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TOXICOLOGY

Specific Antibody Modulates Absorptive Transport and Metabolic Activation of Benzo[a]pyrene across Caco-2 Monolayers

Institute of Immunology, National Health Institute of Luxembourg, Luxembourg, Luxembourg (S.S.D.B., C.P.M.); and Laboratory for Pharmacotechnology and Biopharmacy, Catholic University of Leuven, Leuven, Belgium (P.A.)

Received November 18, 2004; accepted January 20, 2005.

	Abstract

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It has been shown that oral anticarcinogen antibodies can decrease intestinal absorption of procarcinogens. So far, no attempts have been made to understand the potential modulatory effect of such antibodies on metabolic activation at mucosal surfaces. Moreover, the influence of naturally induced serosal-specific antibodies on absorptive transport of carcinogens remains unknown. In this study, the prototype food carcinogen benzo[a]pyrene (B[a]P) and a specific monoclonal antibody were used to address these questions in a bicompartmental model of polarized intestinal cells (Caco-2). Apical (i.e., luminal) administration of a 30-fold molar excess antibodies increased about 25-fold recovery of unmetabolized B[a]P, concomitantly with a decrease of 80% in both absorptive transport and formation of phenol metabolites. Interestingly, when metabolism was slowed down by antibodies, cross-reactive antibodies also increased at least 5-fold the extracellular levels of the 7,8-diol-B[a]P, interrupting subsequent activation steps. On the other hand, basolateral antibodies changed by 8-fold the rate of carcinogen appearance in the basolateral compartment, albeit without interfering with the apical cellular uptake or sequestration of either B[a]P or 7,8-diol-B[a]P by apical antibodies. Furthermore, basolateral antibodies reduced exposure of Caco-2 monolayers to B[a]P as demonstrated by a 50% decrease in apical efflux of 3-OH-B[a]P. These data show for the first time that both luminal and serosal antibodies may reduce the carcinogenic process by preventing high local concentrations, which would overload DNA repair mechanisms. This study also sheds light on the relevance of both naturally induced and immunoprophylactic antibodies against polycyclic aromatic hydrocarbon carcinogens.

DNA adducts trigger the carcinogenic process; therefore, interruption or modulation of metabolic activation pathways should be beneficial. Numerous intervention strategies have been developed to prevent GI absorption of toxic low-molecular-weight compounds, including nonspecific adsorbents such as zeolitic minerals and clays (Smith et al., 1994; Phillips, 1999b). However, nonspecific adsorption technologies may inhibit nutrient absorption. One approach that so far has received little attention relies on active or passive antibodies that bind and sequester carcinogens. In particular, specific IgA antibodies might decrease carcinogen absorption and metabolic activation by mucosal epithelium. Silbart and Keren (1989) showed that passive transfer of intestinal hapten-specific antibodies to the ileum of naive rabbits resulted in a more than 50% reduction in intestinal absorption of carcinogens. More recently, Rasmussen and Silbart (1998) and Rasmussen et al. (2001) demonstrated that oral antibodies survive their passage through the GI tract and enhance fecal excretion of a radiolabeled model carcinogen.

Although these studies have shown that anticarcinogen antibodies can decrease intestinal absorption of procarcinogen, no attempts have been made to understand the potential of such antibodies to modulate metabolic activation by enterocytes. We have shown previously that anticarcinogen antibodies may modulate carcinogen-induced immunosuppression and metabolism both at the level of the procarcinogen and its activated metabolite (De Buck et al., 2005). In this study, we used an in vitro model to investigate mechanisms by which luminal carcinogen-specific antibodies may modulate the intestinal activation and detoxification pathway of B[a]P. Caco-2 cells were selected because after differentiation, they display morphological and biochemical characteristics of human enterocytes. In addition, they express the major phase 1 enzymes, mainly CYP1A1 and CYP1B1, responsible for the metabolic activation of PAH, as well as the relevant phase 2 enzymes required for conjugation [e.g., UDP glucuronosyltransferase (Munzel et al., 1999), sulfotransferase 1A1 (Baranczyk-Kuzma et al., 1991), and glutathione S-transferase (Peters and Roelofs, 1989)]. The types of B[a]P metabolites produced by Caco-2 cells are similar to those found by Autrup et al., who incubated human duodenum tissue with B[a]P (Autrup et al., 1978; Buesen et al., 2002). We also investigated the influence of serosal specific antibodies on absorptive transport of B[a]P.

