Modulation of the metabolism and adverse effects of benzo[a]pyrene by a specific antibody: a novel host factor in environmental carcinogenesis?

Stefan S. De Buck, Fabienne B. Bouche, Annick Brandenburger1 and Claude P. Muller*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The influence of specific antibodies on molecular and cellular mechanisms of activation, detoxification and biological activity of the ubiquitous carcinogen benzo[a]pyrene (B[a]P) was investigated using a monoclonal antibody. The antibody was shown to decrease cellular uptake and metabolic activation of B[a]P as demonstrated by higher recovery of unmetabolized B[a]P and decreased formation of end-point phenol metabolites in two types of target cells. Furthermore, strong antibody reactivity with 7,8-diol-B[a]P provided a second chance for interrupting metabolic activation by sequestration of this intermediate metabolite in the extracellular space. The biological relevance of B[a]P and 7,8-diol-B[a]P redistribution by antibody was demonstrated by reversion of B[a]P-induced inhibition of proliferation of human peripheral blood lymphocytes and by inhibition of CYP 1A1 induction in HepG2 cells. Remarkably, the antibody was still protective against B[a]P-induced immunotoxicity even after delayed addition, suggesting a more important role of metabolites in immunotoxicity than has been appreciated so far. Although B[a]P is activated to 7,8-diol-B[a]P in the same cells that are inhibited by this metabolite, the antibody completely restored lymphocyte proliferation indicating that extracellular trapping of the 7,8-diol-B[a]P is biologically highly effective. Thus, repartitioning of both B[a]P and its metabolites by the antibody may reduce their effective concentration in susceptible target organs and therefore relieve overloaded DNA repair mechanisms and inhibit carcinogen-induced P450 induction. These in vitro data also suggest that a natural or prophylactic antibody response against carcinogens may be associated with a reduced risk of cancer.

Abbreviations: AhR, cytosolic arylhydrocarbon receptor; ANF, {alpha}-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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Polymorphisms of gene products involved in carcinogen activation, detoxification (1) and DNA repair (2), and many other factors such as nutrition (3), drugs (4), cigarette smoking (5) and age, affect the host response to chemical carcinogens. Antibodies against polycyclic aromatic hydrocarbons (PAH) have been found in humans exposed to high levels of these carcinogens (6,7). However, the significance of such antibodies is poorly understood. There are no sero-epidemiological studies in exposed populations to assess the impact of carcinogen-specific antibodies on their risk of tumor development. Also, few attempts have been made in vivo or in vitro to understand the implications of an antibody response for metabolic activation of carcinogens and carcinogenesis. A recent study provided evidence that specific humoral immunity may modulate the genotoxic effect induced by subsequent carcinogen exposure (8). However, the mechanisms involved remain largely unexplored.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Assessment of antibody cross-reactivity by competition ELISA
The B[a]P-specific mouse IgG1 antibody (mAb-13) was produced according to Scharnbweber et al. (18) and ascites fluid was purified by sequential precipitation with caprylic acid and ammonium sulfate solution (Exbio Praha, Praha, Czech Republic). A molecular weight of 150 kDa was used to convert micrograms to picomoles. The amount of antibody was also expressed as fold-molar excess, which describes the ratio of antibody/B[a]P molecules.

Specificity of the mAb-13 antibody was determined by competition ELISA using B[a]P–ovalbumin 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]P–ovalbumin 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 (0–128 µM). A pyridine derivative was used as an irrelevant competitor (0% competition). Each mixture was then added to the B[a]P–ovalbumin-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 Tris–HCl buffer (pH 7.4) containing 250 mM sucrose and 1 mM EDTA using a glass–teflon Potter–Elvehjem 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 10–15 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 Tris–HCl (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 BPDE–DNA (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, 35–100% 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 (2–100 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 351–364 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 {alpha}-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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cross-reactivity of the monoclonal antibody
To identify potential levels of interference in B[a]P metabolism, binding properties of different metabolites to the B[a]P-specific monoclonal antibody (mAb-13) were investigated. Table I shows that all metabolites cross-reacted with the antibody, albeit with different affinities. As shown in Table I, the relative percentage binding of each metabolite to mAb-13 was calculated from their average IC50 values compared with the IC50 of B[a]P, which was 2.1 x 10–6 M. Results were independent of whether B[a]P conjugated to ovalbumin or to diphtheria toxoid was used to coat the ELISA plates. Interestingly, the activated metabolite 7,8-diol-B[a]P bound three times more efficiently than B[a]P or 3-OH-B[a]P and about 10 times more efficiently than the detoxified 9-OH-B[a]P.


