Antibodies to carcinogen-DNA adducts in mice chronically exposed to polycyclic aromatic hydrocarbons

Immunology Letters, 36 (1993) 117-124

0165 - 2478 / 93 / $ 6.00 © 1993 ElsevierSciencePublishers B.V. All rights reserved IMLET 01948

Antibodies to carcinogen-DNA adducts in mice chronically exposed to polycyclic aromatic hydrocarbons B y u n g M . Lee* a n d P a u l T. S t r i c k l a n d Department of Environmental Health Sciences, The Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland, USA

(Received 25 February 1993; accepted 25 February 1993)

1.

Summary

Antibodies specific for polycyclic aromatic hydrocarbon (PAH)-DNA adducts have previously been reported in human sera. In this study, we examined the association between mixed PAH exposure and P A H - D N A adduct specific antibodies in BALB/c mice. Mice were treated either by i.p. injection or by intragastric (i.g.) intubation with a mixture of seven different PAHs [benzo(a)pyrene (BP), benz(a)anthracene (BA), fluoranthene (FA), dibenz(a,h)anthracene (DBA), 3-methylcholanthrene (MC), chrysene (Ch), benzo(b)fluoranthene (BF)] at three doses (0, 15, 150 /~g of each PAH) twice a week for 8 weeks. Sera were screened by direct ELISA for antibodies recognizing D N A modified by diolepoxides or epoxides of each PAH injected. In i.p. treated mice, the sera were slightly more reactive to DNAs modified with diolepoxides of BP, BA, or Ch or an epoxide of DBA than to unmodified DNA. In i.g. treated mice, the sera were more reactive to DNAs modified with diolepoxides of BA or BF than to unmodified DNA. For some PAHs, a dose-response effect was observed between sera reactivity to PAH metabolites and the dose of Dr. P.T. Strickland, Department of Environmental Health Sciences,Rm 2712, Johns Hopkins School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205, USA. Tel.: 410-955-4456. Correspondence to."

*Present address: Sung Kyun Kwan University, College of Pharmacy, Suwon City, Kyunggi-Do440-746, South Korea.

PAH administered. However, there was considerable variation in the immune responses among individual mice within each treatment group. When tested by competitive ELISA, none of the sera could discriminate between modified and unmodified DNA. This animal study suggests that an assessment of previous carcinogen exposure by measuring D N A adduct-specific antibodies requires further validation prior to its application to the human monitoring of carcinogen exposure.

2.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment, carcinogenic in some animal species, and suspected carcinogens in humans [1]. In the past decade, polyclonal and monoclonal antibodies that recognize the covalent addition products (adducts) formed between PAHs and D N A have been experimentally produced in animals [2-6]. These antibodies have been used in immunoassays to measure the levels of P A H - D N A adducts in animals and human D N A samples [7-14]. Chronic adduct formation due to carcinogen exposure might also induce antibodies to carcinogen-DNA adducts in humans. Antibodies specific for P A H - D N A adducts have been detected in sera from humans possibly exposed to PAH compounds [9,11,15]. Specific antibodies to benzo(a)pyrene diolepoxide (BPDE)-DNA were found in the sera of 27% of coke oven plant workers tested, although 67% (18 of 27) of the 117

samples had detectable BPDE-DNA adducts in their lymphocytes by USERIA [9]. In another study of coke oven workers, antibodies to BPDEDNA were found in the sera of about one-third of the workers, and BPDE-DNA adducts in the lymphocytes were detectable in about one-third of workers [11]. It is known that the DNA-damaging drugs procainamide and hydrazine can produce antibodies in humans [16-18]. Antibodies to methylisocyanate have also been investigated in people exposed to this compound during the recent accidental release in Bophal, India [19]. Antibodies were detected in 12 of 144 exposed persons. The induction of penicilloyl antibodies in patients exposed to a single dose of penicillin and of amiodarone antibodies in antiarrhythmic patients receiving this drug have also been reported [20,21]. However, in other studies of foundry workers, roofers, and controls, no significant difference in the titers of antibodies to PAH-DNA adducts was seen between people exposed to PAHs and controls ([22] and unpublished). In the present study, we examined the association between PAH-DNA adduct antibodies in sera and previous PAH exposure in mice. 3.

