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Pharmacological and physiological functions of polyspecific drug transporters

Jonker, J.W.

Publication date 2003

Link to publication

Citation for published version (APA): Jonker, J. W. (2003). Pharmacological and physiological functions of polyspecific drug transporters.

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UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:01 Oct 2021 Chapterr 4

Thee breast cancer resistance protein protects against a major chlorophyll-derivedd dietary phototoxin andd protoporphyria

Johann W. Jonker, Marije Buitelaar, Els Wagenaar, Martin A. van der Valk, Georgee L. Scheffer, Rik J. Scheper, Torsten Plösch, Folkert Kuipers, Ronaldd P.J. Oude Elferink, Hilde Rosing, Jos H. Beijnen and Alfred H. Schinkel

Proc.Proc. Natl. Acad. Set. USA (2002) 99: 15649-J5654

BCRPBCRP protects against a dietary phototo.xin

Thee breast cancer resistance protein protects against aa major chlorophyll-derived dietary phototoxin andd protoporphyria

Johann W. Jonker*, Marije Buitelaar*. Els Wagenaar*, Martin A. van der Valkf, George L. Scheffer', Rik J. Scheper4, Torstenn Plösch5, Folkert Kuipers1, Ronald P. J. Oude Elferink1, Hilde Rosing , Jos H. Beijnen, and Alfred H. Schinkel***

Divisionss of * Experimental Therapy and 'Experimental Animal Pathology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands; tt of Pathology, Free University Hospital, 1081 HV Amsterdam, The Netherlands; ^Center for Liver, Digestive, and Metabolic Diseases, Laboratoryy of Pediatrics, University Hoipital Groningen, 9713 GZ Groningen, The Netherlands; 'Laboratory of Experimental Hematology, Academicc Medical Center, 1105 AZ Amsterdam, The Netherlands; and Department of Pharmacy and Pharmacology, Slotervaart Hoipital,, 1066 EC Amsterdam, The Netherlands

Communicatedd by P. Borst, The Netherlands Cancer Institute, Amsterdam, The Netherlands, October 8, 2002 (received for review September 10, 2002)

Thee breast cancer resistance protein (BCRP ABCG2) is a member of logicallyy important in processes involving handling of porphy- thee ATP-binding cassette family of drug transporters and confers rins,, and we expect that a partial or complete deficiency for resistancee to various anticancer drugs. We show here that mice BCRPP may contribute to several porphyrin-related phototoxici- tackingg Bcrpl ..Abcg2 become extremely sensitive to the dietary tiess in humans and animals. chlorophyll-breakdownn product pheophorbide a, resulting in severe,, sometimes lethal phototoxic lesions on light-exposed skin. Materialss and Methods Pheophorbidee a occurs in various plant-derived foods and food Animals.. Mice were housed and handled according to institu- supplements.. Bcrpl transports pheophorbide a and is highly effi- tionall guidelines complying with Dutch legislation. Animals used cientt in limiting its uptake from ingested food. Bcrpl'- mice also inn this study were Bcrpl " and wild-type mice of a comparable displayedd a previously unknown type of protoporphyria. Erythro- geneticc background (FVB or mixed 129/Ola and FVB) between cytee levels of the heme precursor and phototoxin protoporphyrin 99 and 14 weeks of age. Animals were kept in a temperature- IX,, which is structurally related to pheophorbide a, were increased controlledd environment with a 12-h light/12-h dark cycle. They 10-fold.. Transplantation with wild-type bone marrow cured the receivedd a standard (AM-II) or semisynthetic (reference protoporphyriaa and reduced the phototoxin sensitivity of Bcrp1'~ 4068.02)) diet (Hope Farms, Woerden, The Netherlands) and mice.. These results indicate that humans or animals with low or acidifiedd water ad libitum. absentt BCRP activity may be at increased risk for developing protoporphyriaa and diet-dependent phototoxicity and provide a Materials.. Topotecan and [14C]topotecan [56 Ci/mol (1 Ci = 37 strikingg illustration of the importance of drug transporters in GBq))) were from GlaxoSmith Kline (King of Prussia, PA). protectionn from toxicity of normal food constituents. Pheophorbidee a was from Frontier Scientific/Porphyrin Prod- uctss (Logan, UT).

