Supplementary Material : Dual CYP1A and CYP3A Screening Incompatibility

1 Supplementary material: Dual CYP1A and CYP3A screening incompatibility

2 Materials

3 Dual screening for CYP1A and CYP3A activity (EROD and BFCOD)

4 In order to test the ability of screening for both enzymatic activities in the same

5 individuals, we dosed F. grandis embryos in solutions containing BaP (0, 10, 50, 100 µg/L) and

6 both ER (21 µg/L) and BFC (20 µg/L) with DMSO (0.1%) as a carrier. The experimental setup

7 for this procedure was identical to the one described above for F. grandis embryos.

8 Dual CYP1A and CYP3A activity fluorescence measurement

9 F. grandis embryos dosed with both ER and BFC were imaged as described for single

10 BFC stain, but each embryo was imaged using both rhodamine and GFP filters, respectively, to

11 measure response of both enzymatic activities within the same organism.

12 Results

13 Dual CYP1A and CYP3A screening assay

14 Including ER in the dosing solution for F. grandis embryos, in addition to BFC, caused

15 the BFCOD signal (i.e. HFC fluorescence) to be lower than that observed in embryos only

16 incubated with BFC (Figure S1A). Also, ER by itself caused an increase in the BFCOD signal

17 (Figure 4). On the other hand, adding BFC and ER together caused a higher EROD signal than

18 ER by itself, while BFC alone did not result in an increase in EROD signal (Figure S1B).

19 Discussion

of 5 20 The ability to simultaneously measure multiple enzymes in the same individuals would

21 be quite beneficial. Previous in vitro studies have suggested that dual assays for CYP1A and

22 CYP3A activities may be problematic and should be verified (Johansson et al., 2012).

23 Unfortunately, our testing suggests that both assays should not be used in tandem (Figure S1).

24 We show that ER alone is able to produce a BFCOD signal (Figure S1A). This result suggests

25 that the emission spectrum of resorufin, the product of CYP1A biotransformation of ER,

26 overlaps with the emission spectrum of HFC, the metabolite of BFC after biotransformation by

27 CYP3A (Figure 4). In addition, we show that addition of ER to BFC reduces overall BFCOD

28 response and increases EROD response (Figure S1B). We suggest that this may be due to

29 fluorescence resonance energy transfer (FRET) interaction where the emission spectrum of HFC

30 overlaps with the absorption spectrum of resorufin, thus causing false increase of the EROD

31 signal due to higher intensity of light in the absorption spectrum of resorufin (Figure S1B). The

32 multiple interferences shown by these data suggest that it is highly unlikely that a simple

33 correction factor would allow for the use of both EROD and BFCOD to be used in tandem. Thus,

34 we recommend that this in vivo BFCOD assay be used in separate individuals from those used

35 for in vivo EROD assays.

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37 Figure S1: CYP1A activity is induced by BaP alone, but is less induced by a combination of

38 BaP + FL, which is contrary to the CYP3A pattern shown in Figure 4.

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46 Figure S2: Individual responses of CYP3A activity in control and 10 μg/L BaP reveal a small

47 overlap between the two populations.

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52 Figure S3: Activities of A) CYP3A (BFCOD as measured by GFP filter) and B) CYP1A (EROD

53 as measured by rhodamine filter) showed dependence on whether both BFC and ER were added

54 in the same solution when dosed with BaP. A) ER by itself produced a concentration-response

55 curve, which can be attributed to spillover from the emittance spectrum of resorufin into the GFP

56 detection spectrum. In addition, ER added to BFC reduced the HFC fluorescence, likely due to

57 FRET transfer of HFC signal, exciting resorufin and thus being itself quenched. B) The higher

58 resorufin emission when BFC was added to ER, supports the FRET explanation for quenching

59 HFC by exciting resorufin signal. A minimum of two independent experiments with at least 20

60 embryos per concentration are represented in each panel.