3.1.

Methods

ide] were purchased from the NCI Chemical Carcinogen Repository (Chemsyn Science Laboratories, Lenexa, KS). These are carcinogenic chemicals and must be handled according to acceptable NCI guidelines [23]. ABC Vectastain kit was purchased from Vector Laboratories, Burlingame, CA. 3.2.

BALB/c mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA) and maintained on Agway Prolab 1000 Lab Chow and tap water ad libitum. Eight-week-old mice were treated either by i.p. injection or by intragastric (i.g.) intubation with a mixture of seven different PAHs [benzo(a)pyrene, benz(a)anthracene, benzo(k)fluoranthene, chrysene, fluoranthene, dibenz(a,h)anthracene, 3-methylcholanthrene] at three doses (0, 15, 150/~g of each PAH in 200 #1 corn oil) twice a week for 8 weeks. With the exception of 3-methylcholanthrene, these compounds are produced by combustion processes in various environmental and occupational settings. Control groups (0 dose treatment) were treated with 200/~1 corn oil. Each treatment group contained 5-6 mice. Sera from mice were collected after 8 weeks of treatment and 10 days after the final treatment (8 weeks plus 10 days).

Chemicals 3.3.

Benzo(a)pyrene, fluoranthene, benz(a)anthracene, dibenz(a,h)anthracene, chrysene, 3-methylcholanthrene, and benzo(k)fluoranthene were purchased from Sigma Chemical Co., St. Louis, MO. Calf thymus DNA, p-nitrophenyl phosphate (Sigma 104), and bovine serum albumin were also purchased from Sigma Chemical Co. BPDE [7,8dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene], BFDE [trans-8,9-dihydroxy-lO,11epoxy-8,9,10,11-tetrahydrobenzo(k)fluoranthene], ChDE [trans-l,2-dihydroxy-anti-3,4-epoxy-l,2,3,4-tetrahydrochrysene], BADE [benz(a)anthracene-trans-8,9-dihyrodiol-lO,11-epoxide (anti)], DBAE [dibenz(a,h)anthracene-5,6-dihydroepoxide], IPE [indeno(1,2,3-c,d)pyrene-l,2-epoxide], FADE [trans-2,3-dihydroxy-l,lOb-epoxy-l,2,3,10b-tetrahydrofluoranthene (syn and anti)], and MCE [3-methylcholanthrene- 11,12-dihydroepox118

Animal treatment with PAHs

Modification of DNA in vitro with P A H metabolites

Several PAHs, including benzo(a)pyrene, benz(a)anthracene, fluoranthene, and chrysene, are known to be activated through their respective diol-epoxides to form DNA adducts [24]. In other cases, the active metabolite is unknown or has been hypothesized to be an epoxide. Calf thymus DNA (10 mg DNA, 1 mg/ml in 0.01 M Tris pH 7.5) was modified in vitro by treatment with PAH metabolites (BPDE, BADE, BFDE, FDE, IPE, MCE, DBAE, ChDE) as described previously [25]. Each compound (1 mg) was dissolved in ethanol:tetrahydrofuran (24:1, 5 ml) and reacted in the dark overnight at room temperature. After incubation, unreacted PAH metabolites were extracted eight times with diethyl-ether and four times with water-saturated isoamyl alcohol; mod-

ified DNAs were isolated by ethanol precipitation. Modified DNAs were redissolved in 0.01 M Tris buffer, and the modification levels were estimated from UV absorption spectra using extinction coefficients provided by Chemsyn Science Laboratories. The modification level of BPDED N A was determined to be 0.7% from the absorbance at 347 nm of BPDE-deoxyguanosine (E = 29 000) and the absorbance of D N A at 260 nm (E = 6500). Those of other PAH metabolite-DNAs were less than 0.1%.

plates with PBS-Tween, 100 pl biotinylated antimouse IgG-alkaline phosphatase was added and the plate incubated for 1 h. Vectastain ABC reagent (100/~l/well) was added to the wells and incubated for 30 min as described by the manufacturer. Finally, 100/A p-nitrophenyl phosphate in 1 M diethanolamine pH 8.6 was added. In order to stop the reaction, 0.4 M N a O H was added to the wells. Absorbance at 405 nm was measured with a Microplate Reader (Series 700; Cambridge Technology, Cambridge, MA).