emberss of the ATP-binding cassette (ABC) family of drug - - M transporterss actively export many drugs and toxins from Generationn of Bcrpl ' Mice. By using Bcrpl cDNA probes, a cells.. Their presence at strategic sites in the body such as the 129/Olaa mouse genomic sequence containing exons 1-8 of intestine,, blood-brain barrier, and placenta protects the organ- BcrplBcrpl was identified. A 5.1-kb fragment containing exons 3-6, ismm by limiting the systemic penetration and tissue toxicity of encodingg most of the ATP-binding domain, was deleted and xenotoxinss {1-3). The breast cancer resistance protein (BCRP/ replacedd with a 1.8-kb pgk-hygnt cassette in reverse-transcrip- ABCG2)) and its mouse homologue Bcrpl transport various tionall orientation. Electroporation and selection for recombi- anticancerr drugs including topotecan, mitoxantrone and doxo- nantt E14 embryonic stem cells was done as described (7), Of 161 rubicin,, thus causing multidrug resistance in cancer cells (3). We hygromycin-resistantt clones, 18 were targeted correctly as con- previouslyy found that application of BCRP inhibitors to mice firmedd by Southern analysis of .Sea I-digested genomic DNA with enhancedd the oral uptake and fetal penetration of topotecan, 3'' and 5' Bcrpl probes (Fig. \a). The absence of additional suggestingg that Bcrpl provides an efficient pharmacologic bar- pgk-hy^ropgk-hy^ro cassettes inserted elsewhere in the genome was con- rierr at these sites (4). Coadministration of BCRP inhibitors has firmedd by hybridization with a hygro-specific probe. Chimeric alsoo been tested in patients for its ability to improve anticancer micee were generated by microinjection of two independently chemotherapyy by enhancing oral uptake and possibly tumor targetedd embryonic stem cell clones into blastocysts. Chimeric penetrationn of BCRP substrate drugs. The first results are offspringg were backcrossed to FVB mice. By using this approach, promising,, but they also revealed unanticipated toxicity (5). It twoo independent Bcrpl' mouse lines were established. thuss is important to establish the risks associated with chronic inhibitionn of BCRP. Clinicall Chemical Analysis of Plasma. Standard clinical chemistry Too study the physiological and pharmacological functions of analysess on plasma were performed on a Hitachi 911 analyzer to BCRP,, we generated Bcrpl knockout mice. Absence of Bcrpl determinee levels of bilirubin, alkaline phosphatase, aspartate aminotransferase,, alanine aminotransferase, lactate dehydroge- resultedd in a striking sensitivity to the dietary chlorophyll : catabolitee pheophorbide a, which made these mice extremely nase,, creatinine, urea, Na'. K', Ca ', Ci , phosphate, total photosensitive.. Moreover, Bcrpl knockout mice displayed a protein,, and albumin. previouslyy uncharacterized type of protoporphyria, a group of metabolicc disorders frequently associated with skin photosensi- Abbreviations.. ABC, ATP-t.,nding cassette. BCRP ABCC2. breast cancer resistance protein. tivityy in patients (6). Pheophorbide a and protoporphyrin are Bcrpll Abtgi. murine BCRP; PPIX. protoporphyrin IX structurallyy related and belong to the porphyrins, a broad class ** *To whom correspondence should be addressed at. Division of Experimental Therapy. The off molecules that include the "pigments of life": chlorophyll, Netherlandss Cancer Institute. Presmanlaan 121. 10*6 CX Amsterdam. The Netherlands heme,, and cobalamin (6). Our data show that BCRP is physio- E-mail:: iKhinkeienkinl.

