Institute of Immunology, Laboratoire National de Santé, Rue Auguste Lumière 20A, L-1011 Luxembourg, Luxembourg and 1 IRIBHM-IBMM, Université Libre de Bruxelles, Rue des Professeurs Jeener et Brachet 12, B-6041 Gosselies, Belgium
* To whom correspondence should be addressed. Tel: +352 490604; Fax: +352 490686; Email: claude.muller{at}INS.ETAT.IU
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Abstract |
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Abbreviations: AhR, cytosolic arylhydrocarbon receptor; ANF, -naphthoflavone; B[a]P, benzo[a]pyrene; BPDE, 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; 7,8-diol-B[a]P, 7,8-dihydro-7,8-dihydroxybenzo[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; P450, human cytochrome P450; PAH, polycyclic aromatic hydrocarbons; PBMC, peripheral blood mononuclear cells; PHA, phytohemagglutinin; trans-anti-B[a]P-tetrol, r-7,t-8,t-9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene
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Introduction |
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In vivo studies of chemical carcinogenesis conveniently use high doses of carcinogens mostly given as a single bolus. Such models may provide simple surrogate end-points, but they poorly reflect chronic exposure to the low levels of carcinogens that are found in the environment. Moreover, excessive doses may overload the binding capacity of available binding sites and therefore underestimate the modulatory effect of such antibodies (9,10).
Here, the value of a carcinogen-specific antibody is for the first time assessed in a metabolism study using low pathophysiological doses of benzo[a]pyrene (B[a]P) in different metabolically competent in vitro test systems. B[a]P is frequently used to model exposure to and effects of PAH. After, ingestion, inhalation or dermal absorption (1113), B[a]P acquires its mutagenic and carcinogenic properties in a multi-step process (14,15). Members of the cytochrome P450 superfamily (P450) activate B[a]P to B[a]P-7,8-epoxide, which is hydrolyzed to 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (7,8-diol-B[a]P). 7,8-diol-B[a]P is further metabolized by P450 to the mutagenic anti- and syn-diasteriomers of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). The (7R,8S,9S,10R) enantiomer of anti-BPDE is the most abundant and most active carcinogen, forming a major adduct with the exocyclic amino group of deoxyguanosine (16). anti-BPDE can undergo spontaneous hydrolysis to two products, r-7,t-8,t-9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-anti-B[a]P-tetrol) and r-7,t-8,t-9,t-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene. These two tetrol metabolites are indicative of anti-BPDE formation (17). B[a]P detoxification proceeds via the formation of phenol metabolites such as 1-hydroxy- (1-OH-B[a]P), 3-hydroxy- (3-OH-B[a]P) and 9-hydroxybenzo[a]pyrene (9-OH-B[a]P) (14,15). Detoxification of 7,8-diol-B[a]P and the phenol metabolites is further assisted by phase II enzymes, which convert the metabolites to highly soluble species.
We dissected the molecular and cellular mechanisms by which a B[a]P-specific antibody may modulate cellular uptake, metabolic activation and detoxification of both B[a]P and its activated metabolite 7,8-diol-B[a]P. The biological relevance of carcinogen redistribution due to the antibody was further assessed in two different target cells. The B[a]P-specific antibody was investigated for its potential to reverse B[a]P-induced P450 induction in a human liver cell line and for its potential to restore B[a]P-induced inhibition of proliferation in human blood lymphocytes.