	Materials and Methods

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Reagents and Antibodies. B[a]P was purchased from Sigma-Aldrich (Bornem, Belgium). Its metabolites were purchased from the National Cancer Institute Chemical Carcinogen Repository, Midwest Research Institute (Kansas City, MO). Tissue culture media and reagents were obtained from Cambrex Bio Science Verviers S.p.r.l. (Verviers, Belgium). All other reagents were of analytical or HPLC grade. The B[a]P-specific mouse IgG antibody (mAb-13) was produced as described by Scharnweber et al. (2001), and ascites fluid was purified by sequential precipitation methods with caprylic acid and ammonium sulfate solutions (Exbio Praha, Praha, Czech Republic). This antibody binds the activated metabolite 7,8-diol-B[a]P 3 times more efficiently than B[a]P or 3-OH-B[a]P, and about 10 times more efficiently than the 9-OH-B[a]P (De Buck et al., 2005). The mouse IgG antibody (R63, against infectious bursal disease virus, ATCC no. HB 9490) was used as irrelevant control. A molecular mass of 150 kDa was used to convert micrograms to picomoles.

Cell Culture. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% minimal essential medium nonessential amino acids, 100 IU/ml penicillin, and 100 µg/ml streptomycin ("Caco-2 medium") in a humidified incubator (5% CO₂; 37°C). Monolayers were subcultured every 3 to 4 days when they reached confluence of 80% by treatment with 0.05% trypsin and 0.02% EDTA. Cultures were mycoplasma-free.

Transport Experiments. Passages 54 to 77 of Caco-2 cells (Cambrex Bio Science Verviers S.p.r.l.) were seeded at a density of 40,000 cells/cm² on Costar Transwell inserts (0.4-µm pore diameter, 12-mm diameter; Corning Glassworks, Corning, NY). Confluence was reached within 3 to 4 days after seeding, and the monolayers were allowed to grow for 21 days. Before the experiments, cells were provided with new medium in both the apical and basolateral compartment and kept for 1 to 2 h under standard tissue culture conditions. The integrity of the monolayers was checked before and after each experiment by measuring the transepithelial electrical resistance (TEER) with an epithelial end-ohm voltohmmeter (WPI, Aston, England). Only monolayers with TEER values higher than 325 cm² were used. The inclusion of mAb-13, BSA, or irrelevant antibody did not affect integrity of the Caco-2 monolayers for at least 24 h.

Transport was initiated by adding 800 µl of fresh Caco-2 medium to the basolateral compartment and 300 µl of B[a]P in Caco-2 medium to the apical compartment (10 µM, 1 µM, or 100 nM). B[a]P solutions were prepared by stepwise dilution of a 10 mM stock solution in 100% DMSO. The final concentration of DMSO in the apical compartment was 1% in all samples irrespective of B[a]P concentration. Over a concentration range of 0.17 to 3% DMSO, the bidirectional transport of talinolol was not affected, illustrating that 1% DMSO does not influence the functionality of efflux carriers.

mAb-13 antibodies were added in either the apical and/or basolateral compartments. After 24 h, the total content of each compartment was harvested to obtain the required analytical sensitivity. Toxic effects of B[a]P or antibody were excluded by 24-h TEER measurements. Results are expressed as concentration of B[a]P or its metabolites in both the acceptor and donor compartment at the end of each incubation.

Uptake Experiments. For uptake experiments, Caco-2 cells (passages 30–40; ECACC 86010202; European Collection of Cell Cultures, Porton Down, UK) were plated at a density of 25,000 cells/cm² on collagen type I-coated 24-well plates (Greiner Bio-one, Wemmel, Belgium). Experiments were conducted using monolayers 21 days after seeding, when Caco-2 cells are fully differentiated and express the highest level of CYP1A1 (Boulenc et al., 1992). Monolayers were rinsed with Caco-2 medium before adding B[a]P (300 µl) as described above. After 24 h, supernatants were harvested and frozen at –20°C until further processing for HPLC analysis.