59 9 ChapterChapter 4

tionn of the fusion protein, immunization of rats, and fusion ee allele nn 1 2 3 4 5 protocolss were as described (10, 11). Results are shown for mAbb BXP-9 or BXP-53. which worked well on immunoblots +33 1 'fii, andd in immunohistochemistry.

Westernn Analysis. Crude membrane fractions from tissues were preparedd as described (12). Western blotting was performed as describedd (7). Blots were probed with mAb BXP-9 (1:10). mAb bindingg was detected by using peroxidase-conjugated rabbit anti-ratt IgG (1:1.000. DAKO).

Histologicall Analysis and Immunohistochemistry. Tissues were fixed inn 4% phosphate-buffered formalin, embedded in paraffin, sectionedd at 4 p.m, and stained with hematoxylin and eosin accordingg to standard procedures. For immunohistochemistry. tissuess were deparaffinized in xylene and rehydrated. Endoge- nouss peroxidase activity was blocked by using 3% (vol/vol) H2O2 inn methanol for 10 min. Before staining, paraffin sections were pretreatedd by heat-induced epitope retrieval. Slides were incu- batedd with 5T normal goat serum/PBS for 30 min, and subse- quentlyy sections were incubated overnight with a 1:400 dilution off BXP-53 at 4°C. mAb immunoreactivity was detected with the streptavidin-biotinn immunoperoxidase (sABC) method by using biotinylatedd goat anti-rat IgG (Dako. 1:100) as secondary anti- body,, and diaminobenzidine substrate for visualization. After counterstainingg with hematoxylin, slides were mounted. For negativee control, the primary mAb was omitted.

InIn Vitro Pheophorbide a Accumulation Assay. Exponentially growing cellss were incubated for 1 h at 37°C in normal medium in the presencee of 10 p.M pheophorbide a with or without 10 p:M Kol43.. Cells were trypsinized. washed, and suspended in Hanks' solutionn with \ri FCS. Light exposure was minimized, and after trypsinizationn all procedures were done at 4°C. Relative cellular accumulationn of pheophorbide a was determined by flow cy- tometryy using a FACScan (Becton Dickinson) with excitation at 4888 nm and emission detection at 650 nm.

