The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria

Abstract

The breast cancer resistance protein (BCRPABCG2) is a member of the ATP-binding cassette family of drug transporters and confers resistance to various anticancer drugs. We show here that mice lacking Bcrp1Abcg2 become extremely sensitive to the dietary chlorophyll-breakdown product pheophorbide a, resulting in severe, sometimes lethal phototoxic lesions on light-exposed skin. Pheophorbide a occurs in various plant-derived foods and food supplements. Bcrp1 transports pheophorbide a and is highly efficient in limiting its uptake from ingested food. Bcrp1(-/-) mice also displayed a previously unknown type of protoporphyria. Erythrocyte levels of the heme precursor and phototoxin protoporphyrin IX, which is structurally related to pheophorbide a, were increased 10-fold. Transplantation with wild-type bone marrow cured the protoporphyria and reduced the phototoxin sensitivity of Bcrp1(-/-) mice. These results indicate that humans or animals with low or absent BCRP activity may be at increased risk for developing protoporphyria and diet-dependent phototoxicity and provide a striking illustration of the importance of drug transporters in protection from toxicity of normal food constituents.

Fig 1.

Generation and analysis of Bcrp1^−/− mice. (a) A 5.1-kb fragment containing exons 3–6 (exons are indicated by filled boxes) was replaced with an inverted pgk-hygro cassette. Restriction sites: S, ScaI; A, Asp718; N, NheI. For Southern analysis, 5′ and 3′ probes were used on ScaI-digested genomic DNA. Diagnostic restriction fragments are indicated by double-headed arrows. (b) Western analysis on crude membrane fractions from liver, kidney, and small intestine (20 μg per lane). (c) Bile from wild-type and Bcrp1^−/− mice. (d–g) Immunohistochemical detection (×40) of Bcrp1 in small intestine (d), liver (e), kidney (f), and placenta (g).

Fig 2.

Pharmacologic effects of Bcrp1. (a) Plasma concentration versus time curve after oral administration of 1 mg/kg topotecan to mice (means ± SD, n = 5–6; P < 0.001 for area under the curves, Student's t test). (b) Ratio of [¹⁴C]topotecan concentration in fetus over maternal plasma at 15 min after i.v. administration of 0.2 mg/kg [¹⁴C]topotecan to pregnant dams (gestation day 15.5; mean ± SD, n = 3 dams and 11 Bcrp1^−/−, 13 Bcrp1^+/−, and 7 Bcrp1^+/+ fetuses; *, P < 0.001 compared with Bcrp1^+/+ fetuses, Student's t test).

Fig 3.

Phototoxicity and transport of pheophorbide a. (a) Normal ear. (b–e) Progression of phototoxic ear lesions in a period of 3–5 days in Bcrp1^−/− mice. (f) Incidence of phototoxic ear lesions on diet containing 10% and 20% alfalfa (n = 10 mice). (g) Chlorophyll breakdown in plants, reactions, and enzymes are indicated with an arrow. Also shown is the structure of PPIX. (h) Pheophorbide a accumulation in cells exposed to pheophorbide a (10 μM) for 1 h at 37°C in the presence or absence of Bcrp1/BCRP-inhibitor Ko143 (10 μM) (mean fluorescence ± SD, n = 3; **, P < 0.01, Student's t test).

Fig 4.

Effect of bone marrow transplantation on protoporphyria and phototoxicity. (a) Erythrocyte levels of PPIX in Bcrp1^−/− and wild-type mice receiving normal, phototoxic (Phototox.), or semisynthetic (Synth.) diet (n = 5). (b) Erythrocyte levels of PPIX after bone marrow transplantation (*, P < 0.05, Student's t test). KO, knockout. (c) Incidence of phototoxic ear lesions in wild-type and Bcrp1^−/− mice (recipients) transplanted with Bcrp1^−/− or wild-type (donors) bone marrow. To induce phototoxicity, mice were fed a 20% alfalfa diet (n = 3–5; P < 0.01 for difference between knockout/knockout and knockout/wild type, Student's t test).