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Materials and methods |
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Specificity of the mAb-13 antibody was determined by competition ELISA using B[a]Povalbumin as immobilized antigen and B[a]P (Sigma-Aldrich, Bornem, BE) and its metabolites 7,8-diol-B[a]P, 3-OH-B[a]P and 9-OH-B[a]P as competitors (National Cancer Institute Chemical Carcinogen Reference Standard Repository, Midwest Research Institute, Kansas City, MO). 386-well microtiter plates (Greiner, Wemmel, BE) were coated overnight with 125 nM/well B[a]Povalbumin in 0.1 M bicarbonate buffer (pH 9.6). After washing, plates were blocked for 120 min with 1% bovine serum albumin in Tris-buffered (15 mM) saline. MAb-13 (65 ng/ml) in Tris-buffered saline containing 0.1% Tween-20 and 1% bovine serum albumin was preincubated for 30 min with increasing concentrations of competitor (0128 µM). A pyridine derivative was used as an irrelevant competitor (0% competition). Each mixture was then added to the B[a]Povalbumin-coated plates for 90 min at room temperature. Alkaline phosphatase-conjugated goat anti-mouse IgG (1/750; Southern Biotechnology Associates, Birmingham, AL) and the corresponding substrate were used to measure antibody binding. Absorbance was measured after 60 min at 405 nm. The absorbance (A = 0.087 ± 0.02) obtained with 128 µM B[a]P as competitor was similar to background levels (A = 0.078 ± 0.02) without mAb-13. This value was considered 100% competition and was subtracted from all values. The concentration of competitor that decreased the mAb-13 signal by 50% (IC50) was calculated and compared to the IC50 of B[a]P (considered to be 100%) to give the relative percentage binding.
Preparation of rat liver microsomes
Twelve-week-old female Wistar rats were fasted overnight and killed by cervical dislocation. Livers were removed, rinsed in an ice-cold solution of isotonic KCl and homogenized in 4 vol of 20 mM TrisHCl buffer (pH 7.4) containing 250 mM sucrose and 1 mM EDTA using a glassteflon PotterElvehjem homogenizer (Fisher Bioblock, Doornik, Belgium). The homogenate was filtered and centrifuged for 10 min at 600 g and for 30 min at 10 000 g to sediment nuclei, cell debris and mitochondria. The supernatant was again centrifuged at 170 000 g for 1 h to sediment the microsomes. The microsomal pellet was resuspended in homogenization buffer to a protein concentration of 1015 mg/ml. Microsomes were frozen at 80°C until use.
DNA adduct formation by microsomes
An aliquot of 7,8-diol-B[a]P (50 nM) and 0.4 mg calf thymus DNA were incubated with microsomes in a total volume of 0.4 ml of 0.1 M TrisHCl (pH 7.4), 1 mM EDTA, 3 mM MgCl2 and an NADPH-generating system (5 mM NADP+, 25 mM glucose 6-phosphate and 1.75 U glucose 6-phosphate dehydrogenase) (Boehringer Mannheim, Mannheim, Germany). Incubations were carried out for 1 h at 37°C in the presence of specific (mAb-13) or irrelevant antibody (control). DNA was extracted twice with a mixture of phenol/chloroform/isoamyl alcohol (25:24:1). DNA in the aqueous phase was precipitated by adding 40 µl of 3 M sodium acetate and 0.8 ml of ethanol and centrifugation for 15 min at 4°C. The pellet was washed once with 70% ethanol, resuspended in 0.5 ml of buffer and further purified by treatment with proteinase K (20 µg/ml). The phenol/chloroform/isoamyl alcohol extractions were repeated and the DNA precipitated with sodium acetate and ethanol. The pellet was washed again with 70% ethanol. DNA was first analyzed for unbound trans-anti-B[a]P-tetrol and subsequently hydrolyzed as described below.
Hydrolysis of DNA
Prior to hydrolysis, DNA adducted by incubation with microsomes was analyzed for unbound trans-anti-B[a]P-tetrol. The portion of DNA to be hydrolyzed was rinsed with 100% ethanol, then the ethanol was removed and mixed with water to make a final concentration of 20% ethanol. The ethanol wash was analyzed for trans-anti-B[a]P-tetrol as described below. The DNA, free of unbound tetrols, was dissolved in water and the DNA concentration and purity were determined by UV analysis. The DNA was hydrolyzed for 4 h at 85°C in 0.1 N HCl, releasing tetrols from BPDEDNA (19,20). Hydrolyzed DNA was stored at 20°C until analyzed for trans-anti-B[a]P-tetrol by HPLC.