Hydrolysis of Conjugated Metabolites. To detect and quantify by HPLC total B[a]P metabolites, including glucuronidated and sulfated species, the conjugated metabolites were first enzymatically hydrolyzed. Cell culture supernatants were buffered with 30 mM sodium acetate buffer, pH 5.0, and incubated at 37°C with 5 µl of -glucuronidase (131,000 units/ml; G0876; Sigma-Aldrich) as well as 5 µl of sulfatase (4100 units/ml; S9751; Sigma-Aldrich). After 60 min, proteins were precipitated with chilled HPLC-grade methanol to a final concentration of 70%. Samples were incubated on ice for at least 30 min. After centrifugation (10,000g for 30 min), supernatants containing metabolites were transferred to new Eppendorf vials and HPLC-grade water was added to a final volume of 1.1 ml. Samples were stored at 4°C until HPLC analysis. Extraction of metabolites from culture medium was highly efficient (yield >70%) and reproducible (coefficient of variation <3%) for each metabolite, both in the presence or absence of specific antibodies. Specific antibodies also did not influence nonspecific adsorption of B[a]P to plastic ware.

HPLC Analysis of Hydrolyzed B[a]P Metabolites in the Culture Media. HPLC analysis was performed on the Akta Explorer 10S (Amersham Biosciences AB, Uppsala, Sweden) equipped with an Agilent 1100 Series fluorescence detector. After deconjugation by glucuronidase and sulfatase, B[a]P metabolites were separated on a 201TP54 reversed-phase C18 analytical column (250 x 4.6 mm i.d.; Vydac; Alltech Associates, Lokeren, Belgium). The column assembly was kept at 40°C in a column heater (Jet Stream 2 Plus; Knauer, Berlin, Germany). Samples (1 ml) were eluted using methanol/water (50:50) for 5 min followed by a linear gradient to 97% methanol over 25 min and by a final 15 min at 97% methanol. The flow rate was 1 ml/min. Upon excitation at 380 nm, fluorescence was monitored at 430 nm. The metabolites were identified and quantified by comparing retention times and peak heights with standards, eluting in the following order: 7,8-diol-B[a]P (18.6 min), 9-OH-B[a]P (26.8 min), 1-OH-B[a]P (28.6 min), 3-OH-B[a]P (31.3 min), and B[a]P (34.5 min). The limit of detection of all metabolites was about 10 pg. For all metabolites, linear calibration curves (R² > 0.999) between 10 and 1000 pg were obtained under the described experimental conditions.

Statistical Analysis. Mean and standard deviations of duplicate or triplicate samples of representative experiments are shown. Levels of significance were determined by paired t test using SigmaStat software (SPSS Inc., Erkrath, Germany). Values represent means ± S.D. of two or three independent experiments.

	Results

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Partitioning of B[a]P Metabolites. To investigate the modulation of B[a]P partitioning and metabolism by specific antibodies, a sensitive in vitro model of the intestinal mucosa is required because maximal concentrations of carcinogens are limited by the stoichiometry between antibody and carcinogen (Silbart and Keren, 1989; Silbart et al., 1996; Rasmussen and Silbart, 1998; Rasmussen et al., 2001). Caco-2 cells were cultured in the two-chamber Transwell system and incubated with increasing concentrations of B[a]P. Incubations were performed for 24 h, the time required for the appearance of B[a]P metabolites in supernatant (Buesen et al., 2002). Figure 1 shows that in Caco-2 cells, 10 µMB[a]P is metabolized to both the activated 7,8-diol-B[a]P and inactivated phenols. High levels of 3-OH-B[a]P, 1-OH-B[a]P, and to a lesser extent 9-OH-B[a]P were secreted into the apical compartment (Fig. 1A). At a 10-fold lower concentration, both phenols and 7,8-diol-B[a]P were reduced by a factor of 10. At physiologically relevant concentrations of 100 nM B[a]P (Phillips, 1999a), only the metabolites 3-OH-B[a]P and 1-OH-B[a]P were detectable. If the above-mentioned dose-dependent reduction in metabolite formation is extrapolated to physiological B[a]P concentrations, the 7,8-diol-B[a]P would be below detection limit, as indeed shown in Fig. 1A. Because the diol is not an endpoint metabolite, extracellular levels are lower due to further P450-mediated metabolism. At this concentration, recovery of apically administered B[a]P was also below the limit of detection in the apical compartment (Fig. 1A).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Concentrations of B[a]P and metabolites in the apical medium (A) and the basolateral medium (B) after apical incubation for 24 h of Caco-2 cells with decreasing concentrations of B[a]P in the absence of antibodies. Scale is truncated at 80 nM. (<0.15 nM, level below detection limit).