Pharmacokineticc Experiments. Pharmacokinetic experiments were performedd as described (4. 7). For fetal accumulation studies, Bcrpl'Bcrpl' /Mdrla/b mice were obtained by appropriate back- Fig.. 1. Generation and analysis of Bcrpl mice, (a) A 5.1 -kb fragment containingg exons 3-6 (exons are indicated by filled boxes) was replaced with crossingg of Bcrpl mice to mice deficient for the drug trans- ann invertedpgk-hygro cassette. Restriction sites: S. Seal; A, Asp718; N, Nhel. For portingg /'-glycoproteins Mdrla and Mdrlb (Mdrla/b mice). Southernn analysis. 5 and 3 probes were used on Seal-digested genomic DNA. Subsequently,, Bcrpl' I Mdrla lb mice were crossed to gen- Diagnosticc restriction fragments are indicated by double-headed arrows, (b) eratee Bcrpl' . Bcrpl' . and Bcrpl fetuses in a Mdrla/b Westernn analysis on crude membrane fractions from liver, kidney, and small background. . intestinee (20 jig per lane), (c) Bile from wild-type and Bcrpl mice, (d-g) Immunohistochemieall detection ( 40) of Bcrpl in small intestine (d). liver (e), kidneyy (f), and placenta (g). Photosensitivityy Experiments. Mice were exposed to standard fluorescentt light (Philips TLD50W/84HF) with a 12-h light/12-h darkk cycle. The distance from the light source was 130 cm. Hematologicall Analysis. I lemoglobin, mean corpuscular volume, resultingg in an exposure of = 1.000 lux. These light conditions are redd and white blood cells, and platelets were determined in comparablee to those in an artificially well lit indoor office space. peripherall blood on a Cell Dyn 1200 analyzer (Abbott). Bonee Marrow Transplantation. Bone marrow was harvested from femurss and suspended in Hanks' solution with \'i FCS. Bone HPLCC Analysis, levels of topotecan. pheophorbide a. and proto- marroww cells (3 x 10'') were transplanted via the lateral tail vein porphyrinn IX (PPIX) were determined by HPLC analysis as too recipient mice that had received 9 Gy of whole-body irradi- describedd (4. 8. 9). The method described for the determination ation.. After transplantation mice were kept for 8 weeks in off pheophorbide a was modified slightly. A Luna CI8(2) column filter-topp cages. The genotype of peripheral blood was confirmed (2500 x 4.6-mm i.d.. 5-/xm particle size, Phenomenex. Torrance, byy PCR analysis. CA)) protected with a guard column (10 x 3-mm i.d.) packed withh reversed-phase material (Varian Chrompack) was used for Results s thee separation. Generationn and Analysis of Bcrpl ~ Mice. To study the in vivo roles off Bcrpl. we generated constitutive Bcrpl knockout mice (Fig. Generationn of mAbs. A fusion gene consisting of the gene for the la).. Northern and Western blots confirmed the absence of Bcrpl EscherichiaEscherichia colt maltose-binding protein and a fragmenmRNt enAA- (not shown) and protein in Bcrpl mice (Fig. \b). codingg amino acids 221-394 of the mouse Bcrpl gene was Immunohistochemically.. Bcrpl was detected in apical mem- constructedd in the pMAL-c vector. Production and purifica- braness of epithelial cells of small intestinal villi and renal

60 0 BCRPBCRP protects against a dietary phototoxin

—•—— Wild-lype Bcrp"" (-/-) 300 • ^ ^ // \ 1!i i / / 1C C :" (" ^^ . * * 0.00 0.5 1.0 1.5 2 0 2.5 3.0 3.5 4.1 timee (h) Bcrpll genotype

Fig.. 2. Pharmacologic effects of Bcrpl. (a) Plasma concentration versus time curve after oral administration of 1 mg 'kg topotecan to •nicee (means SD. n 5-6;; P 0.001 for area under the curves. Student's t test), (fa) Ratio of [""dtopotecan concentration in fetus over maternal plasr iaa at 15 min after i.v. administrationn of 0.2 mg kg ['"Cltopotecan to pregnant dams (gestation day 15.5; mean : SD. n 3damsand 11 8crpt ' , 13 Bcrpr' ,jn dd 7 Bcrpl ~'~ fetuses; *,, P 0.001 compared with Bcrpl fetuses. Student's t test). proximall tubules, hepatic bile canalicular membranes, and pla- butt only when fed with one particular batch of food. Phototoxic centall labyrinth cells of wild-type (Fig. 1 d-g) but not Bcrpl earr lesions developed 1 week after feeding with this "phototoxic" (nott shown) mice. batch,, and in some cases lesions also appeared on the tail, snout. BcrplBcrpl mice were fertile, their life spans and body weightans dd rims of the eyes. Phototoxicity was never observed in weree not different from wild-type, they were born at the ex- wild-typee mice. pectedd Mendelian ratio, and they did not demonstrate pheno- Diet-dependentt photosensitization is common and can be typicc aberrations under standard housing conditions. Unlike causedd by a variety of chemicals including drugs and pesticides somee other members of the ABCG subfamily (13), Bcrpl butt also by natural toxins derived from plants and fungi. The seemedd not to be critically involved in lipid metabolism, because primaryy plant component present in our standard mouse diet wee did not observe changes in plasma levels of cholesterol and wass alfalfa (Medicago saliva) leaf concentrate. Outbreaks of phospholipids.. Hematological and plasma clinical chemical anal- photosensitizationn in cattle are reported frequently and are often ysiss revealed no abnormalities except that unconjugated biliru- associatedd with the ingestion of alfalfa. This phototoxicity has binn was increased in Bcrpl mice (1.50 0.55 pM in wild type beenn attributed to biochemical conversion products or myco- versusversus 6.50 1.05 pM in Bcrpl ). Surprisingly, the bile otoxinf ss generated in humidly stored alfalfa (14). To investigate Bcrpl'Bcrpl' mice was red instead of yellow (Fig. i<). Both thwhethee rr alfalfa was the source of the phototoxicity in Bcrpl increasedd levels of unconjugated bilirubin and red-colored bile mice,, we fed them diets containing increased amounts of alfalfa. weree diet-dependent, because they disappeared in mice that Whereass no phototoxicity was observed at normal (59?) levels of receivedd a semisynthetic diet consisting of purified nutrients. alfalfa,, all Bcrpl ' (but not wild-type) mice progressively However.. HPLC analysis indicated that the red bile color was not developedd phototoxic lesions at higher (10% and 20%) alfalfa causedd by bilirubin or its conjugates. As will be described below, dosagess (Fig. 3/). Thus, twice the normal amount of this batch itt is likely to be caused by a chlorophyll catabolite. off alfalfa already induced phototoxicity in Bcrpl mice.