Metabolism of B[a]P and 7,8-diol-B[a]P in HepG2 cells
HepG2 cells, a human hepatoblastoma cell line, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (v/v), 100 IU/ml penicillin and 100 µg/ml streptomycin under standard tissue culture conditions (5% CO2, 37°C). Stock solutions of B[a]P and 7,8-diol-B[a]P were prepared in dimethyl sulfoxide. An aliquot of 20 µl of phosphate-buffered saline with or without antibody was added to 100 µl of cell suspension (106 cells/ml). Then, 30 µl of B[a]P or 7,8-diol-B[a]P in fully supplemented medium was added to a final concentration of 150 or 100 nM, respectively. The final dimethyl sulfoxide concentration was 1% in all cultures. Antibody was present during the complete incubation period and samples were taken after 18 or 48 h for incubation with 7,8-diol-B[a]P or B[a]P, respectively. Addition of non-specific protein (e.g. bovine serum albumin) or control antibody did not influence metabolism.
Hydrolysis of B[a]P and 7,8-diol-B[a]P metabolites in cell culture medium
In order to detect and quantify total B[a]P metabolites, including glucuronidated and sulfated species, by HPLC these 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 ß-glucuronidase (131 000 U/ml) (G0876; Sigma-Aldrich, Bornem, Belgium), as well as 5 µl of sulfatase (4100 U/ml) (S9751; Sigma-Aldrich). After 60 min the proteins were precipitated by addition of chilled HPLC grade methanol to a final concentration of 70%. Samples were incubated on ice for at least 30 min. After centrifugation (10 000 g for 30 min) the supernatants containing metabolites were transferred to new Eppendorf vials and HPLC grade water was added to a final volume of 1.1 ml. The extraction of metabolites from culture medium was highly efficient (yield >70%) and reproducible (coefficient of variation <3%) for each metabolite, both in the presence and absence of specific antibody. Samples were stored at 4°C until HPLC analysis.
HPLC analysis of trans-anti-B[a]P-tetrol and 7,8-diol-B[a]P in culture medium and adducted DNA
trans-anti-B[a]P-tetrol and 7,8-diol-B[a]P were analyzed by a modification of the method of Alexandrov et al. (20). HPLC analysis was performed in an Amersham Pharmacia Biotech Äkta Explorer 10S equipped with an Agilent 1100 Series fluorescence detector. All solvents were degassed under vacuum prior to use and continuously degassed under vacuum throughout the analyses. Prior to analysis cell culture supernatants or adducted DNA samples were hydrolyzed as described above by enzymatic or acid treatment, respectively. Samples (1 ml) were concentrated by an initial 10 min isocratic elution with 35% methanol in water over a 4.6 x 45 mm Beckman Ultrasphere ODS precolumn at 1.0 ml/min. The sample was then eluted from the precolumn by a 30 min, 35100% methanol/water linear gradient. The gradient solvents were switched by a column switching valve to flow over a 4.6 x 250 mm Beckman Ultrasphere ODS analytical column. The excitation wavelength was set at 344 nm and emission was measured at 398 nm. The level of each metabolite was determined by comparison with a standard curve (2100 pg) generated from the fluorescence areas of authentic standards (National Cancer Institute Chemical Carcinogen Reference Standard Repository).
HPLC analysis of B[a]P metabolites in culture medium
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 ID) (Vydac). The column assembly was kept at 40°C in a column heater (Jet Stream 2 Plus; Knauer, 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, metabolite fluorescence was detected 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, 9-OH-B[a]P, 1-OH-B[a]P, 3-OH-B[a]P and B[a]P. The limit of detection of all metabolites was 10 pg. For all metabolites highly linear calibration curves (R2 > 0.999) between 10 and 1000 pg were obtained under the described experimental conditions.