Figure 1B shows that at all concentrations of apical B[a]P, the recovery at 24 h of unmetabolized B[a]P at the basolateral side was only about 1% of the initial B[a]P added to the apical chamber. Independent of B[a]P input, the concentration of all monohydroxylated metabolites was 15 to 60 times higher in the apical chamber than in the basolateral chamber, which illustrates preferential excretion toward the apical compartment. In comparison, the concentration of 7,8-diol-B[a]P was only about 5 times higher in the apical chamber than in the basolateral chamber. This suggests that the intermediate metabolite was less efficiently effluxed to the apical compartment, probably because of less efficient phase 2 coupling compared with phenols (Fig. 1B). These experiments show that extracellular 7,8-diol-B[a]P can only be detected at high B[a]P concentrations. However, at physiologically relevant B[a]P concentrations that can be matched by antibodies (Silbart and Keren, 1989), metabolism can only be monitored by the formation of phenol metabolites as a surrogate for cellular uptake and metabolic activation of B[a]P.

Modulation of the Generation of B[a]P Metabolites by Antibodies. The dose-dependent effect of specific antibody (mAb-13) on both the appearance of B[a]P metabolites and disappearance of B[a]P was first investigated in an uptake experiment using differentiated Caco-2 cells cultivated in collagen-coated plates. Cells were incubated for 24 h with B[a]P (100 nM; 33 pmol) in the presence of increasing concentrations of specific antibodies. In the presence of irrelevant control antibody (Fig. 2, B and D) or nonspecific protein (BSA fraction V; data not shown), B[a]P was quantitatively metabolized after 24 h. In the presence of a 30-fold molar excess of specific antibody (2.9 µM; 900 pmol; 135 µg), 20% of B[a]P was recovered after 24 h (Fig. 2A). Recovery of B[a]P decreased rapidly when lower antibody concentrations were used. Figure 2C shows that binding of B[a]P by specific antibodies resulted in a concomitant dose-dependent decrease of phenol endpoint metabolites 1-OH and 3-OH-B[a]P. At 100 nM B[a]P, both 9-OH-B[a]P and 7,8-diol-B[a]P were not detectable (<0.15 nM; Fig. 1). In the presence of a 4-fold molar excess of antibody (0.4 µM; 120 pmol; 18 µg), metabolite formation was reduced by about 50% (Fig. 2C). Remarkably, at the highest antibody concentrations, up to 1 nM 7,8-diol-B[a]P was detected after 24 h. This finding suggests that antibody-mediated extracellular sequestration of this intermediate metabolite may inhibit its further metabolic activation toward the highly reactive BPDE. These experiments show that a small molar excess of specific antibody modulates procarcinogen activation by extracellular sequestration of both the parent compound and its activated metabolite 7,8-diol-B[a]P.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Levels of B[a]P and its metabolites in the medium after incubation of differentiated Caco-2 cells in monocompartmented wells for 24 h with B[a]P (100 nM; 33 pmol) in the presence of increasing amounts of specific antibody (0–900 pmol; 0–135 µg) (A and C) or irrelevant control antibody (0–900 pmol; 0–135 µg) (B and D). *, significance of recovery of B[a]P (p < 0.001) or inhibition of metabolism (p < 0.001) by specific antibody compared with no antibody as determined by paired t test (<0.15 nM, level below detection limit).