3.4.

4.

E L I S A for antibodies to P A H metaboliteD N A adducts

Results

Sera from each animal were tested by direct ELISA for antibodies to unmodified D N A or DNAs modified by diolepoxides or epoxides of eight different PAHs (Figs. 1 and 2). Sera from i.p. treated mice were slightly more reactive to DNAs modified with BPDE, BADE, ChDE, or DBAE than to unmodified D N A (Fig. 1). Sera from i.g. treated mice were more reactive to DNAs modified with BADE or B F D E than to unmodified D N A (Fig. 2). In some cases, the level of sera reactivity to individual PAH metabolites increased with the dose of PAH administered. The reactivity of sera with unmodified D N A was unaffected by the treatment of mice with PAHs. The relative serum reactivity to

Competitive and noncompetitive (direct) ELISAs were carried out as described [26]. Polystyrene 96-microwell plates (Immulon 2, Dynatech Lab., McLean, VA) were coated with 50 ng per well of heat-denatured BPDE-DNA, BADEDNA, F A D E - D N A , DBAE-DNA, MCE-DNA, IPE-DNA, B F D E - D N A , or ChDE-DNA. Wells were incubated with 200 #1 1% BSA in phosphate-buffered saline containing 0.05% Tween (PBS-Tween) for I h (37°C) to minimize the nonspecific binding of protein to the plate. The plates were washed 3 times with PBS-Tween, and diluted sera (100/~l/well) were added to each test well and incubated for 1.5 h at 37°C. After washing the 0.8

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Fig. 1. Noncompetitive ELISA for binding of antisera from i.p. treated mice. Antisera were tested for binding to unmodified D N A or D N A modified by BPDE, BADE, F A D E , DBAE, MCE, ChDE, BFDE, or 1PE as described in Methods. Each group represents the mean of 4-6 mice. Error bars are SEM. Significant increases (P < 0.05) from untreated controls are indicated by an asterisk.

119

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Fig. 2. N o n c o m p e t i t i v e E L I S A for b i n d i n g of antisera from i.g. treated mice. A n t i s e r a tested as in Fig. 1.

PAH-metabolite modified DNA was examined by normalizing to unmodified DNA reactivity for individual mice (Figs. 3 and 4). In i.p. treated mice, increased reactivity of greater than 50% relative to unmodified DNA (> 1.5 relative reactivity in Fig. 3) was observed in only three mice, all in the

150-/~g dose group. In i.g treated mice, increased reactivity greater than 50% relative to unmodified DNA was observed in two mice in the 15-/~g dose group and four mice in the 150-#g dose group (Fig. 4). Thus, 3 of 9 i.p. treated and 6 of 11 i.g. treated mice demonstrated increased reac-

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Fig. 3. Relative b i n d i n g of antisera from i n d i v i d u a l i.p. treated mice. A n t i s e r a tested as in Fig. 1 by n o n c o m p e t i t i v e E L I S A . Results for each m o u s e are n o r m a l i z e d to b i n d i n g to u n m o d i f i e d D N A .

120

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Fig. 4. Relative binding of antisera from individual i.g. treated mice. Antisera tested as in Fig. 1 by noncompetitive EL1SA. Results for each mouse are normalized to binding to unmodified DNA.

tivity of 50% or more (Table 1). In general, there was no difference in antibody titers between sera from the first bleeding (after 8 weeks of treatment) and from the second bleeding (8 weeks plus 10 days). Antibody specificity was further examined by competitive ELISA (data not shown). Sera from two i.g. treated mice shown to recognize DBAED N A by noncompetitive ELISA were tested on the plates coated with 50 ng of D B A E - D N A by competitive ELISA. The binding of both antisera was inhibited to a similar extent by DBAE-DNA, BPDE-DNA, or unmodified DNA. Thus, specific