Quantification of CYP 1A1 mRNA by real-time PCR
HepG2 cells (1 x 105) were added in 100 µl to 96-well plates and allowed to attach for 4 h at 37°C. Supernatants were subsequently removed and cells were incubated for 6 h with a mixture containing 500 nM B[a]P and the specific antibody (mAb-13) in culture medium. Controls contained no antibody. Total RNA was extracted using a High Pure RNA isolation kit (Boehringer Mannheim). Total RNA was reverse transcribed in two steps. RNA (8 µl) was mixed with 0.5 µl of oligo(dT)16 (500 µg/ml), 2 µl of dNTPs (10 mM each), heated to 72°C for 10 min and subsequently cooled on ice. The solution was mixed with 4 µl of 5x reverse transcriptase buffer, 2.5 mM MgCl2, 10 mM dithiothreitol, 10 U RNase inhibitor and 50 U Multiscribe transcriptase enzyme (Applied Biosystems, Lennik, Belgium). Reverse transcription was performed for 80 min at 42°C. PCR amplification was carried out in a 25 µl final volume containing 5 µl of cDNA sample (1/10 dilution), 2.5 µl of 10x PCR Gold Buffer, 3 mM MgCl2, 200 µM dNTPs, 100 nM each primer, SYBR Green 0.5x and 0.625 U AmpliTaqGold (Applied Biosystems). Each cycle consisted of 95°C for 15 s, 60°C for 30 s and 72°C for 30 s, the fluorescence being read at the end of this step. The following primers were used: CYP1A1 forward, 5'-GCTGACTTCATCCCTATTCTTCG-3', reverse, 5'-TTTTGTAGTGCTCCTTGACCATCT-3'; ß-actin forward, 5'-GGCCACGGCTGCTTC-3', reverse, 5'-GTTGGCGTACAGGTCTTTGC-3'.
Flow cytometry of B[a]P in peripheral blood mononuclear cells
Human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque gradient and resuspended in RPMI 1640 medium (Cambrex Bioscience, Verviers, Belgium). B[a]P (12 pmol) was preincubated for 5 min at room temperature with specific (mAb-13) or irrelevant antibody in 40 µl of phosphate-buffered saline (Cambrex Bioscience). A sample of 105 cells was added and incubated for 60 min at 37°C in a final volume of 200 µl. Cells were analyzed on an Epics Elite ESP Flow Cytometer (Beckman-Coulter, Hailey, FL). B[a]P was excited at 351364 nm using an Innova Enterprise Ion laser (Coherent Inc., Santa Clara, CA) and emission was measured between 400 and 450 nm by blocking primary light with dichroic 400 and 450 nm long-pass filters (Omega Optical, Brattleboro, VT). Live cells were detected by gating on dual scatter profiles generated with the 488 nm band of the argon laser. Data acquisition of this two-laser system was triggered on the 488 nm forward scatter signals.
PBMC metabolism and mitogenesis
Heparinized venous blood was obtained from different donors in the laboratory. Gradient-isolated PBMC were resuspended at 106 cells/ml in RPMI 1640 supplemented with 10% fetal bovine serum (v/v), 100 IU/ml penicillin and 100 µg/ml streptomycin. A sample of 105 cells (100 µl) was added to 20 µl of antibody or -naphthoflavone (ANF) (Sigma-Aldrich) in phosphate-buffered saline (Cambrex Bioscience). Then, 20 µl of B[a]P or 7,8-diol-B[a]P in medium were added, as well as 60 µl of phytohemagglutinin (PHA) (Sigma-Aldrich) to a final concentration of 5 µg/ml. Stock solutions of B[a]P, 7,8-diol-B[a]P and ANF were prepared in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide was 0.1% in all cultures, which did not influence mitogenesis (data not shown). For metabolism studies samples were taken at the indicated time points, hydrolyzed and analyzed by HPLC as described above. For proliferation assays all cultures were incubated for 72 h at 37°C, pulsed for 18 h with [3H]thymidine (1 µCi/well), harvested with a cell harvester (ICN Biomedicals) onto filter paper disks and assessed by scintillation counting. Results are reported both as counts per minute and as a percentage of the control proliferation in the absence of B[a]P.