Modulation of Transepithelial Transport by Antibodies. We further investigated in the Transwell system how the 30-fold molar excess of antibody taken from Fig. 2 would modulate the partitioning of apical B[a]P as well as the appearance of its metabolites after 24 h. First, the influence of apical antibody alone was tested. As expected from the experiment of Fig. 2, Fig. 3 shows that the coaddition of B[a]P (100 nM; 33 pmol) with a 30-fold molar excess of specific antibodies (2.9 µM; 900 pmol; 135 µg) to the apical compartment resulted after 24 h in a recovery of about 25% of B[a]P, corresponding to apical concentrations of B[a]P that were about 25-fold higher than in the absence of antibody. In the presence of irrelevant antibody (2.9 µM) or nonspecific protein (BSA fraction V) (data not shown), the partitioning of B[a]P and its metabolites was not influenced compared with no antibody (Fig. 3). Specific apical antibodies also reduced B[a]P in the basolateral compartment to about 20% of those found in the absence of antibody. Sequestration of B[a]P by apical antibodies was accompanied by an equally strong concomitant decrease of 80% of apical concentrations of both 1-OH and 3-OH-B[a]P. Apical antibodies also reduced basolateral 3-OH-B[a]P levels by 35%. Although 7,8-diol-B[a]P was undetectable (<0.15 nM) in the absence of antibody, specific antibody increased apical 7,8-diol-B[a]P concentrations at least 5-fold up to 0.85 nM.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Modulation of transepithelial transport and metabolism of apically administered B[a]P (100 nM; 33 pmol) by apical (AP; 2.9 µM) and/or basolateral (BL; 2.2 µM) specific antibody (mAb-13) in Caco-2 medium. Amounts of B[a]P and its metabolites were found in apical medium (A) and basolateral medium (B) after 24 h in the presence or absence of antibody. *, significance of recovery of B[a]P (p < 0.001) or inhibition of metabolism (p < 0.001) by antibody compared with no antibody as determined by paired t test (<0.15 nM, level below detection limit).

In a second set of experiments, the influence of basolateral antibody was tested. Interestingly, Fig. 3 shows that the addition of similar specific antibody concentrations (2.2 µM; 1760 pmol; 264 µg) to the basolateral chamber increased by 18-fold the levels of B[a]P found after 24 h in this compartment in comparison with no antibody. Similar basolateral concentrations of irrelevant control antibody or nonspecific protein (BSA fraction V) showed no effect on absorptive transport (data not shown) compared with no antibody (Fig. 3). The sequestration of B[a]P in the basolateral chamber was associated with a reduction of about 40% of phenol metabolites in the apical compartment, suggesting that sequestration of carcinogens by specific antibodies results in enhanced absorptive transport and reduces the retention time as well as the metabolic activation of B[a]P inside Caco-2 cells.

Finally, specific antibody was added simultaneously to both compartments. Yet, in the apical compartment, recovery of B[a]P by apical antibodies was not influenced by the simultaneous presence of antibodies in both chambers, compared with apical antibodies alone. Metabolite levels in the apical compartment were not significantly reduced by applying antibody in both chambers compared with apical antibodies alone. When antibodies were present in both chambers, levels of basolateral B[a]P were 3 times lower compared with basolateral antibodies alone.

Modulation of Kinetics of B[a]P Transport by Basolateral Antibodies. The experiment of Fig. 3 suggests that systemic antibodies may enhance the absorptive transport of B[a]P from the gut. In the following experiment, we investigated how basolateral antibodies may influence the kinetics of absorptive transport of B[a]P. Increasing concentrations of antibody were added to the basolateral compartment, whereas antibody was absent in the apical compartment. A high concentration of irrelevant antibody (2.5 µM) (Fig. 4A) or nonspecific protein BSA fraction V (2.5 µM) (data not shown) showed no increase in absorptive transport compared with no antibody (data not shown). Figure 4A shows that at the highest antibody concentration of specific antibody (2.5 µM; 2000 pmol; 300 µg), about 45% of the initial B[a]P was recovered after 4 h in the basolateral compartment. This was about 8 times more than in above-mentioned control conditions. A rapid decrease of B[a]P was observed in the apical compartment, but this was independent of basolateral antibody (Fig. 4B). This further confirms that basolateral antibodies did not influence the uptake of apical B[a]P by Caco-2 cells. These data show that basolateral antibodies enhance the accumulation of B[a]P into the basolateral chamber, without enhancing cellular uptake from the apical compartment.

