Carcinogen-modified DNA and specific humoral immunity toward carcinogen-DNA adducts. A review.

Carcinogenesis is a complex and multistep process starting from initiation to tumor progression. Damage to DNA, induced by the covalent binding of chemical carcinogens on critical DNA segments, reflects exposure and is directly related to tumor formation. For this reason it's very important detect and quantify DNA-adducts by using highly sensitive methods. During the last 30 years sophisticated methods have been developed, in particular immunoassays that have a widespread application in monitoring animal models and human tissues for evidence of carcinogen exposure. In this paper we describe the work done in our laboratory, from the production of antibodies specific for two different carcinogens, 2-Acetylaminofluorene and Benzo[a]pyrene, to their application in chemical carcinogenesis studies. Moreover, we describe as immunological methods can be used for detecting the presence of specific antibodies in sera of exposed individuals.

Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization.

Using phage-display technology, a recombinant single-chain Fv antibody fragment (scFv) was rapidly generated from the K16-16 hybridoma secreting mouse monoclonal antibody (MAb) that binds to acetylaminofluorene-labeled DNA (AAF-DNA). The selected A4 phage-scFv specifically bound to AAF-DNA. The anti-AAF scFv gene was then recloned into a fusion vector for the production of a hybrid protein comprising the antibody fragment fused to a potent bacterial alkaline phosphatase variant (PhoAv). The anti-AAF scFv-PhoAv hybrid protein was bifunctional and possessed both antigen binding capacity and PhoA activity. The recombinant conjugate was directly used, without further purification, for one-step immunodetection in dot-blot hybridization. The detection limit was identical and the test was quicker than the conventional two-step procedure with the purified anti-AAF MAb revealed with a secondary enzyme-labeled antibody. To assess the value of this new reagent for the immunodetection of genomic nucleic acids, genomic DNAs of Campylobacter jejuni and Campylobacter coli were then one-step immunodetected with non-purified recombinant scFv-PhoAv conjugate in a Southern-blot hybridization experiment. The present study shows that the genetic fusion with PhoAv provides a new tool for immunodetection which presents easier and quicker production and use with the same sensitivity and specificity as classical reagents. The recombinant anti-AAF scFv-PhoAv conjugate is a promising alternative reagent for applications involving the immunodetection of specific DNA or RNA sequences, such as the detection and characterization of microorganisms.

Induction of humoral immunity toward 2-acetylaminofluorene in mice: modulation of DNA binding after 4 weeks dietary exposure to the carcinogen.

In order to investigate the modulatory effect of the immune response induced by recurrent carcinogen exposure, anti-2-acetylaminofluorene (anti-2-AAF) IgG were elicited in Swiss mice before subsequent carcinogen administration. The immunization schedule consisted of three weekly i.p. injections of 2-acetylaminofluorene (2-AAF)-gelatin conjugate, followed by a final immunogen injection 14 days later. At the end of treatment, the presence of specific anti-2-AAF antibodies in blood serum of all immunized animals was demonstrated. The immunization procedure did not affect liver metabolic activities, as evaluated using liver homogenates for the exogenous activation of 2-AAF to mutagen. After immunization, mice were fed 2-AAF pelleted in the diet at 50 and 150 p.p.m. for 4 weeks and killed at the end of treatment. The determination of DNA adducts by ELISA in liver and spleen of treated animals revealed significantly (P < 0.01-0.001) lower 2-AAF adduct levels in both tissues of immunized mice with respect to non-immunized animals (both naive and pretreated with the adjuvant alone). This result suggests that the specific humoral immunity elicited by repeated carcinogen exposure may be able to modulate the genotoxic effect induced by subsequent carcinogen administration.

Modulation of DNA binding in vivo by specific humoral immunological response: a novel host factor in environmental carcinogenesis?

