Transport of the cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) across the human intestinal Caco-2 cell monolayer: role of efflux pumps

U.Kristina Walle and Thomas Walle1

Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Avenue, PO Box 250505, Charleston, SC 29425, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cooked-food mutagens formed when frying meat have been suggested to contribute to the etiology of colon, breast and prostate cancer. The most prevalent of these mutagens is 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which after absorption is bioactivated by both phase I and phase II enzymes. Although available data suggest absorption of PhIP in humans, the extent and mechanism of absorption are unknown. In the present study we examined the transport of [3H]PhIP through the human Caco-2 intestinal epithelial cell monolayer, a well-accepted model of human intestinal absorption. The influx, or absorption, was extensive and linear for 2 h and up to a PhIP concentration of 5 µM. Still, the basolateral to apical efflux [apparent permeability coefficient (Papp) 54.2 ± 0.7x10–6 cm/s, mean ± SEM, n = 24] was 3.6 times greater than the apical to basolateral influx (Papp 15.1 ± 0.6x10–6 cm/s, n = 21, P < 0.0001). Equilibrium exchange experiments demonstrated the efflux to be a true active process. Preincubations with verapamil, an inhibitor of P-glycoprotein-mediated transport, or MK-571, an inhibitor of multidrug resistance-associated protein-mediated transport, stimulated influx and reduced efflux of PhIP, suggesting that PhIP is a substrate for both of these transporters. These findings should be considered when determining exposure to the cooked food mutagens.

Abbreviations: HBSS, Hanks' balanced salt solution; MRP, multidrug resistance-associated protein; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most prevalent of the heterocyclic amines formed during the cooking process of various meats (1,2), is mutagenic in the Salmonella typhimurium assay (3) and, in particular, in Chinese hamster ovarian cells (4,5). In vivo it produces tumors in the colon of male rats (6) and in the mammary gland of female rats (6,7). Recently, it has also been shown to induce tumors of the prostate (8). PhIP, which is not a mutagen per se, is bioactivated to the N-hydroxylated procarcinogen species, mainly by cytochrome P450 1A2 in the liver (9,10). N-Hydroxy-PhIP is further bioactivated to the ultimate carcinogen by sulfation (1113) and/or acetylation (1416). Specifically, in human breast cells N-hydroxy-PhIP has been shown to bind to native DNA after activation by both of these conjugation reactions (13,16).

The amounts of PhIP formed during normal cooking are highly variable, dependent on the cooking method, temperature and cooking time (1,2). Several studies have attempted to assess how much of an ingested dose of PhIP and other cooked food mutagens is actually absorbed in humans. This has been difficult, as plasma measurements have not been successful. The recovery of unchanged PhIP in human urine after a meal of beef cooked at high temperature was <2% of the dose; 4–10% after hydrolysis of unknown conjugates (1722). It has been speculated that PhIP is fairly well absorbed and that the poor recovery of parent compound in urine is due to extensive metabolism (22). However, this remains to be demonstrated more directly.

In the present study we examined the transport of PhIP through the Caco-2 human intestinal cell monolayer which is a well-established model of human intestinal absorption (2325). It would, however, not be unexpected to find protective, outwardly directed, transport of PhIP, a toxic xenobiotic. P-Glycoprotein and MRP2, members of the ATP-binding cassette (ABC) multidrug transport family (2628) have previously been localized in Caco-2 cells (24,29). The potential role of these efflux mechanisms for PhIP was established with selective transport inhibitors.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Tritium-labeled PhIP (2.1 Ci/mmol) was synthesized by Chemsyn Science Laboratories (Lenexa, KS) for the Cancer Research Program of the National Cancer Institute (Division of Cancer Cause and Prevention, Bethesda, MD). The radiochemical purity was >=98%. Unlabeled PhIP hydrochloride (>99% purity) was obtained from the Midwest Research Institute (Kansas City, MO) (National Cancer Institute Chemical Carcinogen Reference Standard Repository). [14C]Mannitol (52 mCi/mmol) was purchased from Amersham Corp. (Arlington Heights, IL). Verapamil hydrochloride was obtained from Knoll Pharmaceutical Co. (Whippany, NJ). MK-571 was a generous gift from Dr A.W.Ford-Hutchinson (Merck-Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Canada).