TABLE 1 Antisera response in mice treated with PAHs for 8 weeks. Dose (~g)b

Number of mice responding/number of mice treated a i.p. injection

i.g. intubation

0 15

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0/4 c 2/6

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3/5

4/5

aSera from responding mice demonstrate at least 50% more reactivity to PAH-metabolite modified D N A than to unmodified D N A by noncompetitive ELISA. bDose of each PAH twice weekly (see Methods). CTwo mice died during study.

binding to D B A E - D N A was not confirmed by competitive ELISA. Sera from five i.g. treated mice shown to recognize B A D E - D N A by noncompetitive ELISA were also tested on the plates coated with B A D E - D N A by competitive ELISA (data not shown). Four of the five sera tested on B A D E - D N A plates showed no consistent difference in inhibition between modified D N A and unmodified DNA. 5.

Discussion

In previous studies, antibodies against BPDED N A were detected in sera from humans occupationally exposed to high levels of PAH compounds or in a random sample of an urban population [9,11,15]. Antibodies have also been detected in sera from people exposed to the genotoxic compounds procainamide and hydrazine. However, in general, only 10-35% of exposed individuals showed positive antibody responses in these human studies. More recently, groups of workers occupationally exposed to PAHs, such as foundry workers and roofers, as well as presumedly unexposed controls were tested for the presence of antibodies against B P D E - D N A in sera, but no significant difference was seen between the control and exposed groups ([22] and unpublished). It may not be surprising to see positive antibody responses in the sera of 'controls' since humans are

exposed to PAHs from various sources (food, environment, passive smoking, etc.), although one might expect fewer positive responses in control sera than in occupationally exposed sera. Another confounding factor is the interindividual differences in the immune response to specific antigens. In this study, we examined antibody profiles in mice exposed to a mixture of PAHs. PAHs were administered by either i.p. injection or i.g. intubation twice a week for two months. Sera from 9 of 20 animals treated with PAHs showed positive antibody responses to PAH-modified D N A by noncompetitive ELISA. Thus, in animals exposed to relatively high doses of PAHs, 45% showed increased binding to one or more PAH-metabolite modified DNAs compared with unmodified DNA. However, the antibody titers for modified D N A were only marginally increased over titers for unmodified DNA. Individual mice demonstrated distinct binding profiles; enhanced binding to B A D E - D N A was more common than to other modified DNAs. Although B P D E - D N A was more highly modified than the other PAH metabolite-modified DNAs, it was no more reactive to antisera. Competitive inhibition experiments indicated that in most cases the mouse antisera could not distinguish between modified and unmodified DNA. These results suggest that low-affinity polyspecific antisera with a slight preference for modified D N A may form in treated mice. However, the antibodies formed are not highly specific for individual PAH-metabolite modified D N A s and demonstrate some cross-reactivity to unmodified DNA. This is in contrast to some, but not all, of the human studies, in which antisera specificity was confirmed by competitive ELISA [9,15]. It should be noted that the metabolic activation pathway for some PAHs is not completely understood [24]. Therefore, the metabolites used to produce the modified DNAs may not be the most relevant metabolites in all cases. In addition, the metabolism of PAHs may differ in mice and humans. In this animal model, a uniform specific antibody response to PAH exposure was not observed. This indicates that the level of antibodies to P A H - D N A adducts in inbred animals treated 122

with known doses of PAH is an imprecise measure of previous (or current) PAH exposure. Given the heterogeneity in the immune response among individual mice, as well as among humans, it would appear that the use of serum antibodies to P A H - D N A adducts as an indicator of PAH exposure in humans is qualitative at best. However, the previous reports of P A H - D N A specific antibodies, confirmed by competitive ELISA, in human sera indicate that PAHs (or compounds with similar structures) were at some time inhaled, ingested, or absorbed by the individual. Conversely, the absence of detectable specific antibodies cannot be taken as proof of non-exposure. More extensive examination of the factors governing carcinogen-modified D N A antibody formation in humans and animal models may improve the usefulness of this approach to the human biomonitoring of genotoxicity.

Acknowledgement Supported in part by D H H S grants ES06052 and ES03819.

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