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Table I. Relative percentage binding of B[a]P and its metabolites to mAb-13

 
Inhibition of DNA alkylation by 7,8-diol-B[a]P
The ability of the specific antibody (mAb-13) to inhibit DNA adduct formation was investigated in rat liver microsomes spiked with calf thymus DNA and 7,8-diol-B[a]P (Figure 1). Under the experimental conditions of Figure 1A, 50–400 µg of microsomes generated measurable BPDE–DNA adduct levels (measured as trans-anti-B[a]P-tetrol), even at very low 7,8-diol-B[a]P concentrations (50 nM, 20 pmol), which could be matched by a molar excess of antibody. Figure 1A shows that the maximal level of NADPH-dependent DNA adduct formation was reached with 50 µg microsomal protein. A 25-fold molar excess (500 pmol, 75 µg) of specific antibody inhibited DNA adduct formation up to 75% (Figure 1B). As expected, inhibition was lower at higher microsome concentrations, probably because of increasing competition of P450 and antibody. In the controls with dimethyl sulfoxide alone or in the absence of microsomal protein no adducts were detectable.



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Fig. 1. (A) DNA adduct formation by rat liver microsomes spiked with 7,8-diol-B[a]P (50 nM, 20 pmol) and calf thymus DNA. (B) Effect of mAb-13 (500 pmol, 75 µg) on DNA adduct formation by 7,8-diol-B[a]P (50 nM, 20 pmol). Incubations were carried out at 37°C for 60 min. DNA was extracted, hydrolyzed and analyzed by HPLC. Values are means ± SD of triplicates of a representative experiment repeated three times. *, Significant inhibition of DNA adduct formation (P < 0.005) by mAb-13 as determined by paired t-test.

 
Modulation of B[a]P and 7,8-diol-B[a]P metabolism in HepG2 cells
In order to determine the effect of the antibody on procarcinogen activation, HepG2 cells were incubated for 48 h with B[a]P (150 nM, 22.5 pmol). Both the activated metabolite 7,8-diol-B[a]P and the detoxified phenol metabolites were monitored (Figure 2A–D). Metabolites are expressed in femtomoles as well as as a percentage of metabolites in the control cultures without specific antibody (mAb-13). Recovery of B[a]P is expressed in femtomoles as well as a percentage of initial input. The specific antibody slowed down B[a]P metabolism and recovery of B[a]P after 48 h was ~40% of the initial input and at least 35 times higher than without specific antibody (Figure 2A). In the absence of the specific antibody only very little 7,8-diol-B[a]P was detected (Figure 2B), in contrast to significant amounts of 1-OH-B[a]P (Figure 2C) and 3-OH-B[a]P (Figure 2D). In the presence of a 6-fold molar excess of specific antibody (125 pmol, 18.7 µg) total metabolite formation was reduced by >50% (Figure 2E). Production of end-point metabolites (1-OH-B[a]P and 3-OH-B[a]P) was strongly inhibited by the specific antibody, indicating decreased bioavailability of B[a]P (Figure 2C and 2D). However, using a 22-fold molar excess (500 pmol, 75 µg) of the specific antibody up to 10-fold more 7,8-diol-B[a]P was detected, indicating extracellular sequestration of this intermediate metabolite and inhibition of further metabolic activation (Figure 2B). These experiments show that measurements of total metabolites can be misleading and highlight the need to evaluate both the intermediate activated metabolites and end-point detoxification metabolites separately.



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Fig. 2. Modulation of (AE) B[a]P (48 h, 150 nM) and (FH) 7,8-diol-B[a]P (18 h, 100 nM) metabolism by specific antibody mAb-13 (0–500 pmol, 0–75 µg) in HepG2 cells. Metabolites are expressed in fmol of metabolite recovered in supernatants (right scale) as well as as a percentage of control cultures without specific antibody (left scale) (B–E, G and H). Recovery of parent compound is expressed in fmol (right scale) as well as as a percentage of initial input amount (left scale) (A and F). Values are means ± SD of triplicates of a representative experiment repeated two or three times. *, Statistically different metabolite titers (P < 0.01) in the presence of mAb-13 as determined by paired t-test.

