Human CYP2S1 metabolizes cyclooxygenase- and lipoxygenase-derived eicosanoids

Abstract

CYP2S1 is a recently described dioxin-inducible cytochrome P450. We previously demonstrated that human CYP2S1 oxidizes a number of carcinogens but only via the peroxide shunt. In this article, we investigated whether human CYP2S1 can metabolize cyclooxygenase- and lipoxygenase-derived lipid peroxides in a NADPH-independent fashion. Human CYP2S1 metabolizes prostaglandin G(2) (PGG(2)) (K(m) = 0.267 ± 0.072 μM) into several products including 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid (12-HHT). It also metabolizes prostaglandin H(2) (PGH(2)) (K(m) = 11.7 ± 2.8 μM) into malondialdehyde, 12-HHT, and thromboxane A(2) (TXA(2)). The turnover to 12-HHT by human CYP2S1 (1.59 ± 0.04 min(-1)) is 40-fold higher than that of TXA(2) (0.04 min(-1)). In addition to PGG(2) and PGH(2) metabolism, human CYP2S1 efficiently metabolizes the hydroperoxyeicosatetraenoic acids (5S-, 12S-, and 15S-) and 13S-hydroperoxyoctadecadienoic acid into 5-oxo-eicosatetraenoic acid (turnover = 16.7 ± 0.3 min(-1)), 12-oxo-eicosatetraenoic acid 1 (11.5 ± 0.9 min(-1)), 15-oxo-eicosatetraenoic acid (16.9 ± 0.8 min(-1)), and 13-octadecadienoic acid (20.2 ± 0.9 min(-1)), respectively. Other cytochromes P450 such as CYP1A1, 1A2, 1B1, and 3A4 underwent similar conversions but at slower rates. The fatty acid hydroperoxides were also converted by human CYP2S1 to several epoxyalcohols. Our data indicate that fatty acid endoperoxides and hydroperoxides represent endogenous substrates of CYP2S1 and suggest that the enzyme CYP2S1 may play an important role in the inflammatory process because some of the products that CYP2S1 produces play important roles in inflammation.

Fig. 1.

Metabolism of PGG₂ by recombinant human CYP2S1. HPLC chromatograms of 133 μM PGG₂ incubated with 0.1 μM human CYP2S1 (line I) or without CYP2S1 (line III) or with heat-treated human CYP2S1 (line II) with detection set at 275 nm (A) or at 220 nm (B). 1, unknown, but probably either 12-oxo-HT or 15-oxo-PGG₂; 2, unknown; and 3, 12-HHT, which was identified with an authentic standard. mAU, milli-arbitrary unit.

Fig. 2.

Metabolism of PGG₂ and PGH₂ by recombinant human CYP2S1 (h2S1). PGG₂ and PGH₂ incubated without enzyme were used as controls. The concentrations of (A) TXB₂ and (B) PGE₂ and PGD₂ were determined by LC-ESI-MS/MS MRM in the negative ion mode. Data are presented as means ± S.D. *, p < 0.05; **, p < 0.01, respectively. Turnover of TXB₂ from metabolism of PGH₂ by human CYP2S1 was determined to be 0.04 min⁻¹.

Fig. 3.

Metabolism of PGH₂ by recombinant human CYP2S1. PGH₂ was incubated for 5 min with human CYP2S1 (line I) or heat-treated human CYP2S1 (line II) or no enzyme (line III). 12-HHT was detected at 230 nm and identified using an authentic standard. Samples were analyzed by HPLC method 2. mAU, milli-arbitrary unit.

Fig. 4.

Metabolism of PGH₂ by extracts of mammalian cells expressing CYP2S1. Lysates of c33-MSCV or c33-h2S1 cells incubated with PGH₂. Concentrations of PGE₂ and PGD₂ were determined by LC-ESI-MS/MS using MRM negative ion mode. The data are representative of two separate experiments.

Fig. 5.

Reaction scheme for PGG₂ metabolism by CYP2S1. *, these products are speculative and have not been confirmed with authentic standards. Bold lines indicate reactions catalyzed by CYP2S1; dashed lines represent spontaneous reactions and/or reactions catalyzed by other enzymes.

Fig. 6.

Metabolism of fatty acid hydroperoxides by recombinant human CYP2S1. 5-HpETE (A), 12-HpETE (B), 15-HpETE (B), or 13-HpODE (D) was incubated with human CYP2S1 (line I), without CYP2S1 (line II), with 5-, 12-, and 15-oxoETEs or 13-oxoETE product standards (line III), or with heat-treated human CYP2S1 (line IV). 1, 5-oxoETE; 2, 12-oxoETE; 3, 15-oxoETE; and 4, 13-oxoODE. Detection was set at 279 nm. A to C were separated using HPLC method 1. D was separated with HPLC method 2. Arrows indicate unknown products. mAU, milli-arbitrary unit.

Fig. 7.

Other products from the metabolism of 5- and 15-HpETE by recombinant human CYP2S1. Samples were separated with HPLC method 2 and detected at 205 nm. 15-HpETE was incubated with human CYP2S1 (line I) or without human CYP2S1 (line II). 5-HpETE was incubated with human CYP2S1 (line II) or without human CYP2S1 (line IV). The unknown products are indicated by arrows. mAU, milli-arbitrary unit.

Fig. 8.

Product detection of 15-HpETE metabolism by recombinant human CYP2S1 using mass spectrometry. LC-ESI-MS/MS using SIM with a negative ion mode [M − H]⁻ on a 15-HpETE sample incubated with purified CYP2S1 (A and C) or without CYP2S1 (B and D). Products were detected by selection of m/z 335 [M − H]⁻ (A and C) and m/z 353 [M − H]⁻ (B and D). The m/z [M − H]⁻ of 15 HpETE is 335, the m/z [M − H]⁻ of epoxyalcohol or hepoxilin is also 335, and the m/z [M − H]⁻ of trihydroxy derivatives or trioxilin is 353. The arrows indicate 15-HpETE and the circles indicate epoxyalcohols and trioxilins.

Fig. 9.

CYP2S1 reduces the generation of 5-HETE and 15-HETE from 5-HpETE and 15-HpETE. 5-HpETE or 15-HpETE (50 μM) was incubated with 0.1 μM purified recombinant human CYP2S1 or without enzyme for 10 min. Concentrations were determined by LC-ESI-MS/MS with the MRM negative ion mode. Data are presented as means ± S.D. of triplicate determinations. *, p < 0.05.

Fig. 10.

Reaction scheme for fatty acid hydroperoxide metabolism by CYP2S1. Bold lines indicate reactions catalyzed by CYP2S1; dashed lines represent spontaneous reactions and/or reactions catalyzed by other enzymes.