Limited modulation of the transport activity of the human multidrug resistance proteins MRP1, MRP2 and MRP3 by nicotine glucuronide metabolites

doi:10.1016/j.toxlet.2004.12.014

Toxicology Letters

Volume 157, Issue 1, 16 May 2005, Pages 9-19

https://doi.org/10.1016/j.toxlet.2004.12.014 Get rights and content

Abstract

The human ATP-binding cassette proteins MRP1 (ABCC1), MRP2 (ABCC2) and MRP3 (ABCC3) are active transporters of antineoplastic drugs as well as conjugated metabolites and other organic anions. In addition to being substrates, many glucuronide, glutathione and sulfate conjugates can also inhibit the transport activities of these MRP-related proteins, sometimes in a glutathione (GSH)-dependent manner. Nicotine is the major addictive component of cigarette smoke. Three glucuronide metabolites of this compound have been identified in vivo: nicotine-N-glucuronide, cotinine-N-glucuronide and trans-hydroxycotinine-O-glucuronide. In this study, we first chemically synthesized trans-hydroxycotinine-O-glucuronide and then tested the ability of this compound, nicotine-N-glucuronide and cotinine-N-glucuronide to modulate the vesicular transport of several organic anions by MRP1, MRP2 and MRP3. We observed that none of the three metabolites at concentrations up to 100 μM significantly affected organic anion transport by MRP1 or MRP2, either in the absence or presence of GSH. MRP3-mediated transport of 17β-estradiol 17-(β-d-glucuronide) and methotrexate were partially inhibited by trans-hydroxycotinine-O-glucuronide (300 μM) (by 70% and 50%, respectively), whereas nicotine-N-glucuronide and cotinine-N-glucuronide had no effect. We conclude that the physiological functions of MRP1, MRP2 and MRP3 are not likely to be substantially affected by nicotine glucuronide metabolites at concentrations achievable in human serum.

Introduction

The human multidrug resistance proteins MRP1, MRP2 and MRP3 belong to subfamily C of the ATP-binding cassette (ABC) superfamily of proteins and share 48% to 58% amino acid homology with one another (Haimeur et al., 2004). These transport proteins mediate the ATP-dependent efflux of a broad range of substrates from the cytoplasm to the extracellular space. MRP1 causes resistance by pumping many natural product drugs out of tumor cells, often via a co-transport mechanism with GSH. Other drugs such as the antimetabolite methotrexate (MTX) are transported without GSH (Cole et al., 1994, Haimeur et al., 2004, Hooijberg et al., 1999, Loe et al., 1996b, Loe et al., 1998, Rappa et al., 1997). Tumor cells expressing elevated levels of MRP2 are resistant to a similar spectrum of oncolytic agents and like MRP1, MRP2 is capable of effluxing GSH out of cells (Cui et al., 1999, Haimeur et al., 2004). In contrast, MRP3 confers resistance to relatively few anticancer drugs and there is no evidence that it can transport GSH (Zelcer et al., 2001).

In addition to anticancer drugs, MRP1 is a primary active transporter of organic anions conjugated to glutathione (e.g. leukotriene C₄ (LTC₄)), glucuronate (e.g. 17β-estradiol 17β-(d-glucuronide) (E₂17βG)) and sulphate (e.g. estrone 3-sulphate (E₁3SO₄)) (Haimeur et al., 2004). MRP2 and MRP3 also transport conjugated organic anions but there are important differences in the affinities and transport capacities of the three transporters for a given substrate as well as in their sensitivities to inhibition and/or stimulation by various modulators (Bakos et al., 2000, Bodo et al., 2003a, Haimeur et al., 2004, Konig et al., 2003, Zelcer et al., 2003). Collectively, MRP1 and MRP3 (which are localized on the basolateral membranes of polarized epithelial cells) and MRP2 (which is localized on apical membranes) are thought to play an important role in the absorption, disposition and/or elimination of drugs and other organic anions (Haimeur et al., 2004).

Previous studies have shown that many, but not all, glucuronide conjugates can inhibit MRP1-mediated uptake of organic anions into inside-out membrane vesicles (Leslie et al., 2001, Loe et al., 1996a). For example, E₂17βG is an effective inhibitor of LTC₄ transport by MRP1 while E₂3βG is not (Loe et al., 1996a). In addition, LTC₄ transport is inhibited by the 3β-O-glucuronide conjugate of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) but E₂17βG transport is not (Leslie et al., 2001). Thus, it is not possible to predict based on structure alone whether or not a particular glucuronide conjugate will be an effective inhibitor of MRP1-mediated transport and furthermore, even if inhibition is observed, transport of some but not necessarily all substrates may be affected.