View larger version (14K): [in this window] [in a new window]   Fig. 4. A, cumulative absorptive transport of B[a]P in the presence of specific and irrelevant control antibody in the basolateral (BL) medium. B, decline of apical B[a]P levels in the presence of specific or irrelevant control antibody in the BL medium. A and B, B[a]P (100 nM; 33 pmol) was administered apically in Caco-2 medium at time 0 (*, p < 0.01 and **, p < 0.001, significance of increase in absorptive transport of B[a]P by specific antibody compared with control antibody as determined by paired t test).

Decrease of Metabolic Activation of B[a]P by Basolateral Antibodies. In a last set of experiments, different concentrations of antibody were added to the basolateral compartment but not to the apical compartment. Transport was initiated by adding 100 nM B[a]P to the apical side. Figure 5A clearly shows the antibody-dependent decrease of apical 3-OH-B[a]P levels, suggesting that basolateral antibodies modulate metabolic activation of apically administered B[a]P. In the presence of basolateral antibody (2.5 µM; 2000 pmol; 300 µg), apical 3-OH-B[a]P titers were about 50% lower at 24 h than in the presence of irrelevant control antibody (2.5 µM). The rapid decrease of B[a]P in the apical compartment was independent of basolateral antibody (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 5. A, apical efflux of 3-OH-B[a]P in the presence of specific or irrelevant control antibody in the BL medium. B, absorptive transport of B[a]P in the presence of specific or irrelevant control antibody in the BL medium. A and B, B[a]P (100 nM; 33 pmol) was administered apically in Caco-2 medium at time 0. *, significance of decrease in apical efflux of 3-OH-B[a]P (p < 0.001) by specific antibody compared with control antibody as determined by paired t test.

Figure 5B confirms that antibodies enhance the accumulation of B[a]P into the basolateral chamber. After 4 h, however, basolaterally sequestered B[a]P slowly declines, suggesting that it is released from its antibody and repartitions into the cell. These results further support our hypothesis that basolateral antibodies may decrease the residence time of B[a]P at the monolayer, resulting in a decreased local metabolic activation.

	Discussion

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Control over GI absorption of foodborne carcinogens by oral antibodies could be an attractive strategy to prevent cancer. In this study, the prototype carcinogen B[a]P was selected to assess the ability of hapten-specific antibodies to prevent GI uptake of PAH. The average daily intake of PAH and B[a]P by the general population, including professionally exposed individuals and cigarette smokers, is estimated to be in the lower microgram range per day (Hecht, 1999; Phillips, 1999a). These quantities can be matched on a molar basis by passive transfer of intestinal specific antibody (Rasmussen and Silbart, 1998) or even active immunization (Silbart et al., 1996). The current study extends previous observations of antibody-mediated carcinogen sequestration within the GI tract (Rasmussen and Silbart, 1998; Rasmussen et al., 2001) by demonstrating that antibodies may not only decrease absorptive transport but also modulate carcinogen metabolism, in particular metabolic activation. Metabolic activation of B[a]P proceeds via the intermediate metabolite 7,8-diol-B[a]P to the ephemeral DNA alkylating diol-epoxide BPDE, the ultimate carcinogen. Small but significant amounts of these harmful byproducts of B[a]P detoxification are catalyzed by the same set of P450 enzymes. Unfortunately, at physiologically relevant concentrations of B[a]P (100 nM), extracellular 7,8-diol-B[a]P levels are below the detection limit (Fig. 1).

Since stoichiometric considerations between antibody and carcinogen dictate the use of the low physiologically relevant B[a]P concentrations, we assessed phenol formation to investigate how antibodies could modulate metabolism of B[a]P. Phenols are useful surrogates because their appearance in supernatant is normally associated with the intracellular formation of activated 7,8-diol-B[a]P. Apical (i.e., luminal) administration of antibodies increased recovery of unmetabolized B[a]P, concomitantly with a decrease in both absorptive transport and formation of phenol metabolites. Although a small molar excess of antibodies (5-fold, corresponding to a 10-fold excess of antibody binding sites) already strongly decreased generation of phenol metabolites, sequestration of unmetabolized B[a]P was only observed at higher concentrations of antibodies. Although antibodies slowed down metabolism (Fig. 2C), irreversible sequestration cannot be obtained, even at high molar excess of antibody binding sites (Figs. 2A and 5B). B[a]P is a highly lipophilic compound (log P_ow = 6.04) with high-affinity intracellular binding sites (Piskorska-Pliszczynska et al., 1986); hence, intracellular partitioning may be difficult to fully prevent.