Rolee of Bcrpl in Oral Availability and Fetal Accumulation of Topotecan. Bcrpl-'"" Mice Are Extremely Sensitive to the Phototoxin Pheophor- Wee studied the pharmacologic role of Bcrpl by determination bidee a. It has been shown that high levels of pheophorbide a. a off the oral availability and fetal accumulation of topotecan phototoxicc porphyrin catabolite of chlorophyll, can be formed by (Fig.. 2). The oral availability of topotecan was increased alfalfaa chlorophyllase depending on the treatment of the alfalfa =6-foldd in Bcrpl mice, indicating that intestinal Bcrpl duringg storage and processing (refs. 15 and 16; Fig. 3g). To test limitss the uptake of topotecan. Fetal accumulation of topote- whetherr Bcrpl mice were sensitive to pheophorbide a. we cann was measured in fetuses from a cross between Bcrpl determinedd its phototoxicity after oral administration (Table 1). andd Mdrla/b mice to avoid confounding effects of Mdrla/b Whereass phototoxicity (or other toxicity) was never observed in /'-glycoproteinn (4). Fig. 2b shows that the ratio of fetal wild-typee mice up to 200 mg/kg/day. the Bcrpl mice dis- topotecann concentration to maternal plasma concentration playedd a dramatic hypersensitivity. The lowest dose at which wass «2-fold higher for Bcrpl fetuses compared with phototoxicityy occurred in Bcrpl mice was 2 mg/kg/day. BcrplBcrpl fetuses, whereas Bcrpl' fetuses showed an interindicatin- gg that Bcrpl mice are at least 100-fold more sensitive mediatee accumulation. These results show that Bcrpl in the too pheophorbide a. At 16 mg/kg/day. ear lesions developed placentaa can limit the penetration of substrate drugs from the alreadyy after 2 days, and after 3 days mice developed severe maternall plasma into the fetus. edemaa of the head and became moribund. The hypersensitivity off the Bcrpl mice to pheophorbide a corresponded with Diet-Dependentt Phototoxicity in Bcrpl ' Mice. The Bcrpl mice highlyy increased plasma levels. Plasma levels of pheophorbide a hadd not displayed any visible phenotypical aberration until a few weree 17-fold (10.40 2.74 ug/ml) and 24-fold (14.54 2.50 BcrplBcrpl mice suddenly developed severe necrotic ear lesionug/mls )) higher, respectively, in Bcrpl mice fed with photo- (Fig.. 3 a-e). Only mice housed on the top shelf, closest to the toxicc or 209? alfalfa food compared with a "normal" food batch lightt source, developed these lesions, suggesting some form of (0.611 0.47 u.g/ml). In wild-type mice, plasma levels of pheo- phototoxicity.. Further analysis showed that all Bcrpl mice phorbidee a were undetectable on any of these diets. developedd ear lesions when exposed to standard fluorescent light

