / GCGTTGGCCTCCCTAGCAGC / 157 / na
Vitellogenin / VG / X92805 / AGCCCATCCACGAACTTGCTGTT / AGGGCCAAAACTGCATCAGCCT / 190 / na
Estradiol receptor β2 / ER / NM_001124570 / CTGACCCCAGAACAGCTGATC / TCGGCCAGGTTGGTAAGTG / 125 / Nagler et al. (2007)
Reference gene
Elongation factor I α / EFIα / BG933853 / GAATCGGCTATGCCTGGTGAC / GGATGATGACCTGAGCGGTG / 141 / Bower and Johnston (2009)
RNA polymerase I / RPL / CB516726 / ACTATGGCTGTCGAGAAGGT
GCT / TGTACTCGAACAGTCGTGGGTCA / 120 / Jorgensen et al. (2006)
Prolylpeptidyl isomerase I / PPIA / DY727143 / CATCCCAGGTTTCATGTGC / CCGTTCAGCCAGTCAGTGTT / 203 / Bower and Johnston (2009)
Hypoxanthine phosphoribosyl transferase I / HPRT / EG866745 / CCGCCTCAAGAGCTACTGTAAT / GTCTGGAACCTCAAACCCTATG / 255 / Bower and Johnston (2009)
18s rRNA / 18S / AF308735 / CACGCGAGATGGAGCAATAA / CGCAGAGTAGACACACGCTGAT / 96 / Salaberria et al. (2009)
Note: na, not applicable. Primers were designed by the authors.

Table 2s. Selection of positive controls for this hepatocytes gene expression tests.

Positive control / Toxic end point or
gene expression changes / Response factors compared to controls (mean±2 stand. Deviation)
Copper sulphate
10 μg/mL / Cytotoxicity
MT-2
SOD / 2±0.2
1.8±0.3
1.5±0.3
Estradiol-17β
1 μM / Vtg
ERβ / 3±1
2±1
β-naphthoflavone
1 μg/mL / CYP1A1 / 1.8±0.3
Carbamazepine / CYP3A4 / 1.5±0.2