To investigate the possible modulatory effect of the immune response induced by recurrent carcinogen exposure, a specific humoral immune response toward 2-acetylaminofluorene (2-AAF) was elicited in Swiss mice with repeated intraperitoneal injections of a 2-AAF-gelatin conjugate. The immunization procedure resulted in the production of specific anti-2-AAF antibodies in all treated animals. Groups of immunized and nonimmunized mice were subsequently fed 2-AAF pelleted in the diet at 50 and 150 ppm for 4 weeks. At the end of 2-AAF administration, animals were sacrificed and the content of 2-AAF-adducts in liver DNA was determined by enzyme-linked immunoadsorbent assay using a polyclonal rabbit antiserum. The comparison of the adducts levels in immunized and nonimmunized mice (receiving either the vehicle or the adjuvant alone during pretreatment) demonstrates a highly significant (p < 0.001) difference among groups, with far lower adduct levels in immunized animals. No significant difference in food consumption or liver metabolic activities was observed among experimental groups, suggesting the absence of external bias. The mechanism underlying the result observed is not yet clear; however, the experimental data strongly suggest that the specific immunological response induced by recurrent carcinogen exposure may exert a modulatory effect and act as a relevant host factor in chemical carcinogenesis.

Selective induction of mucosal immune responses to 2-acetylaminofluorene.

Mucosal vaccination with chemical carcinogens coupled to enterotoxins such as cholera toxin (CT) can elicit carcinogen-specific immunoglobulin secretion into the intestinal lumen. The present study examines the ability of several related bacterial enterotoxins and their subunits to act as adjuvants or carrier proteins in stimulating an intestinal secretory IgA (S-IgA) response to 2-acetylaminofluorene (AAF). Using Thiry-Vella loops in rabbits, CT, cholera toxin B subunit (CTB) and the recombinant B subunit of the heat labile enterotoxin from E. coli (rLTB) were all found to be effective carrier proteins and adjuvants for eliciting S-IgA anti-AAF. However, marked differences in the ratio of mucosal S-IgA to serum IgG production were observed. CT elicited the highest luminal S-IgA anti-AAF titers as well as the highest ratio of intestinal S-IgA/serum IgG when used as an adjuvant. Conversely, rLTB elicited a high serum IgG anti-AAF titer but only a modest intestinal S-IgA response. Dialysis studies using monoclonal IgA versus IgG anti-AAF on opposing sides of a semipermeable membrane demonstrated the potential importance of the intestinal S-IgA/serum IgG ratio. A high "intestinal" IgA/"serum" IgG ratio abolished carcinogen transfer to the "serum" side of the membrane, while a low ratio enhanced transfer. Thus, to generate an active mucosal immune response capable of blocking carcinogen absorption, the carrier protein or adjuvant should be selected to optimize the intestinal S-IgA/serum IgG ratio.

Development of a bispecific monoclonal antibody for use in molecular hybridisation.

A mouse hybrid hybridoma (tetradoma) was prepared by fusing hybridomas producing monoclonal antibody to acetyl-aminofluorene with hybridomas producing antibody against calf intestine alkaline phosphatase. The tetradoma line established secreted immunoglobulin manifesting parental and bispecific binding characteristics. Bispecific monoclonal antibody was purified and used for a one-step immunodetection assay of non-radioactive DNA and RNA probes. The immunoassay developed was able to detect 5 pg DNA within 2 h and gave low background noise.

Characterization of the mucosal immune response to 2-acetylaminofluorene-protein conjugates.