61 1 ChapterChapter 4

g g Chlorophylll a :...... •;HII H

• •

timee (days) ' '

Pii ' : irphynn IX

CHCHH CH

CC : 3H - .•.;; ••

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 Bcrpl ' mice, (f) Incidencee of phototoxic ear lesions on diet containing 10% and 20% alfalfa (n 10 mice). (g> Chlorophyll breakdown in plants, reactions, and enzymes are indicatedd with an arrow. Also shown is the structure of PPIX. (h) Pheophorbide a accumulation in cells exposed to pheophorbide a {10 ,M) for 1 h at C in the presencee or absence of Bcrpl BCRP-inhibitor Ko143 (10 ,iM) (mean fluorescence SD, n n 3; »*, P 0.01, Student's t test).

Pheophorbidee a Is Transported by Bcrpl BCRP. To determine Interestingly,, in plants, degradation of pheophorbide a by whetherr pheophorbide a is transported by Bcrpl/BCRP, we cleavagee of the porphyrin ring yields a red chlorophyll catabolite measuredd its accumulation in cell lines by flow cytometry (Fig. (ref.. 21: Fig. 3g). In Bcrpl mice that received a semisynthetic 3/i).. We used the mouse fibroblast line MEF3.8 and its deriva- diett and thus had yellow bile, the red bile color reappeared after tivess T6400 and A2 that overexpress Bcrpl by drug selection or orall administration of (dark-green) pheophorbide a. suggesting transductionn with Bcrpl cDNA. respectively (17. 18). Accumu- thatt the red compound excreted in the bile of Bcrpl mice (Fig. lationn of pheophorbide a was reduced 18-fold in Tt>400 cells 1<)) is red chlorophyll catabolite or a related pheophorbide a comparedd with MEF3.8. This effect could be largely reversed by metabolite. . thee specific Bcrpl/BCRP inhibitor Kol43 (19). We obtained similarr results for the A2 cell line and the human IGROV1 ovariann cancer cell line and its ZJCW-overexpressing derivative Noo Abnormalities in Differentiation of Bone Marrow Cells in BcrpJ T88 (20). indicating that pheophorbide a is transported efficiently Mice.. It has been shown that Bcrpl is also expressed in hema- byy both murine Bcrpl and human BCRP. Thus. Bcrpl efficiently topoieticc stem cells and erythroid precursor cells in (he bone reducess the bioavailability of dietary pheophorbide a by pre- marroww and that it is responsible for the "side-population" (SP) ventingg its uptake from the intestine and possibly by mediating phenotype,, associated with primitive slem cells and caused by itss elimination via liver and kidney. Without this efficient barrier, activee extrusion of the fluorescent dye Hoechst 33342 (22). We (natural)) fluctuations of pheophorbide a in the diet can lead to alsoo detected Bcrpl in mature murine erythrocytes (not shown). phototoxicity.. The phototoxic food batch must have contained Althoughh the physiologic function of Bcrpl in hematopoietic relativelyy high pheophorbide a levels, most likely resulting from (stem)) cells is still unknown. Zhou el ill. (22) speculated that it variationss in treatment of the alfalfa ingredient. mightt be involved in hematopoietic differentiation. However, by

62 2 RCRPRCRP protects against a dietary plwtoto.xin

Tablee 1. Pheophorbide a phototoxicity in mice precursorss apparently protects these cells from excessive accumulationn of PPIX. possibly by extrusion of this compound. Dosage,, mg kg day Wild-type Bcrpl Bcrpl