Cells and culture conditions
The human colon adenocarcinoma cell line Caco-2 (American Type Culture Collection, Rockville, MD) was grown as monolayers in Eagle's minimum essential medium with Earle's salts, 10% fetal calf serum, 1% non-essential amino acids and penicillin (100 U/ml) and streptomycin (0.1 mg/ml) in a humidified 37°C incubator with 5% carbon dioxide. Stock cultures were split 1:12 when just confluent, using trypsin/EDTA (3032). For transport studies, the Caco-2 cells were seeded at a density of 100 000 cells/insert in 1 cm2 Transwells® containing 0.4 µm pore size permeable polycarbonate membranes (Corning Costar, Cambridge, MA) which were placed in 12-well tissue culture plates. The volume of cell culture medium in the inserts was 0.5 ml (apical side) and in the surrounding wells 1.5 ml (basolateral side). The medium on both sides of the cell layer was changed every 2 days for the first 2 weeks, then daily. The integrity of the cell monolayers was evaluated by measuring the transepithelial electrical resistance values with a Millicell-ERS volt/ohmmeter (Millipore, Bedford, MA). Only cell inserts with resistance values exceeding 300 cm2 were used for transport experiments. The transport of [14C]mannitol, a marker of paracellular transport, was also measured in parallel inserts. The cell monolayers were considered tight when the mannitol transport was <0.3% of the dose per hour, corresponding to an apparent permeability coefficient (Papp) (23) of 0.4x10–6 cm/s. The inserts were used for experiments at 24–41 days after seeding. During this time the resistance and mannitol transport values did not change. Also, there was no difference in PhIP transport rate between young and old cells. The presence of unlabeled PhIP on either the apical or the basolateral side of the insert did not affect [14C]mannitol transport.

To ensure that the radioactivity measured was related purely to PhIP and not PhIP metabolites or degradation products, 3 h transport medium from both sides of the cell monolayer after apical or basolateral loading of 5 µM PhIP was subjected to HPLC analysis. A Symmetry C18 column (Waters Corp.) was used with a mobile phase of 22% acetonitrile in 50 mM ammonium acetate buffer (pH 5) with a flow rate of 0.9 ml/min and UV detection at 315 nm. Fractions were collected at 1 min intervals and subjected to liquid scintillation spectrometry. PhIP had a retention time of 14 min and >99% of the radioactivity coincided with the PhIP HPLC peak.

PhIP transport
Prior to experiments the cells were washed twice for 30 min with warm Hanks' balanced salt solution (HBSS) buffered to pH 7.4 with 25 mM HEPES (31,32). The buffer was then replaced with fresh HBSS buffer on one side of the cell layer and [3H]PhIP in HBSS buffer on the other side. The apical side of the cell layer (insert) contained 0.5 ml and the basolateral side (well) contained 1.5 ml. In each experiment two inserts were used for each treatment. The [3H]PhIP solution (0.5–5 µM) contained <1% ethanol. Although this concentration of ethanol slightly increased the Papp of mannitol from 0.16 to 0.26 x 10–6 cm/s (data not shown), it had no effect on the transport of PhIP. When [3H]PhIP was added to the apical side, the inserts were moved to a well with fresh buffer at each time point. At the end of the experiment (3 h), the radioactivity in aliquots from each well and insert was measured by liquid scintillation spectrometry after the addition of Biodegradable Counting Scintillant (Amersham Corp.). When [3H]PhIP was added on the basolateral side, the buffer in the insert was replaced with 0.5 ml of fresh buffer at each time point. The radioactivity in each sample and an aliquot of the 3 h basolateral solution was determined. In separate experiments with loading of [3H]PhIP (1 and 5 µM) on both sides of the inserts, 50 µl samples were taken from both sides at 1, 2 and 3 h. In transport inhibition experiments, verapamil hydrochloride (50 µM) or MK-571 (50 µM) was added to the buffer on both sides of the cell layer. The [3H]PhIP concentration used in these experiments was 5 µM.

Calculations
The apparent permeability coefficients (Papp), expressed in cm/s (23), were calculated as {Delta}Q/{Delta}tx1/60x1/Ax1/C0, where {Delta}Q/{Delta}t is the permeability rate (µg/min), A is the surface area of the membrane (cm2) and C0 is the initial concentration in the donor chamber (µg/ml). The statistical significance of differences between different transport directions or treatments was evaluated using a two-tailed paired Student's t-test with a significance level of P < 0.05.