Nicotine, the main component of tobacco, is responsible for the addictive properties of cigarette smoking, and its metabolism and elimination have been the subject of considerable investigation (Tricker, 2003). To date, three different glucuronide metabolites of nicotine have been identified in vivo. Thus, most of the ingested nicotine is metabolized to cotinine (mainly by cytochrome P450 followed by the action of a cytosolic aldehyde oxidase) and both nicotine and cotinine can be conjugated to form nicotine-N-glucuronide and cotinine-N-glucuronide, respectively (Fig. 1A) (Benowitz et al., 1994, Tricker, 2003). Cotinine can also be hydroxylated to (3′R, 5′S)trans-hydroxycotinine which can then be further conjugated to form trans-hydroxycotinine-O-glucuronide (Byrd et al., 1992, Schepers et al., 1992).

In the present study, we have investigated the ability of the three nicotine glucuronide metabolites (nicotine-N-glucuronide, cotinine-N-glucuronide and trans-hydroxycotinine-O-glucuronide) (Fig. 1B), to modulate the organic anion transport activities of MRP1-3. We found that none of the three metabolites inhibited MRP1- or MRP2-mediated transport of any of four different organic anion substrates tested to a significant degree. On the other hand, MRP3-mediated transport of E₂17βG and MTX was partly inhibited by trans-hydroxycotinine-O-glucuronide, but nicotine-N-glucuronide and cotinine-N-glucuronide had no significant effect. We also found that, unlike our earlier study of NNAL-O-glucuronide transport (Leslie et al., 2001), the capacity of the nicotine metabolites to modulate the transport activities of any of these MRP-related transporters tested was not affected by GSH.

Section snippets

Materials

[14,15,19,20-³H]LTC₄ (158 Ci mmol⁻¹), [6,7-³H] E₂17βG (40.5 Ci mmol⁻¹) and [6,7-³H(N)]E₁3SO₄ (43.5 Ci mmol⁻¹) were purchased from Perkin-Elmer Life Sciences (Woodbridge, ON, Canada). [3′,5′,7′-³H(N)]MTX sodium salt (28 Ci mmol⁻¹) was from Moravek Inc. (Brea, CA). LTC₄ was purchased from CalBiochem (LaJolla, CA). AMP, ATP, GSH, E₂17βG, E₁3SO₄ and DTT were purchased from Sigma Chemical Co. (St. Louis, MO). MTX sodium salt was purchased from Faulding (Vaudreuil, QC, Canada). MK-571 was obtained from

Expression of MRP1, MRP2 and MRP3 in membrane vesicles prepared from transfected cells

MRP1, MRP2 and MRP3 proteins in membrane vesicles prepared from transfected HEK293T cells were detected by immunoblotting with isoform-specific MAbs (Fig. 2). In all cases, significant levels of MRP1, MRP2 and MRP3 were detected. There was no evidence of endogenous MRP1, MRP2 or MRP3 in control membrane vesicles prepared from untransfected HEK293T cells.

Nicotine-N-glucuronide, cotinine-N-glucuronide and trans-hydroxycotinine-O-glucuronide do not modulate MRP1 transport activity

To determine whether nicotine-N-glucuronide, cotinine-N-glucuronide or trans-hydroxycotinine-O-glucuronide could modulate LTC₄ or E₂17βG

Discussion

Like most other conjugated organic anions, glucuronide metabolites are generally considered to be non-toxic (Kroemer and Klotz, 1992). However, some glucuronide conjugates such as morphine-6-glucuronide remain pharmacologically active and in other cases, the glucuronide moiety can be removed by the action of cellular β-glucuronidases, leading to regeneration of the biologically active parent compounds. Therefore, it is important for cells to have a mechanism for extruding Phase II conjugates of

Acknowledgements

We thank Drs. Elaine Leslie and Curtis Oleschuk for helpful advice and discussions, and Kathy Sparks for tissue culture support. Supported by grant MOP-10519 from the Canadian Institutes of Health Research (CIHR). I.J. Létourneau is the recipient of an Ontario Graduate Scholarship and a CIHR Doctoral Award. Dr. S.P.C. Cole is the Canada Research Chair in Cancer Biology.

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Limited modulation of the transport activity of the human multidrug resistance proteins MRP1, MRP2 and MRP3 by nicotine glucuronide metabolites

Abstract

Introduction

Section snippets

Materials

Expression of MRP1, MRP2 and MRP3 in membrane vesicles prepared from transfected cells

Nicotine-N-glucuronide, cotinine-N-glucuronide and trans-hydroxycotinine-O-glucuronide do not modulate MRP1 transport activity

Discussion

Acknowledgements

Med. Clin. North Am.

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J. Pharm. Biomed. Anal.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Toxicology

J. Biol. Chem.

Transport activity of human MRP3 expressed in Sf9 cells: comparative studies with rat MRP3

Pharm. Res.

Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions

Mol. Pharmacol.

Analysis of the contribution Mrp3 to in vivo chemosensitivity using knock out mice

Proc. Am. Assoc. Cancer Res.

Nicotine metabolic profile in man: comparison of cigarette smoking and transdermal nicotine

J. Pharmacol. Exp. Ther.