Protoporphyriaa Can Be Cured by Bone Marrow Transplantation. We 22 na +" nextt transplanted lethally irradiated wild-type and Bcrpl 44 na +8 micee with bone marrow from either genotype and determined 88 - +5 thee effects on protoporphyria and photosensitivity. (light weeks 166 — afterr transplantation, erythrocyte levels of PPIX in Bcrpl 322 — na micee with wild-type transplants were comparable with those of 644 — na wild-typee mice, whereas wild-type mice with Bcrpl trans- 1000 — na plantss were comparable with Bcrpl mice (Fig. 4/)). The 2000 — na BcrplBcrpl protoporphyria is thus a bone marrow-autonomous phenotypee that can be cured by transplantation with wild-type Pheophorbidee a was administered orally for up to 12 days to mice fed with bonee marrow, and that does not depend on Bcrpl activity "normal"" food (n 3 per group), na, Not analyzed; —, no phototoxicity elsewheree in the body. Wild-type mice transplanted with observed;; •, phototoxicity observed; superscript numbers, average number of BcrplBcrpl (or wild-type) bone marrow were not photosensitive dayss until phototoxicity was first observed. whenn fed a 20% alfalfa diet, whereas Bcrpl recipients of •Moribundd mice were killed after 3 days. Light conditions are specified in eitherr bone marrow: genotype did display photosensitivity (Fig. MaterialsMaterials and Methods. Ac).Ac). However. Bcrpl mice with Bcrpl transplants (with protoporphyria)) were more photosensitive than Bcrpl mice withh w ild-typt' transplants (no protoporphyria: Fig. 4c). suggest- floww cytometry we found no abnormalities in the relative ingg that PPIX may have contributed to the photosensitivity in numberss of erythroid precursors (Terll9). granulocytes BcrplBcrpl mice. (Grll ). macrophages (Mad), or B cells (B220) in bone marroww of Bcrpl mice. In addition, no significant hemato- logicc differences were observed in peripheral blood (not shown). Discussion n Ourr data provide a striking illustration of the importance of the ABCC transporter Bcrpl in protection from a normal food Bcrpll ' Mice Display a Previously Unknown Type of Protoporphyria. constituentt such as pheophorbide a and at the same time Inn humans, well known causes of phototoxicity are genetic emphasizee the unpredictability of exposure to such constituents. defectss in the heme biosynthetic pathway that result in the Givenn the amount of chlorophyll ingested by most omnivoresand accumulationn of photosensitizing porphyrins in the skin. These herbivores,, it is not surprising that they are normally well porphyrinss are structurally highly related to pheophorbide a. We protectedd from its toxic breakdown products. In humans, pheo- thereforee determined levels of various endogenous porphyrins, phorbidee a-induced phototoxicity has been reported after inges- includingg PFIX (Fig. 3g), the immediate precursor of heme, in tionn of chlorella tablets (23). a dried algae preparation taken as erythrocytes,, plasma, liver, bile, and urine. We found that ""naturall health supplement" by millions of people. Several erythrocytee levels of PPIX were increased 10-fold in Bcrpl pickledd vegetables also contain substantial amounts of pheo- mice.. This increase was seen in mice kept on all diets (Fig. Ati) phorbidee a and have been shown to cause phototoxicity in rats includingg the semisynthetic diet, with which the mice do not (23).. In the human population there are many incompletely displayy obvious photosensitivity. This result implies that the understoodd incidences of idiosyncratic food and drug hypersen- increasedd level of PPIX was independent of the diet, and that this sitivitiess including phototoxicity (24-26). In light of our data it levell in itself was not sufficient to cause marked photosensitivity. willl be of great interest to investigate whether part of these Plasmaa PPIX was also increased, but no significant differences hypersensitivitiess could be explained by partial or complete inn levels of PPIX in liver or bile were observed between the deficienciess in the activity of BCRP. /'-glycoprotein, or related groupss (not shown). Bcrpl expressed in erythrocytes and their ABCC transporters.