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 Materials and methods
 Results
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 References
 
The influence of time on the flux of PhIP (0.5–5 µM) across the Caco-2 cell monolayer is shown in Figure 1Go.Figure 1AGo shows the cumulative PhIP transport after apical loading of PhIP (absorption) and Figure 1BGo shows the corresponding data after basolateral loading (exsorption or efflux). The time course was linear at all PhIP concentrations for ~2 h in both directions. The basolateral to apical transport rate was significantly higher than the apical to basolateral transport rate at all time points and PhIP concentrations.



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Fig. 1. Cumulative transepithelial flux of PhIP across the Caco-2 cell monolayer versus time. (A) Apical to basolateral flux (Ap->BL); (B) basolateral to apical flux (BL->Ap). The PhIP concentrations used were 0.5 ({blacksquare}), 1 ({blacklozenge}), 2 (•) and 5 ({blacktriangleup}) µM. Each point is the mean ± SEM of 4–8 experiments. In most cases the SEM is contained within the symbol.

 
The influence of the concentration of PhIP on its transport rate, i.e. amount of PhIP transported across the cell monolayer per hour, as calculated from the 0.5–2 h time points, is shown in Figure 2Go. The transport rate was essentially linear with PhIP concentration both for the apical to basolateral and the basolateral to apical transport (correlation coefficients 0.999). At each PhIP concentration, the efflux was 3.0- to 3.9-fold higher than the absorption (P < 0.001), suggesting the involvement of an active process, although the low PhIP concentrations used (<=5 µM) did not show any saturation kinetics. The mean Papp values calculated from these data were 54.2 ± 0.7x10–6 cm/s (mean ± SEM, n = 24) for efflux versus 15.1 ± 0.6x10–6cm/s (n = 21) for absorption. For comparison, the Papp value for the paracellular transport marker mannitol in parallel inserts was 0.27 ± 0.04x10–6 cm/s (n = 9).



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Fig. 2. Transepithelial flux of PhIP across the Caco-2 cell monolayer as a function of PhIP concentration. •, Basolateral to apical flux; {circ}, apical to basolateral flux. The transport rate is calculated as the average rate for the 0.5, 1 and 2 h time points for each experiment. Each point is the mean ± SEM of 4–8 experiments. *Significantly higher than with apical loading at the same PhIP concentration (P < 0.001).

 
In order to establish that an active transport mechanism is responsible for the efflux of PhIP, equal concentrations of PhIP (1 or 5 µM) were loaded on both sides of the cell monolayers and aliquots were withdrawn from both sides at various time points. As shown in Figure 3Go, the PhIP concentrations on the apical side significantly exceeded those on the basolateral side at each time point with both 1 and 5 µM PhIP (P < 0.01–0.001, n = 3). It should be noted that, due to the 3-fold larger volume of basolateral than apical fluid, the changes in basolateral concentration were smaller than those in apical concentrations. At the end of the 3 h experiments the apical/basolateral concentration ratio was 2.7 at 1 µM and 2.0 at 5 µM initial PhIP concentration.



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Fig. 3. Basolateral to apical efflux of PhIP in the Caco-2 cell monolayer. Equal concentrations (1 or 5 µM) of [3H]PhIP were loaded on both sides. At 1, 2 and 3 h, samples were withdrawn for measurements of radioactivity. •, {blacksquare}, Apical concentration; {circ}, {square}, basolateral concentration. Means ± SEM are shown (n = 3). *,**Significantly higher than the corresponding basolateral concentrations (P < 0.01 and 0.0001, respectively).

 
When 50 µM of the P-glycoprotein inhibitor verapamil was added to both sides of the inserts, the apical to basolateral flux (absorption) of 5 µM PhIP increased from a Papp of 14.9 ± 0.3x10–6 to 21.0 ± 0.5x10–6 cm/s (P < 0.0001, n = 6), i.e. 141% of control, whereas the efflux Papp decreased slightly, from 56.4 ± 1.5x10–6 to 44.4 ± 1.6x10–6 cm/s (P < 0.0001), i.e. 70% of control (Figure 4AGo). The effect of 50 µM of the MRP inhibitor MK-571 on the transport of 5 µM PhIP is shown in Figure 4BGo. The absorption more than doubled from a Papp of 14.5 ± 1.1x10–6 to 31.3 ± 3.0x10–6 cm/s (P < 0.001, n = 6), whereas the efflux Papp decreased slightly, from 54.6 ± 0.7x10–6 to 42.6 ± 2.0x10–6 cm/s (P < 0.001), i.e. 78% of control.