 
We further tested whether the antibody would interfere with metabolic activation of 7,8-diol-B[a]P (100 nM, 15 pmol) in HepG2 cells (Figure 2F–H). Metabolites are expressed in femtomoles as well as a percentage of the control cultures without specific antibody (mAb-13). Recovery of 7,8-diol-B[a]P is expressed in femtomoles as well as as a percentage of initial input. In the absence of specific antibody (mAb-13) the diol was metabolized to trans-anti-B[a]P-tetrol (Figure 2G), which is indicative of ephemeral anti-BPDE formation. Other tetrols were not detected in significant quantities. Moreover, in the absence of the specific antibody we detected a metabolite of unknown identity (Figure 2H). A strong dose-dependent inhibition by the specific antibody was observed for trans-anti-B[a]P-tetrol (Figure 2G), as well as for the unknown metabolite (Figure 2H). Recovery of 7,8-diol-B[a]P increased in the presence of the specific antibody, indicating decreased bioavailability (Figure 2F). Up to 80% of the initial input amount was recovered in the presence of a 33-fold molar excess (500 pmol, 75 µg) of specific antibody. These experiments show that specific antibodies can have profound effects on both B[a]P and 7,8-diol-B[a]P activation and detoxification.

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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Fig. 3. Effect of mAb-13 (500 pmol, 75 µg) on induction of CYP1A1 by B[a]P (500 nM, 20 pmol) in HepG2 cells. Results are expressed as relative induction of specific mRNA after 6 h by real-time PCR [comparative Ct method (35)]. Values are means ± SD of triplicates of a representative experiment repeated twice. *, Statistically different (P < 0.05) in the presence of mAb-13 as determined by paired t-test.

 
Inhibition of cellular uptake of B[a]P
To determine intracellular uptake of B[a]P a flow cytometry assay was developed exploiting the natural fluorescence of B[a]P. A number of different cell lines were tested and similar results were obtained. Here we report the data from human PBMC because (i) their autofluorescence was lowest, which increased the sensitivity at low B[a]P concentrations that can be matched by antibodies on a molar basis, and (ii) their proliferation to mitogens is highly sensitive to very low B[a]P concentrations.

Human PBMC were incubated (37°C, 60 min) with different concentrations of B[a]P (0–1.6 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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Fig. 4. Inhibition by antibody mAb-13 of cellular uptake of B[a]P by human PBMC. (A) Natural fluorescence of B[a]P measured by flow cytometry as mean arbitrary fluorescence units (AFU). (B) Dose-dependent inhibition by mAb-13 (0–250 pmol, 0–37.5 µg) of B[a]P (60 nM, 12 pmol) uptake. The control (Con) contains neither carcinogen nor antibody. Values are means ± SD of triplicates of a representative experiment repeated three times. Inserts show histograms of cells incubated with B[a]P (60 nM, 12 pmol) alone (continuous line) or in the presence of both B[a]P (60 nM, 12 pmol) and 120 pmol (18 µg) of mAb-13 (Insert 1, dashed line) or irrelevant antibody (Insert 2, dashed line). The histogram obtained by co-incubation of 120 pmol (18 µg) mAb-13 and B[a]P (60 nM, 12 pmol) is indistinguishable from the histogram of unstained control cells. *, Significant inhibition of B[a]P uptake (P < 0.001) by mAb-13 antibody as determined by paired t-test.

 
PBMC were incubated with B[a]P in the presence or absence of antibody. Figure 4B shows a dose-dependent inhibition by the specific antibody (mAb-13) of B[a]P cellular uptake (60 nM, 12 pmol). Cellular uptake of 12 pmol B[a]P was >50% inhibited with 15 pmol (2.3 µg) mAb-13 and >90% inhibition was obtained with 60 pmol (9 µg) of antibody. Inserts show representative histograms demonstrating the inhibition of cellular uptake of B[a]P (60 nM, 12 pmol) by 120 pmol (18 µg) of specific antibody (Figure 4B, Insert 1), in contrast to a control antibody (Figure 4B, Insert 2), which showed no inhibition. The histogram obtained by co-incubation of 120 pmol (18 µg) mAb-13 and B[a]P (60 nM, 12 pmol) overlapped completely with unstained control cells.

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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Fig. 5. (A) Recovery of input B[a]P (100 nM, 20 pmol) in PHA-stimulated human PBMC in the absence and presence of a 25-fold molar excess (500 pmol, 75 µg) of mAb-13. Concomitant modulation of the kinetics of B[a]P metabolism in the (B) absence and (C) presence of specific antibody, respectively. Results are expressed in fmol of B[a]P or metabolite recovered in supernatants. (D) Percentage inhibition of each metabolite by mAb-13. Values are means ± SD of triplicates. *, **, Significant inhibition of metabolism (P < 0.05 and P < 0.001, respectively) by mAb-13 as determined by paired t-test.

 
mAb-13 (500 pmol, 75 µg) slowed down B[a]P (100 nM, 20 pmol) metabolism and recovery of B[a]P after 72 h was >50% of the input and at least 10 times higher than without the specific antibody (Figure 5A). The specific antibody prevented B[a]P metabolites from reaching plateau values within 72 h (Figure 5C). Metabolism of all phenols was consistently inhibited at all time points (Figure 5D). During the first 24 h the specific antibody inhibited the production of 1-OH-B[a]P, 3-OH-B[a]P and 9-OH-B[a]P up to 78%. At 72 h, however, inhibition of phenol formation was less pronounced. The specific antibody inhibited the production of 7,8-diol-B[a]P during the first 24 h by 40–48%. Remarkably, at 48 h there was no inhibition of this metabolite and at 72 h supernatants contained almost 10 times more 7,8-diol-B[a]P in the presence of the specific antibody. These data further confirm that mAb-13 slows down the metabolism of B[a]P and sequesters 7,8-diol-B[a]P outside the cell.