Statistical analysis
Data were analyzed with Sigmastat software (Jandel Scientific, Erkrath, Germany) using a paired t-test. Representative experiments, repeated at least three times, are shown.
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Results |
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Modulation by the antibody of CYP 1A1 induction by B[a]P
CYP1A1 is the main culprit in metabolic activation of B[a]P to carcinogenic metabolites and CYP1A1 induction by B[a]P and its metabolites is a critical step towards enhanced cancer risk. In order to determine whether a substrate-specific antibody may modulate CYP1A1 induction, HepG2 cells were incubated with 500 nM B[a]P (20 pmol) before total RNA was extracted. This was the minimum concentration of B[a]P required to significantly induce CYP1A1 mRNA and induction of CYP1A1 mRNA was optimal after 6 h (data not shown). Figure 3 shows that B[a]P (500 nM, 20 pmol) induces a 50-fold increase in CYP1A1 mRNA expression. A 25-fold molar excess of specific antibody mAb-13 (500 pmol, 75 µg) inhibited CYP1A1 induction by 50%.
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Human PBMC were incubated (37°C, 60 min) with different concentrations of B[a]P (01.6 µM) (Figure 4A). The uptake of B[a]P was linear between 0 and 200 nM (R2 = 0.9898) and fluorescence reached saturation levels at higher concentrations. This was a sensitive assay which at concentrations as low as 60 nM generated a fluorescence signal which was 10 times higher than the signal of unstained control cells (no B[a]P). Because higher concentrations are difficult to match by equimolar amounts of antibody, this concentration was chosen to investigate the possibility of inhibiting cellular uptake B[a]P with the antibody.
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Modulation of B[a]P metabolism in human PBMC
Stimulated human PBMC can activate B[a]P and their proliferation is highly sensitive to this carcinogen. In the absence of specific antibody (mAb-13), recovery of B[a]P was <5% after 72 h (Figure 5A). 1-OH-B[a]P, 3-OH-B[a]P and 9-OH-B[a]P accumulated in a linear fashion during the first 48 h before reaching plateau values (Figure 5B). In contrast, levels of 7,8-diol-B[a]P in supernatants decreased after maximal values were reached at 48 h. This biphasic curve for 7,8-diol-B[a]P reflects the simultaneous production and further processing of this intermediate metabolite.
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Inhibition of B[a]P- and 7,8-diol-B[a]P-induced immunotoxicity
In an effort to determine the biological relevance of antibody-mediated modulation of B[a]P metabolism, the ability of a B[a]P-specific antibody to inhibit B[a]P-induced immunotoxicity was tested. Human PBMC were stimulated for 3 days with PHA in the presence of B[a]P. B[a]P and 7,8-diol-B[a]P consistently inhibited the PHA-stimulated proliferation of human PBMC from five different donors at very low concentrations. Figure 6 shows the averages of triplicate determinations for one representative donor. Figure 6A shows that B[a]P (100 nM, 20 pmol) suppress the proliferation of human PBMC by 50% (range in the different donors 4560%). At this concentration of B[a]P the specific antibody (mAb-13) inhibited suppression in a dose-dependent manner (Figure 6B). A 12.5-fold molar excess (250 pmol, 37.5 µg) of the specific antibody blocked >90% of the B[a]P-induced suppression of T cell proliferation in all donors. ANF, a known cytosolic arylhydrocarbon receptor (AhR) antagonist and blocker of P450 function was used as a positive control for blocking B[a]P-induced immunosuppression. On a molar level ANF seemed to be a more potent blocker than the B[a]P-specific antibody (data not shown).