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Fig. 4. Effect of (A) verapamil (50 µM) and (B) MK-571 (50 µM) on the transport of PhIP (5 µM) across the Caco-2 cell monolayer. Ap->BL, apical to basolateral flux; BL->Ap, basolateral to apical flux; hatched bars, control, without inhibitor; filled bars, with inhibitor. Each point is the mean 1 h value ± SEM of 4–8 experiments. *Significantly different from control (P < 0.001).

 

    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
This study shows that PhIP is readily absorbed through the Caco-2 intestinal epithelial cell monolayer, consistent with the lipophilic properties of this mutagen (octanol:pH 7.4 buffer logD = 1.32) (33). Thus, the mean Papp of 15x10–6 cm/s greatly exceeded the 1x10–6 cm/s value thought to be sufficient for effective absorption of many drugs in humans (34). As a comparison, the paracellular transport marker mannitol and the hydrophilic drug atenolol (logD –2.1) (23), both incompletely absorbed in humans, had Papp values of 0.3 x 10–6 and 0.2 x 10–6 cm/s, respectively. In contrast, the lipophilic drugs propranolol and alprenolol (logD 1.2 and 1.0), which are completely absorbed through passive diffusion and not subject to efflux mechanisms, have Papp values of 40 x 10–6 cm/s (23), i.e. considerably higher than for PhIP. The transport rate was essentially linear with time, indicating that metabolism of PhIP did not occur to any appreciable extent in the Caco-2 cells (as confirmed by HPLC analysis), in contrast to the flavonoid chrysin (29). These findings support previous notions that although only small amounts of ingested PhIP appear in urine unchanged, gastrointestinal absorption may be extensive (1722). Complete absorption was observed in a study in mice, using accelerator mass spectrometry (35).

In rat panreatic acini and hepatocytes the uptake and efflux of PhIP appeared to be through passive diffusion (33). In contrast, even though the absorption of PhIP by the Caco-2 cells, presumably through passive diffusion, was comparatively high, the finding of extensive active efflux is important. The Papp for efflux of PhIP, 54.2 x 106 cm/s, was even higher than for taxol, 31.8 x 10–6 cm/s, a well known substrate for P-glycoprotein with limited oral absorption (31). Thus, whereas the efflux is the result of active transport plus passive diffusion, the absorption is the result of passive diffusion minus active transport. How this may extrapolate to the in vivo human situation is difficult to predict, as we do not know how the expression of efflux transporters in Caco-2 cells compares with that in the normal human intestine, in particular at the level of the small intestine. A high expression of either P-glycoprotein or, in particular, MRP would obviously be expected to result in much reduced net absorption. The findings of active efflux mechanisms may not only be important for intestinal absorption but also for key target sites for the cancer-initiating effect of PhIP, such as the colon, breast and prostate (68).

The suggestion from this study, based on the effect of verapamil, that PhIP may be a substrate for P-glycoprotein is not surprising. PhIP is a basic, lipid-soluble molecule, properties characteristic of substrates of this transporter (26,36). The finding that PhIP may be a substrate for MRP is more surprising, as the physicochemical properties of PhIP are not typical of the mainly anionic substrates of these transporters (27,37). On the other hand, as the MRP transport family is expanding to include a number of new isoforms (27), we may find that some have selective affinity for heterocyclic amines.

At the present time P-glycoprotein is known to be expressed on the apical membrane of Caco-2 cells (36). However, very little is known about the MRP family in these cells or in the human intestine in general. We recently showed, using specific antibodies, that MRP2, but not MRP1, was expressed in Caco-2 cells (29), presumably at the apical membrane, as is the case for the canalicular membrane of the liver and the proximal tubules of the kidneys (38,39). Our working hypothesis at this time is therefore that PhIP is a substrate mainly for the MRP2 efflux pump.

In summary, the present findings indicate that intestinal absorption of PhIP in humans is effective. However, findings of active efflux, presumably through both P-glycoprotein and MRP2, have to be taken into consideration when estimating exposure to this mutagen/carcinogen at various sites. The Caco-2 cell monolayer is suggested as a useful model to probe membrane penetration of carcinogens in general.


    Acknowledgments
 
This work was supported by National Institutes of Health grants CA69138 and GM55561.


    Notes
 
1 To whom correspondence should be addressed Email: wallet{at}musc.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 2, 1999; revised June 1, 1999; accepted June 17, 1999.