<p> 1 Supplementary material: Dual CYP1A and CYP3A screening incompatibility</p><p>2 Materials</p><p>3 Dual screening for CYP1A and CYP3A activity (EROD and BFCOD)</p><p>4 In order to test the ability of screening for both enzymatic activities in the same </p><p>5 individuals, we dosed F. grandis embryos in solutions containing BaP (0, 10, 50, 100 µg/L) and </p><p>6 both ER (21 µg/L) and BFC (20 µg/L) with DMSO (0.1%) as a carrier. The experimental setup </p><p>7 for this procedure was identical to the one described above for F. grandis embryos.</p><p>8 Dual CYP1A and CYP3A activity fluorescence measurement</p><p>9 F. grandis embryos dosed with both ER and BFC were imaged as described for single </p><p>10 BFC stain, but each embryo was imaged using both rhodamine and GFP filters, respectively, to </p><p>11 measure response of both enzymatic activities within the same organism.</p><p>12 Results</p><p>13 Dual CYP1A and CYP3A screening assay</p><p>14 Including ER in the dosing solution for F. grandis embryos, in addition to BFC, caused </p><p>15 the BFCOD signal (i.e. HFC fluorescence) to be lower than that observed in embryos only </p><p>16 incubated with BFC (Figure S1A). Also, ER by itself caused an increase in the BFCOD signal </p><p>17 (Figure 4). On the other hand, adding BFC and ER together caused a higher EROD signal than </p><p>18 ER by itself, while BFC alone did not result in an increase in EROD signal (Figure S1B).</p><p>19 Discussion</p><p>Page 1 of 5 20 The ability to simultaneously measure multiple enzymes in the same individuals would </p><p>21 be quite beneficial. Previous in vitro studies have suggested that dual assays for CYP1A and </p><p>22 CYP3A activities may be problematic and should be verified (Johansson et al., 2012). </p><p>23 Unfortunately, our testing suggests that both assays should not be used in tandem (Figure S1). </p><p>24 We show that ER alone is able to produce a BFCOD signal (Figure S1A). This result suggests </p><p>25 that the emission spectrum of resorufin, the product of CYP1A biotransformation of ER, </p><p>26 overlaps with the emission spectrum of HFC, the metabolite of BFC after biotransformation by </p><p>27 CYP3A (Figure 4). In addition, we show that addition of ER to BFC reduces overall BFCOD </p><p>28 response and increases EROD response (Figure S1B). We suggest that this may be due to </p><p>29 fluorescence resonance energy transfer (FRET) interaction where the emission spectrum of HFC </p><p>30 overlaps with the absorption spectrum of resorufin, thus causing false increase of the EROD </p><p>31 signal due to higher intensity of light in the absorption spectrum of resorufin (Figure S1B). The </p><p>32 multiple interferences shown by these data suggest that it is highly unlikely that a simple </p><p>33 correction factor would allow for the use of both EROD and BFCOD to be used in tandem. Thus,</p><p>34 we recommend that this in vivo BFCOD assay be used in separate individuals from those used </p><p>35 for in vivo EROD assays. </p><p>Page 2 of 5 36</p><p>37 Figure S1: CYP1A activity is induced by BaP alone, but is less induced by a combination of </p><p>38 BaP + FL, which is contrary to the CYP3A pattern shown in Figure 4.</p><p>39</p><p>40</p><p>41</p><p>42</p><p>43</p><p>Page 3 of 5 44</p><p>45</p><p>46 Figure S2: Individual responses of CYP3A activity in control and 10 μg/L BaP reveal a small </p><p>47 overlap between the two populations.</p><p>48</p><p>49</p><p>50</p><p>Page 4 of 5 51</p><p>52 Figure S3: Activities of A) CYP3A (BFCOD as measured by GFP filter) and B) CYP1A (EROD</p><p>53 as measured by rhodamine filter) showed dependence on whether both BFC and ER were added </p><p>54 in the same solution when dosed with BaP. A) ER by itself produced a concentration-response </p><p>55 curve, which can be attributed to spillover from the emittance spectrum of resorufin into the GFP</p><p>56 detection spectrum. In addition, ER added to BFC reduced the HFC fluorescence, likely due to </p><p>57 FRET transfer of HFC signal, exciting resorufin and thus being itself quenched. B) The higher </p><p>58 resorufin emission when BFC was added to ER, supports the FRET explanation for quenching </p><p>59 HFC by exciting resorufin signal. A minimum of two independent experiments with at least 20 </p><p>60 embryos per concentration are represented in each panel.</p><p>61</p><p>Page 5 of 5</p>