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Multidrug resistance-associated proteins are involved in the transport of the glutathione conjugates of the ultimate carcinogen of benzo[a]pyrene in human Caco-2 cells

  • Toxicokinetics and Metabolism
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Abstract

A wide variety of contaminants are ingested through food, among them the pro-carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) that is resorbed and partially metabolized in the enterocytes of the small intestine. Previous in vitro studies have revealed that BP phenols are excreted as Phase II metabolites including glucuronides and sulfates. This export is mediated by the breast cancer resistance protein (ABCG2). The ultimate carcinogenic Phase I BP metabolite anti-BP-7,8-dihydrodiol-9,10-epoxide (BPDE) can be detoxified by glutathione conjugate formation catalyzed by glutathione S-transferases. In the present study, differentiated human intestinal Caco-2 cells were used as a model for the human small intestine to investigate the detoxification of BPDE and excretion of stereoisomeric glutathione conjugates in the presence of an inhibitor of the glutathione-cleaving enzyme γ-glutamyl transpeptidase at the cell surface. The results indicate that the glutathione conjugates of BPDE are formed and excreted mainly to the apical and to a minor extent to the basolateral side of polarized Caco-2 monolayers. Inhibition studies revealed that the multidrug resistance-associated proteins (ABCCs) are involved in the transport of BPDE glutathione conjugates. Stable ABCC1, ABCC2 and ABCC3 knockdown cell lines were generated, thus making it possible to demonstrate that ABCC1 mediates the basolateral and ABCC2 the apical excretion of BPDE glutathione conjugates. In conclusion, the ultimate carcinogen BPDE is detoxified via glutathione conjugation and subsequently excreted by Caco-2 cells in both apical and basolateral directions. This finding is equivalent to a transport into feces as well as blood system in the in vivo situation.

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Abbreviations

1-MP-SG:

S-(1-Methylpyrenyl)glutathione

ABC transporter:

ATP-binding cassette transporter

BP:

Benzo[a]pyrene

BP-7,8-diol:

(±)-trans-7,8-Dihydroxy-7,8-dihydrobenzo[a]pyrene

BPDE:

BP-7,8-diol-9,10-epoxide

(+)-syn-BPDE:

(7S,8R,9S,10R)-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

(−)-syn-BPDE:

(7R,8S,9R,10S)-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

(+)-anti-BPDE:

(7R,8S,9S,10R)-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

(−)-anti-BPDE:

(7S,8R,9R,10S)-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

(+)-BPD-SG:

(+)-10R-(S-Glutathionyl)-7S,8R,9R-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene

(−)-BPD-SG:

(−)-10S-(S-Glutathionyl)-7R,8S,9S-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene

BCRP:

Breast cancer resistance protein

BSO:

dl-Buthionine-[S,R]-sulfoximine

CYP:

Cytochrome P450 monooxygenase

EPHX1:

Microsomal epoxide hydrolase

GIT:

Gastrointestinal tract

GSH:

Glutathione

GST:

Glutathione S-transferase

γGT:

γ-Glutamyl transpeptidase

MRP:

Multidrug resistance-associated protein

PAH:

Polycyclic aromatic hydrocarbon

qRT-PCR:

Real-time quantitative PCR

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Acknowledgments

We would like to thank Anja Koellner and Maja Tomic for their excellent technical support. This work was supported by the German Research Foundation DFG (grant number: LA 1177/4-2).

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Food Safety, Federal Institute for Risk Assessment, Max-Dohrn-Strasse 8-10, 10589, Berlin, Germany

    Stefanie Hessel & Alfonso Lampen

  2. Biochemical Institute for Environmental Carcinogens, Prof. Dr. Gernot Grimmer Foundation, Lurup 4, 22927, Grosshansdorf, Germany

    Andrea John & Albrecht Seidel

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  1. Stefanie Hessel
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  2. Andrea John
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  3. Albrecht Seidel
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  4. Alfonso Lampen
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Correspondence to Stefanie Hessel.

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Hessel, S., John, A., Seidel, A. et al. Multidrug resistance-associated proteins are involved in the transport of the glutathione conjugates of the ultimate carcinogen of benzo[a]pyrene in human Caco-2 cells. Arch Toxicol 87, 269–280 (2013). https://doi.org/10.1007/s00204-012-0917-0

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