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The antibody reverses B[a]P-induced immunotoxicity even after delayed addition
The observation that the anti-B[a]P antibody slows down 7,8-diol-B[a]P formation and sequesters 7,8-diol-B[a]P outside the cell raises the question of whether both mechanisms contribute to protection. A 25-fold molar excess (500 pmol, 75 µg) of the specific antibody mAb-13 was added to PHA-stimulated PBMC at different time points after addition of B[a]P (100 nM, 20 pmol) (Figure 7). As late as 24 or 48 h after addition of B[a]P proliferation of lymphocytes was restored to 90%, but no protection was obtained when antibody was added after 72 h. This finding suggests that extracellular sequestration of metabolites by antibodies contributes significantly to the inhibition of immunotoxicity, since metabolic activation of B[a]P was not slowed down before the antibody was added to the cultures.
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Discussion |
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Our data indicate that an antibody directed against B[a]P and some of its metabolites can protect the cell in several ways. The antibody decreases cellular uptake and subsequent metabolism of B[a]P, as shown by increased recovery of unmetabolized B[a]P and decreased formation of end-point metabolites (e.g. phenols) in both PBMC and HepG2 cells. However, complete sequestration and inhibition of metabolism was not obtained. B[a]P is a highly lipophilic molecule with high affinity cytosolic binding sites (e.g. AhR) and, hence, its partitioning may be difficult to control even in the presence of excess antibodies (10,21). In contrast, its metabolite 7,8-diol-B[a]P, upstream of the ultimate carcinogen BPDE, is more soluble in water and more easily released from the cell, both as a phase I metabolite and as a detoxified highly soluble phase II metabolite conjugated to glucuronic acid (22). Conjugation of 7,8-diol-B[a]P to glucuronic acid by human PBMC is characterized by strong individual variation (23) and, therefore an important fraction of unconjugated 7,8-diol-B[a]P released from the cell can be further activated to BPDE in a second round of P450-mediated metabolism. Metabolic activation of B[a]P as well as 7,8-diol-B[a]P was severely reduced by the specific antibody in both PBMC and HepG2 cells. Strong antibody reactivity with unconjugated 7,8-diol-B[a]P thus provides a biologically relevant second chance of interrupting metabolic activation by sequestration of this metabolite in the extracellular space. Extracellular trapping of metabolites may even enhance their efflux in the way of sink conditions. Thus, a second protective mechanism may act at the level of activated B[a]P metabolites such as 7,8-diol-B[a]P and, perhaps, BPDE. Unfortunately, direct measurements of antibody reactivity with the ultimate carcinogen BPDE are difficult to perform. Therefore, this highly reactive and short-lived metabolite was measured as trans-anti-B[a]P-tetrol.
It is understood that such metabolism studies alone provide only limited insights into the potentially beneficial long-term effects in vivo. Nevertheless, at least a short-term biological relevance of B[a]P and 7,8-diol-B[a]P redistribution by the antibody was demonstrated by reversal of their immunotoxicity in lymphocytes. High affinity ligation of agonists to AhR may cause immunosuppression in vivo (21,24,25). Both B[a]P and 7,8-diol-B[a]P bind to AhR (21,26). Davila et al. found a significant difference between the immunotoxicity of B[a]P and 7,8-diol-B[a]P only at higher concentrations (24). In our hands, 7,8-diol-B[a]P was a 10-fold more potent inhibitor of T cell proliferation at physiological concentrations and the only immunotoxic B[a]P metabolite at these concentrations (<100 nM). Protection of T cell proliferation even after delayed addition of the antibody confirms a more important role of metabolites in immunotoxicity than was appreciated so far. This could also explain earlier observations of a discordance between PAHAhR affinity and immunosuppressive properties (27), as well as an important role of P450 in immunosuppression in vivo.