Interestingly, even when the metabolism was slowed down by antibodies, significant extracellular levels of the 7,8-diol-B[a]P were detected in comparison with controls. We have previously shown that the antibody mAb-13 binds 7,8-diol-B[a]P with similar efficiency as B[a]P (De Buck et al., 2005).

Extracellular sequestration of this intermediate metabolite by cross-reactive antibody most probably interrupted subsequent activation steps and led to its extracellular accumulation (Fig. 6). These results suggest that cross-reactivity with activated metabolites may provide a second chance of interrupting metabolic activation and therefore would increase the efficiency of immunoprophylactic strategies.

View larger version (26K): [in this window] [in a new window]   Fig. 6. B[a]P-specific antibodies in both the lumen and the serum may modulate B[a]P metabolism at different levels of its activation pathway.

A previous attempt to prevent GI absorption of B[a]P by active immunization seemed to have failed (Moolten et al., 1978) because total radiotracer in feces was not reduced. However, oral B[a]P is almost entirely eliminated via hepatobiliary circulation, and its inactive conjugated phenol metabolites may be actively secreted back into the gut (Buesen et al., 2002). Therefore, a potentially beneficial effect may have been overlooked because the nature of antibody-bound radioactive material was not determined. The preferential secretion of phenol metabolites toward the apical compartment in our cultures is in agreement with Buesen et al. (2003), who suggested this apical efflux as a natural protection mechanism to counteract absorption (Buesen et al., 2002). However, a luminally directed transport of activated 7,8-diol-B[a]P metabolite generated in the gut epithelium would be more beneficial. Our observation of 5-fold higher levels of diol in the apical compartment in the absence of antibody suggests that also 7,8-diol-B[a]P is actively and preferentially returned to the apical compartment (Fig. 1, B[a]P; 1000 nM). However, apical titers of 7,8-diol-B[a]P were less pronounced compared with the phenols (which were about 20 times higher in the apical compartment), most probably because 1) phase 2 conjugation of the diol is less efficient compared with phenols, and 2) the unconjugated intermediate can be reabsorbed and further metabolized to the diol-epoxide BPDE. With low concentrations of B[a]P, the 7,8-diol-B[a]P was not detectable unless antibodies were added to the apical compartment. This finding demonstrates that this metabolite is capable of leaving the cell and that antibodies that bind to unconjugated diol may subsequently inhibit reuptake and further activation (Fig. 6). Extracellular sequestration by specific antibodies would act complementary to active efflux mechanisms.

Modulation of metabolic activation of B[a]P and redistribution of its metabolites may be highly biorelevant because B[a]P is known to be a contact carcinogen rather than a systemic carcinogen after oral administration (Culp et al., 1998). Although the oral bioavailability of B[a]P is thought to be low (Foth et al., 1988), studies have demonstrated that B[a]P is rapidly absorbed in the epithelial cells but is slowly transported into the adjacent capillary bed (Plant et al., 1987). Thus, the epithelium may accumulate high concentrations even at low environmental exposure levels, and long colonic and intracellular residence times may lead to substantial local metabolic activation, even when enzymatic activity is low (Gerde et al., 1997). Local metabolic activation may be further enhanced by induction of P450 as shown by induction of CYP1A1 at mucosal surfaces after the consumption of charcoal-grilled meat diets (Fontana et al., 1999). Although metabolism and its induction have been suggested as natural protection mechanisms to decrease systemic uptake (Brooks et al., 1999; Buesen et al., 2002), local neoplasia may be an unfortunate byproduct of this defense mechanism. In this perspective, oral administration of specific antibodies may 1) avoid the initial interaction of carcinogens with the epithelium; 2) inhibit the reuptake and further metabolic activation of released unconjugated 7,8-diol-B[a]P; 3) inhibit reuptake of phase 2 metabolites after enzymatic cleavage to phase 1 metabolite catalyzed by sulfatases of the gut microflora (Fig. 6); or 4) decrease the effective concentration of parent compound and metabolites and thus modulate the relative pattern of product species; in particular, lower concentrations may favor phase 2 reactions and diminish the availability of oxygenated metabolites for recycling. It should, however, be added that our in vitro model does not take into account the very complex gut environment in which matrix factors (pH, proteolytic enzymes, and bile) and GI cellular factors (Fc receptors and transporters) may be responsible for multiple competing effects.