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 45–60%). 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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Fig. 6. Immunotoxicity of (A) B[a]P and (C) 7,8-diol-B[a]P to PHA-induced lymphocyte proliferation. Effect of mAb-13 (0–500 pmol, 0–75 µg) on (B) B[a]P (100 nM, 20 pmol)-induced and (D) 7,8-diol-B[a]P (100 nM, 20 pmol)-induced suppression. PBMC proliferation is expressed as [3H]thymidine uptake/well as well as as a percentage of control cultures (Con) in absence of B[a]P. Values are means ± SD of triplicates of a representative experiment repeated at least three times. *, Significant inhibition of proliferation (P < 0.005) by B[a]P or 7,8-diol-B[a]P (A and C); *, significant reversal of inhibited proliferation (P < 0.005) by mAb-13 (B and D) as determined by paired t-test.

 
Metabolites, including 7,8-diol-B[a]P, 3-OH-B[a]P and 9-OH-B[a]P, were also tested for their immunotoxicity. The phenol metabolites (3-OH-B[a]P and 9-OH-B[a]P) were not immunotoxic at concentrations up to 100 nM (data not shown). In contrast, 7,8-diol-B[a]P was ~10 times more immunotoxic than B[a]P (Figure 6C). 7,8-diol-B[a]P (100 nM, 20 pmol) suppressed proliferation to <20% of controls for the different donors. Remarkably, this metabolite was still significantly immunotoxic at concentrations as low as 10 nM. As expected, the specific antibody also inhibited 7,8-diol-B[a]P-induced (100 nM, 20 pmol) suppression in a dose-dependent manner (Figure 6D). A 12.5-fold molar excess (250 pmol, 37.5 µg) of the specific antibody blocked >90% of the 7.8-diol-B[a]P-induced suppression of T cell proliferation in all donors. On a molar basis ANF was about 20 times more efficient than the antibody (data not shown). These data show that antibodies are able to reverse both B[a]P- and 7,8-diol-B[a]P-induced immunotoxicity.

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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Fig. 7. Reversal of B[a]P (100 nM, 20 pmol)-induced suppression of PBMC proliferation by mAb-13 (500 pmol, 75 µg) added at different time points. PBMC proliferation is expressed in [3H]thymidine uptake/well as well as as a percentage of control cultures containing neither carcinogen nor antibody. Values are means ± SD of triplicates. *, Significant reversal of inhibited proliferation (P < 0.005) by mAb-13 as determined by paired t-test.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Specific antibodies elicited by recurrent carcinogen exposure may be able to modulate the genotoxic and carcinogenic effects of carcinogens. This hypothesis has not been explored under appropriate conditions of low physiological doses of carcinogens and mechanisms of action of antibodies on carcinogens have never been investigated in vitro (10). In vivo cancer models or models using surrogate end-points are limited to high concentrations, which cannot be matched by antibodies. In this study, using 100–1000 times lower levels of B[a]P, we could show that a specific antibody can modulate cellular uptake and subsequent metabolic activation. More importantly, this is the first study to show that (i) transient sequestration of carcinogens by an antibody inhibits specific biological activities of carcinogens and (ii) metabolism can be interrupted both at the level of the parent procarcinogen and of its metabolites, as shown in two different relevant target cells.

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 PAH–AhR 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.


    Acknowledgments
 
The authors acknowledge the skilful assistance of the following staff members at the Laboratoire National de Santé, Dr Corinne Ensch for bioconjugation of benzo[a]pyrene and Wim Ammerlaan for expert advice and thoughtful suggestions concerning flow cytometry analysis. We thank Prof. Thierry Vélu (Université Libre de Bruxelles) for fruitful discussions and guidance. This research was sponsored by Fonds National de la Recherche grant FNR/01/04/11 and Bourse BFR from the Ministère de la Recherche, Luxembourg and CRP-Santé, Luxembourg.


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 Materials and methods
 Results
 Discussion
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Received October 27, 2004; revised December 13, 2004; accepted December 15, 2004.





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