Wild-type e Untreated d Bcrpll l-'-l (VII ".,' si. ml KOO transplant

XX 4

-- KO.KO -- • N I

:: I r

111 1Wild-typee Bcrpl (-/-) 011 23456789 1C Normall Phototox. Synth Recipient t timee (days)

Fig.. 4. Effect of bone marroDiewt t transplantation on protoporphyria and phototoxicity, (a) Erythrocyte levels of PPIX in Bcrpl ' and wild-type mice receiving normal,, phototoxic (Phototox.), or semisynthetic (Synth.) diet (n = 5). (M Erythrocyte levels of PPIX after bone marrow transplantation (*, P 0.05. Student's ttest).. KO, knockout, (c) Incidence of phototoxic ear lesions in wild-type and Bcrpl mice (recipients) transplanted with Bcrpl 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'ss ttest).

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Ourr results demonstrate unequivocally that Bcrp! affects the otherr genetic or drug-induced protoporphyrias and thus could pharmacologicc behavior of the anticancer drug topotecan, po- explainn some of the highly variable penetrance seen in these tentiallyy limiting the therapeutic efficacy of it and related syndromess (6). clinicallyy used drugs, for instance by restricting their oral avail- Afterr completion of this study, Zhou et at. (27) published an ability.. Our current and previously obtained data indicate that analysiss of an independently generated Bcrpl knockout mouse. thiss problem can be circumvented by strategic application of Inn line with our findings, no abnormalities were observed in the effectivee BCRP inhibitors (4. 5). However, this study also hematopoiesiss of these mice. No mention was made of other indicatess that prolonged inhibition of BCRP activity might reveal physiologicall abnormalities except that hematopoietic cells were unexpectedd sensitivities to compounds that are commonly unablee to extrude the dye Hoechst 33342 and displayed an presentt in the diet. Ongoing clinical trials with BCRP inhibitors increasedd sensitivity to the drug mitoxantrone (27). We note that shouldd therefore be monitored carefully for such unexpected thee phenotypeswc observed in our Bcrpl' ~ mice may well have toxicityy events. beenn missed unless specifically looked for or were only apparent Ourr study further shows that BCRP is important for the underr specific (dietary and light) conditions. handlingg of endogenous porphyrins. The Bcrp] ' mice display Inn conclusion, we have shown that BCRP is involved in aa previously unknown type of genetic porphyria characterized by physiologicallyy important processes involving the handling of increasedd levels of PPIX in erythrocytes. This porphyria is exogenouss and endogenous porphyrins. Reduced BCRP activity uniquee because it is not caused by a defect in one of the enzymes off the heme biosynthetic pathway, in contrast to all previously ass a consequence of mutation, inhibition, or down-regulation identifiedd genetic porphyrias (6). Erythropoietic protoporphyria thuss might contribute to diet-induced phototoxicity, protopor- forr instance also leads to accumulation of PPIX in erythrocytes phyria,, and possibly other porphyrin-related toxicities and butt is caused by a deficiency of ferrochelatase, the enzyme that disorders. . mediatess the conversion of PPIX into heme. Severe erythro- poieticc protoporphyria results in marked photosensitivity. We do Wee lhank our colleagues for critical reading of the manuscript; Eric nott understand the exact mechanism behind the protoporphyria Challa,, Alida Ooslcrloo-Puinkerken, Vincent W. Bloks, Hugo Oppel- causedd by Bcrpl deficiency yet, but the structural similarity with aar,, Rob Lodewijks, and Hans Tensen for excellent technical assistance; Tonn Schrauwers and Muriel Beumkes for animal care; Marlon Tjin-A- pheophorbidee a suggests that excess PPIX (or perhaps another Koeng,, Jurjen Bulthuis, and Kees de Goeij for histotechnical assistance; porphyrin)) may be cleared from erythroid cells by BCRP. Low andd Lutien Groeneveld for assistance in preparing animal diets. This orr absent BCRP activity may exacerbate the consequences of workk was supported by the Dutch Cancer Society.

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