On the other hand, serum antibodies that are induced in individuals heavily exposed to B[a]P (Harris et al., 1985; Haugen et al., 1986; Newman et al., 1988) could enhance systemic carcinogen absorption as suggested by simple dialysis experiments (Silbart et al., 1996). Apically administered B[a]P also reached the basolateral compartment, where it was efficiently sequestered by basolateral antibodies. The latter changed the kinetics of carcinogen appearance in the basolateral compartment (Fig. 4A), albeit without interfering with the sequestration of either B[a]P or 7,8-diol-B[a]P by apical antibodies (Fig. 3). Remarkably, basolateral antibodies did not accelerate the decline of apically administered B[a]P and are therefore not expected to accelerate initial uptake from the luminal side. This finding may be explained by an initial rapid intracellular accumulation as driving force responsible for the disappearance of B[a]P from the apical compartment. These results strongly suggest that sequestration in the basolateral chamber accelerates the partitioning of intracellular B[a]P toward the basolateral chamber and therefore reduces exposure of the Caco-2 monolayer to B[a]P. Reduced exposure and metabolism was indeed demonstrated by a decreased apical efflux of 3-OH-B[a]P when antibodies were present only in the basolateral compartment (Fig. 5). In vivo, the effect would be additive on the sink conditions of the bloodstream and further decrease residence time of PAH in the epithelium, thereby preventing accumulation of high levels of genotoxicant in these susceptible tissues (Fig. 6). Although blood and tissue fluids will further dilute carcinogens and expose peripheral tissues to lower concentrations, the biological outcome of antibody-mediated carcinogen redistribution remains speculative.

The results of this study may be of importance in the development of effective strategies against PAH carcinogenesis, in particular against ingested PAH in contaminated food items. These data show for the first time the possibility of specific antibodies to interrupt metabolic activation of B[a]P at different levels and therefore change the balance between carcinogen activation and detoxification, a major determinant of cancer risk (Hecht, 2002). Antibodies may inhibit the carcinogenic process by preventing high local concentrations that could overload DNA repair mechanisms.

	Acknowledgements

 
We thank Prof. T. Vélu and Dr. A. Brandenburger (Université Libre de Bruxelles, Brussels, Belgium) for fruitful discussions and guidance as well as Dr. D. Knopp (Technische Universität Munich, Munich, Germany) for mAb-13 antibody. We acknowledge the skillful assistance of R. Mols and L. Van Vaeck (Laboratory for Pharmacotechnology and Biopharmacy, Catholic University of Leuven, Leuven, Belgium).

	Footnotes

 
This research was supported by Fonds National de la Recherche Grant FNR/01/04/11 and the Centre de Recherche Public-Santé, Luxembourg. S.D.B. was supported by a fellowship of the Ministère de la Recherche, Luxembourg.

doi:10.1124/jpet.104.081034.

ABBREVIATIONS: GI, gastrointestinal; B[a]P, benzo[a]pyrene; PAH, polycyclic aromatic hydrocarbon; BPDE, 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; mAb, monoclonal antibody; HPLC, high-performance liquid chromatography; 7,8-diol-B[a]P, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; 1-OH-B[a]P, 1-hydroxybenzo[a]pyrene; 3-OH-B[a]P, 3-hydroxybenzo[a]pyrene; 9-OH-B[a]P, 9-hydroxybenzo[a]pyrene; TEER, transepithelial electrical resistance; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; P450, cytochrome P450; BL, basolateral.

Address correspondence to: Dr. Claude P. Muller, Institute of Immunology, Laboratoire National de Santé, Rue Auguste Lumière 20 A, L-1011 Luxembourg. E-mail: claude.muller{at}lns.etat.lu

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