Cytochrome P450 CYP1B1 interacts with 8-methoxypsoralen (8-MOP) and influences psoralen-ultraviolet A (PUVA) sensitivity

Abstract

Background: There are unpredictable inter-individual differences in sensitivity to psoralen-UVA (PUVA) photochemotherapy, used to treat skin diseases including psoriasis. Psoralens are metabolised by cytochrome P450 enzymes (P450), and we hypothesised that variability in cutaneous P450 expression may influence PUVA sensitivity. We previously showed that P450 CYP1B1 was abundantly expressed in human skin and regulated by PUVA, and described marked inter-individual differences in cutaneous CYP1B1 expression.

Objectives: We investigated whether CYP1B1 made a significant contribution to 8-methoxypsoralen (8-MOP) metabolism, and whether individuality in CYP1B1 activity influenced PUVA sensitivity.

Methods: We used E. coli membranes co-expressing various P450s and cytochrome P450 reductase (CPR) to study 8-MOP metabolism and cytotoxicity assays in CYP1B1-expressing mammalian cells to assess PUVA sensitivity.

Results: We showed that P450s CYP1A1, CYP1A2, CYP1B1, CYP2A6 and CYP2E1 influence 8-MOP metabolism. As CYP1B1 is the most abundant P450 in human skin, we further demonstrated that: (i) CYP1B1 interacts with 8-MOP (ii) metabolism of the CYP1B1 substrates 7-ethoxyresorufin and 17-β-estradiol showed concentration-dependent inhibition by 8-MOP and (iii) inhibition of 7-ethoxyresorufin metabolism by 8-MOP was influenced by CYP1B1 genotype. The influence of CYP1B1 on PUVA cytotoxicity was further investigated in a Chinese hamster ovary cell line, stably expressing CYP1B1 and CPR, which was more sensitive to PUVA than control cells, suggesting that CYP1B1 metabolises 8-MOP to a more phototoxic metabolite(s).

Conclusion: Our data therefore suggest that CYP1B1 significantly contributes to cutaneous 8-MOP metabolism, and that individuality in CYP1B1 expression may influence PUVA sensitivity.

Figure 1. 8-MOP influences CYP1B1 catalytic activity.

The influence of 8-MOP on: (A) EROD activity and (B) 17-β-estradiol hydroxylase activity in E. coli membranes co-expressing CYP1B1 and CPR. 17-β-estradiol 4-hydroxylase and 17-β-estradiol 2-hydroxylase activities were determined as described in Materials and Methods and are presented as mean ± SD of % control 17-β-estradiol 4-hydroxylation. (C) Dose-response curve for 8-MOP dependent CYP1B1 inhibition. EROD activity was assessed as described in Materials and Methods and data presented as mean ± SD percentage of control activity of three independent determinations. (D) Lineweaver-Burk plots for inhibition of CYP1B1 activity by 8-MOP. CYP1B1 catalysed EROD activity was determined at varying concentrations of 8-MOP and 7-ethoxyresorufin, as described in Materials and Methods. Data represent linear regression analysis of the mean of transformed data for three independent determinations.

Figure 2. The presence of catalytically active CYP1B1 in human skin.

(A) Inhibition of EROD activity by 8-MOP and CYP1B1 polyclonal antibodies in HaCaT keratinocyte microsomes. EROD activity was determined as described in Materials and Methods. Pre-incubation was performed with 8-MOP (5 µM), and anti-CYP1B1 and anti-CYP3A4 polyclonal antibodies as described in Materials and Methods. (B) Cytochrome P450 CYP1B1 and CPR expression in non-lesional skin of patients with psoriasis was assessed as described in Materials and Methods. (i) Negative (no antibody) control (ii) Hematoxylin-Eosin stain (iii) CYP1B1 antibody (1∶1000) stain and (iv) CPR antibody (1∶2000) stain.

Figure 3. CYP1B1 expression in CHO cells influences psoralen-UVA sensitivity.

(A) Western blot analysis of CYP1B1 and CPR expression in modified CHO cell lines was performed as described in Materials and Methods (lane 1, CHO-1B1/CPR cells; lane 2, CHO-CPR cells; lane 3, CHO cells; lane 4, control E. coli membranes). The influence of (B) UVA and (C) PUVA treatment on CHO cell viability and (D) PUVA treatment on CHO-1B1/CPR and CHO-CPR cells was assessed by NRUA . Data are presented from representative experiments and expressed as mean ± SD of duplicate determinations compared to dark control (for the majority of cell lines, experimental errors were very small and SD values are therefore too small to be visible).