Induction of humoral immunity toward 2 ...

Carclnogenesis vol.17 no.8 pp.1705-1709, 1996

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

A.Verdina, R.Zito, G.Cortese, P.Leopardi1, F.Marcon1, A.Zijno1 and R.CrebelU1-2 Istituto Regina Elena and 'istituto Superiore di Sanita', Rome, Italy 2

To whom correspondence should be addressed

Introduction Several inherited and acquired host factors are known to affect the biological response to carcinogen exposure (1-3). Individual variance in xenobiotic metabolism, for which genetic polymorphism exists (4-5), is known to play a key role in determining the individual susceptibility to chemical carcinogens (6-10). Deficiencies in DNA repair are known to be associated with an increased cancer risk in humans (11-14) and the persistence of carcinogen adducts has been shown to be related to organ and tissue specificity in chemical carcinogenesis (15,16). Other possible modulating factors include age, nutrition, stress, diseases, hormonal status (17,18) and immunological (19) and genetic factors (20,21). Previous investigations demonstrated the occurrence of antibodies directed against benzopyrene-DNA adducts in sera from humans occupationally exposed to high levels of polycyclic aromatic hydrocarbons (PAH*) (22-23), as well as in the urban population (24). Variable levels of antibodies to carcino•Abbreviatlons: PAH, polycyclic aromatic hydrocarbons; 2-AAF, 2-acetylaminofluorene; PBS, phosphate-buffered saline; 2-AAAF, A'-acetoxy-2-acetylaminofluorene. tPreliminary results of this work were presented at the Second International Conference on Environmental Mutagens in Human Populations, Prague, 2025 August, 1995. © Oxford University Press

Materials and methods Animals Male Swiss mice, provided by Charles River Italia (Calco, Como, IT), were used throughout the work. The animals, aged 4 weeks at the beginning of treatment, were maintained on a balanced standard chow (Mucedola, Milan IT) and tap water ad libitum, housed in polycarbonate cages at constant temperature and humidity with a light/dark period of 12/12 h. All animals were acclimatized for 1 week before random allocation to experimental groups. Preparation of the 2-AAF-gelatin earner conjugate The 2-AAF-protein conjugate was prepared by reacting the carcinogen with acetylated gelatin in vitro. Gelatin was chosen as the carrier molecule for its complete lack of immunogenicity (28). 2-Acetylaminofluorene (Aldrich) dissolved in dimethylsulfoxide (5 mg/50 ul) was added to a solution of acetylated gelatin (10 mg/ml) in 0.1 M KH2PO4 (final 2-AAF concentration, 22 mM). The clarified solution was adjusted to pH 4.5 with 1 M HC1, then A'-ethyl-A'-(3-dimethylaminopropyl)carbodiimide (Sigma Chemical Co.) was added to a final concentration of 0.5 M. The pH was continuosly monitored and kept at 4.5 with 5 N HC1. After 4 h of end-over-end mixing, the 2-AAFgelatin conjugate was precipitated with acetone (66% v/v) at -20°C to remove unreacted 2-AAF, redissolved in water and dialyzed against phosphate-buffered saline (PBS). The gelatin-bound 2-AAF was quantitatively determined by UV spectrophotometry with a Varian DMS 100 instrument, using a molar extinction coefficient of 2.75X 10"4 M/cm at 288 nm. The same procedure was used to prepare bovine serum albumin, mouse serum albumin and egg albumin conjugates with 2-AAF. Adducted DNA was obtained by reacting calf thymus DNA with Nacetoxy-2-acetylaminofluorene (2-AAAF; Chemsyn) overnight at 37°C in PBS, removing unreacted 2-AAAF as above. Immunization schedule The immunogen (50 ul 1 mg/ml AAF-gelatin conjugate solution together with 50 ul Freund's adjuvant) was administered by i.p. injection once a week for 3 weeks. Fourteen days after the third treatment, mice received the last immunogen injection. Parallel experimental groups received the adjuvant alone or no treatment at all. 2-AAF treatment One week after the end of the immunization procedure, both immunized and non-immunized mice were fed 2-AAF pelleted in the diet at 50 and 150 p.p.m. for 4 weeks. Controls were fed with the standard diet.