Naïve T cells are unable to activate B[a]P (28). In our assay the main source of 7,8-diol-B[a]P was activated T cells, as well as blood monocytes, which metabolize B[a]P to 7,8-diol-B[a]P mainly by induced P450 [29,30]. Although B[a]P is activated to 7,8-diol-B[a]P in the same cells that are immunosuppressed by this metabolite, the specific antibody completely restored T cell proliferation. This demonstrates that the sequestration of 7,8-diol-B[a]P is biologically highly relevant. The observed protection after delayed addition of the antibody underlines antibody cross-reactivity with metabolites as an important factor contributing to protection against immunotoxicity by carcinogens.
Carcinogen-associated immunosuppression has been implicated as a secondary mechanism in tumorigenesis (31). Thus, reversal of chronic immunosuppression could potentially lower the risk of cancer. However, our results may also have some more direct implications for adduct and tumor formation. Repartitioning of B[a]P and its metabolites by the antibody, reducing the effective concentration, could be beneficial in several ways. (i) Antibodies reduce 7,8-diol-B[a]P availability for further metabolic activation to BPDE, as shown by extracellular sequestration of 7,8-diol-B[a]P and by decreased formation of both trans-anti-B[a]P-tetrol and an unidentified metabolite, which is most probably a less harmful metabolite of the dihydrodiol dehydrogenase pathway. (ii) DNA adducts and tumors are mostly found in organs which activate B[a]P, perhaps because of local formation of high concentrations of 7,8-diol-B[a]P and BPDE, but in non-target tissues adducts are not tumorigenic. Because of long retention times of B[a]P in lung epithelium, metabolic activation can be substantial even at low enzymatic activities and carcinogenic metabolites are also expected to be retained in the airway epithelium (32). Therefore, antibody-mediated redistribution of B[a]P as well as 7,8-diol-B[a]P to non-target organs may be beneficial by lowering effective concentrations in target organs (such as the lung) and by relieving overloaded DNA repair mechanisms. For instance, the ubiquitous dihydrodiol dehydrogenase pathway could process 7,8-diol-B[a]P to quinone metabolites (e.g. 7,8-dion-B[a]P) which is a mild mutagen compared with BPDE (33). (iii) Our results show that the antibody reduced CYP1A1 induction by B[a]P in HepG2 cells. In a smoker's lungs the 100-fold induction of CYP1A1 is considered a major risk factor for lung cancer (5) and therefore reduced CYP1A1 activity is probably beneficial. However, in other organs, such as the liver, the significance of CYP1A1 inhibition must be interpreted with caution, because other enzymes may play a significant role in B[a]P metabolism. (iv) Temporary sequestration and redistribution (e.g. to the liver) may favor more complete conjugation to inactive phase II metabolites. Highly active glucuronidation is known to be associated with reduced cancer susceptibility (22,34). (v) Antibodies may also reduce cancer risk by cross-reacting with and even covalently binding to the highly reactive epoxide moiety of BPDE.
Systemic antibodies against carcinogens seem to have multiple and profound effects on the cellular uptake and subsequent metabolic activation of B[a]P, the kinetics of the different pathways and P450 induction. A carcinogen-specific antibody inhibited B[a]P-induced immunotoxicity by inhibiting its cellular uptake and subsequent metabolic activation. The antibody also modulated further activation of 7,8-diol-B[a]P to the ultimate carcinogen and its interference may protect against B[a]P-induced carcinogenesis. Even if sequestration of B[a]P by the antibody is transient, our results clearly demonstrate that strong interference with the activation pathway may change the balance between activation and detoxification, a major determinant of cancer risk. Although this may turn short-term effects into a lasting beneficial effect on carcinogenesis, this remains to be demonstrated in refined in vivo studies.
Our data suggest that a natural immune response against carcinogens may reduce the risk of cancer. Sero-epidemiological studies in exposed individuals are warranted to investigate this possibility. Furthermore, it may be speculated that populations at risk may benefit from an immunoprophylactic approach against selected carcinogens.
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Acknowledgments |
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References |
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