Many environmental carcinogens gain access to the body only after traversing a mucosal surface. Due to their small size, most carcinogens are not recognized by the immune system and pass unhindered from the external to the internal environment. In previous studies, we demonstrated that secretory IgA directed against the carcinogen 2-acetylaminofluorene (AAF) can be elicited by covalently coupling AAF to the mucosal immunogen cholera toxin (CT). Rabbit intestines receiving secretions containing secretory IgA anti-AAF demonstrated a marked reduction in transmucosal absorption of carcinogen from the intestinal lumen to the mesenteric blood supply. In actively immune animals, however, recent data suggests that the disposition of luminal carcinogen may be influenced by the relative abundance of serum versus mucosally-based immunoglobulins. Our objective was to quantify the amount and isotype distribution of antibodies produced in response to AAF-carrier protein conjugates administered via different routes; using traditional parenteral carrier proteins and routes of administration, compared to mucosal carrier proteins and routes of administration. Administration of AAF-cholera toxin conjugates to isolated ileal (Thiry-Vella) loops in rabbits elicited a vigorous sIgA anti-AAF response in ileal secretions, with low levels of serum or intestinal IgG, or serum-based IgA produced concomitantly. All parenteral immunization protocols generated extremely high titers of serum IgG anti-AAF, with only moderate levels of sIgA produced concomitantly, even when mucosal boosting followed parenteral priming. When AAF-CT mucosal boosts were administered after intraperitoneal priming, a dramatic rise in serum, not secretory IgA was observed.

Reduction of intestinal carcinogen absorption by carcinogen-specific secretory immunity.

A secretory immune response to the carcinogen 2-acetylaminofluorene (AAF) was elicited in rabbits by directly immunizing the small intestine with an AAF-cholera toxin conjugate. High-titer, high-affinity secretory immunoglobulin A (IgA) antibody to AAF was secreted into the intestinal lumen in response to this immunogen. Immune secretions reduced the transepithelial absorption of a 125I-labeled derivative of AAF by more than half. This reduction of absorption by hapten-specific IgA suggests that oral vaccines against carcinogens and toxicants could be developed for humans.

A rapid and sensitive screening method for the detection of anti-2-acetylaminofluorene immunoglobulins.

A method is described in which anti-2-acetylaminofluorene immunoglobulins may be detected using a simple and sensitive screening procedure. The method is based on immunoglobulin binding of an 125I derivatized 2-aminofluorene radiotracer. Tracer binding is not isotype specific, and thus the method is useful for the detection of either IgG or IgA. Competitive binding experiments with the radiotracer were used to determine the specificity of immunoglobulin response by measurement of cross-reactivity with related ligands. This method allows quantitation of the immune response to the carcinogen in serum and other biological fluids (i.e., intestinal secretions).

An ELISA for detection of DNA-bound carcinogen using a monoclonal antibody to N-acetoxy-2-acetylaminofluorene-modified DNA.

We have produced and characterized a murine monoclonal antibody that recognizes DNA modified with N-acetoxy-2-acetylaminofluorene. The effectiveness of competitive binding inhibitors in an ELISA indicates that this antibody binds most strongly to N-(guanin-8-yl)-2-acetylaminofluorene. It also binds to N-(guanin-8-yl)-2-aminofluorene, albeit some 20-fold less avidly. Thus the monoclonal antibody displays specificity generally similar to the polyclonal antisera elicited by other laboratories but having the advantage of stable and precisely defined specificities. We employed a biotin-streptavidin ELISA to demonstrate that the antibody can detect less than 10 fmol of N-(guanin-8-yl)-2-acetylaminofluorene. N-(guanin-8-yl)-2-acetylaminofluorene is a more effective competitive binding inhibitor of the antibody than is N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene or calf thymus DNA modified with N-acetoxy-2-acetylaminofluorene. Thus the antibody should be most useful in quantifying the persistence of N-acetoxy-2-acetylaminofluorene adducts in DNA hydrolyzed with trifluoroacetic acid.

Significance of immune responses to mucosal carcinogens: a hypothesis and a workable model system.

Carcinogens must enter the tissues before neoplastic transformation of cells can occur. The present report describes the 2-AAF model system being used to test our hypothesis that development of mucosal immunity to carcinogens will interfere with their passage through mucosal epithelial cells. The 2-AAF model is ideal for testing this hypothesis. 2-AAF is a carcinogen which passes through the intestinal epithelium following oral administration, is transported to the liver and is there metabolized with the ultimate formation of hyperplastic nodules and neoplasms. Further, neoplasms form at other sites including the ear duct, gastrointestinal tract and the lungs. Our studies indicate that 2-AAF alone given orally does not elicit an immune response. However, 2-AAF is immunogenic when conjugated to carrier proteins and administered in CFA. Such conjugates of 2-AAF are not carcinogenic when administered in doses needed to achieve a systemic immune response in rats. Future studies will document the optimal conditions required to stimulate mucosal immunity to 2-AAF and the effect of such immunity on carcinogenesis in this model.