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In order to investigate the modulatory effect of the immune response induced by recurrent carcinogen exposure, anti2-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 anti2-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 weeeks 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.

gen-DNA adducts were also detected in sera of mice chronically exposed to PAH (25). This indicates that chronic carcinogen exposure may elicit a specific immunological response, with the potential significance of an exposure marker for human biomonitoring studies (26). On the other hand, few or no attempts have been made to assess the biological significance of such an induced immune response, and in particular to assess whether it could modulate in some way the effect induced by the carcinogen itself. Regarding this possibility, an interesting clue was provided by the observation of a protective effect of the secretory immune response to 2acetylaminofluorene (2-AAF) in rabbits, possibly related to a reduction in transepithelial absorption of the carcinogen (27). In order to investigate the biological significance of the immune response induced by carcinogen exposure, in this study a specific humoral immunity toward 2-AAF was elicited in Swiss mice which were subsequently challenged with the carcinogen. The results obtained support the hypothesis of a possible modulating role of this host factor in chemical carcinogenesis.

A.Verdina et aL DNA extraction After sacrifice, liver and spleen were aseptically removed, weighed and quickly frozen at —80°C. For DNA extraction, tissues were thawed and homogenized in extraction buffer (10 mM Tris-HCl, pH 8.0, 100 mM EDTA, 0.5% SDS, 20 Ug/ml RNase) using an Omni-Mix apparatus. The homogenate was incubated for 1 h at 37°C before the addition of proteinase K (Sigma Chemical Co.) to a final concentration of 100 Hg/ml, followed by a further 2 h incubation at 50°C. Further steps of the extraction procedure, based on phenol extraction and ethanol precipitation, were performed according to standard methods (29). DNA extraction was quantitated by measurement of the absorbance of samples at 260 nm (A26o)The A26O:A28O r ^ ' 0 w a s determined to check for protein and RNA contamination. Determination of 2-AAF adducts to DNA Production of the antiserum The anti-2-AAF immune serum was produced by immunization of adult New Zeland white rabbits with weekly subcutaneous injections of 2-AAF-gelatin (1 mg) dissolved in 0.5 ml 0.1 M phosphate buffer, pH 7.2, and emulsified in complete Freund's adjuvant (0.5 ml). This schedule was repeated for six consecutive weeks. A 2 mg booster injection was delivered 15 days after the last inoculation. One week after this the rabbits were bled from the heart.

Bacterial mutagenicity assays Liver post-mitochondrial fractions (S9) from both immunized and nonimmunized mice were used as an exogenous metabolic activation system in bacterial assays with 2-AAF. Either different 2-AAF dosages or microsomal protein contents were applied to compare the efficiency of pooled liver homogenates obtained from groups of five animals. The assays were carried out by the plate incorporation procedure (33) with Salmonella typhimurium strain TA98. Protein content of S9 fractions was determined by the Bradford method (34). All determinations were made on triplicate plates, in at least two independent experiments.

Results The repeated i.p. injection of 2-AAF-gelatin conjugate effectively elicited the production of specific IgG in Swiss mice. The response obtained in direct ELISA with 0.5 ng conjugate is shown in Figure 1. Although inter-individual differences in serum titers were observed, all the immunized mice examined produced humoral antibodies capable of reacting against the 2-AAF conjugate. Conversely, serum pools of both nonimmunized mice and mice treated with the adjuvant alone did not show any reactivity toward 2-AAF-gelatin. In all cases, no reactivity was observed against the carrier protein alone (Figure 1). The content of 2-AAF adducts in liver and spleen DNA was determined by competitive ELISA using a polyclonal rabbit antiserum. Preliminary experiments were performed to check the ability of the serum to recognize 2-AAF adducted to different carrier proteins and to compare the reactivity toward 2-AAF adducted to either DNA or gelatin. The poly1706

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I • Reactivity vs 2-AAF-gelatin

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Not immunized

Immunized with adjuvant only

Fig. 1. Reactivity of mouse sera against 2-AAF-gelatin conjugate as determined by ELISA. Plates were coated with 0.5 ng/well conjugated 2-AAF, using 50 |il 1 % gelatin in water as the blank. Sera were diluted 1:1000 The mean ± SE of the results obtained with immunized mice (n = 37) and the response obtained with pooled sera from 20 nonimmunized and 20 adjuvant-pretreated mice are shown.

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Fig. 2. Reactivity of anti-2-AAF rabbit antiserum (A) and pooled sera of immunized mice (B) against 2-AAF adducted to different carrier proteins: O, mouse albumin; X, bovine serum albumin; • , gelatin; A, ovalbumin. Carrier proteins alone ( • ) gave in all cases values of