Monoclonal antibody detection of 2-acetyl-aminofluorene-modified DNA probes for the specific detection of nucleic acids in hybridization procedures.

We describe the use of acetoxy-acetyl-aminofluorene-modified DNA probes in several hybridization techniques. Hybrids were detected with the help of a monoclonal antibody raised against AAF-guanosine and a second antibody coupled to an enzyme. The sensitivity achieved with AAF-DNA probes routinely detected 0.25 pg DNA bound to a filter. AAF-DNA probes were highly stable and were prepared by simple chemical modification of DNA. Their use as a possible diagnostic tool is discussed.

Monoclonal antibodies to DNA modified by the carcinogen N-acetoxy-N-2 acetyl aminofluorene.

BALB/c mice were immunized against acetyl aminofluorene-substituted guanosine (AAF.Guo). Hybridomas were generated from the immune splenocytes fused with the SP2/0.Ag8 cell line. Four monoclonal antibodies (mAb) were selected. Two of them recognize mainly AAF. substituted DNA but also native DNA, while the others (derived from the hybridoma 16) are specific for the modified DNA. Using a solid phase radioimmunoassay, the fine specificity of mAb 16 was examined. The interaction between purified mAb 16 an AAF.DNA is inhibited by either AAF.GMP, AAF.Guo, or AAF.DNA. Moreover this interaction is slightly inhibited by aminofluorene-substituted guanosine (AF.Guo). By control, free AAF, fluorenamine, guanosine, desoxy GMP and native DNA do not compete in the assay. This mAb 16, which specifically recognizes the AAF-nucleotide complex may be a very useful tool to analyze the carcinogenic processes. Such an antibody can also be used for chromosomal localization of genes by AAF-substituted probes.

Immunological detection and quantification of the reaction products of 2-acetylaminofluorene with guanine in DNA.

Rabbits were immunized with N-(guanosin-8-yl)-N-acetyl-2-aminofluorene bound to bovine serum albumin. The presence of specific antibodies was determined in a radioimmunoassay by competing N-([5'-3H]-guanosin-8-yl)-N-acetyl-2-aminofluorene (specific radioactivity, 25 Ci/mmol) as tracer against various carcinogen-modified and normal DNA components. Highly specific antibodies were obtained (affinity constant, 6.0 X 10(9) l/mol) and have been employed in a sensitive radioimmunoassay to determine N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene directly in enzymatic hydrolysates of DNA modified with N-acetoxy-N-acetyl-2-aminofluorene. In a similar fashion, the modified nucleoside could be detected in hydrolysed rat liver DNA following administration of N-acetyl-2-aminofluorene, with a detection limit of one carcinogen-modified deoxyguanosine moiety per 10(6) deoxynucleotides in 150 micrograms of DNA.

Radioimmunoassays for monitoring exposure to potential carcinogens.

As the numbers and production of environmental pollutants and hazardous industrial compounds increase, the necessity for monitoring the environment and workplace to avoid or to limit human exposure to toxic substances becomes more urgent. In support of the occupational safety program at the National Center for Toxicological Research, very sensitive and specific radioimmunoassays for measuring two known animal carcinogens, 2-acetylaminofluorene and diethylstilbestrol and their metabolites were developed. Radioimmunoassay (RIA) techniques offer unique advantages over ixisting methodologies in the field of toxicology; these include exquisite specificity and sensitivity, simplicity in sample handling and rapid analysis. These RIAs are currently being used for monitoring possible human exposure to carcinogenic compounds.