Immunological Methods for Monitoring Human Exposure to Benzo[a

Chapter 20

Immunological Methods for Monitoring Human Exposure to Benzo[a]pyrene and Aflatoxin B 1

Measurement of Carcinogen Adducts

Downloaded by SUFFOLK UNIV on January 19, 2018 | http://pubs.acs.org Publication Date: December 26, 1990 | doi: 10.1021/bk-1990-0451.ch020

Regina M . Santella, Yu Jing Zhang, Ling Ling Hsieh, Tie Lan Young, Xiao Qing Lu, Byung Mu Lee, Guang Yang Yang, and Frederica P. Perera Cancer Center and Division of Environmental Science, School of Public Health, Columbia University, New York, NY 10032

Immunologic methods have been developed for the measurement of human exposure to environmental and occupational carcinogens by quantitation of carcinogen-DNA or protein adducts. Antibodies recognizing specific adducts have been used in highly sensitive competitive enzyme linked immunosorbent assays (ELISA) to detect femtomole levels of adducts in human samples. Antibodies recognizing benzo(a)pyrene diol epoxide-DNA have been used to quantitate adducts in white blood cells of foundry workers and coal tar treated psoriasis patients and in white blood cells and placenta of smokers and nonsmokers. These same antibodies have been used in indirect immunofluorescence staining of tissues to localize adduct formation to specific cell types. Albumin adducts have also been measured in several of these populations. Antibodies recognizing aflatoxin B-DNA adducts have been used to quantitate adducts in liver tissue from hepatocellular cancer patients. Adduct measurement provides a relevant marker of carcinogen exposure but may also prove useful in identifying individuals at risk for cancer development. 1

Monitoring human exposure to environmental or occupational carcinogens can be carried out at several different levels (1). External exposure can be measured by methods which quantitate carcinogen levels in air, food and water. Internal dose can be estimated by measuring levels of the carcinogen in body fluids such as blood and urine. More recently, methods have been developed to measure the biologically effective dose of the chemical, defined as the amount of chemical reacting with critical cellular targets (2). 0097-6156/91A)451-0229$06.00A) © 1991 American Chemical Society Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


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Covalent binding of the carcinogen to DNA is believed to be the i n i t i a l c r i t i c a l step i n chemical carcinogenesis. Thus, measurement of carcinogen-DNA adducts should be a more relevant marker of exposure to carcinogens than measurement of the chemical i t s e l f i n the environment or body f l u i d s . Such assays take into account individual differences i n absorption and metabolism of carcinogens as well as repair of adducts once they are formed. In addition to being a more relevant marker of exposure, i t i s hoped that such measurements w i l l be useful as markers of individual r i s k for development of cancer. While DNA i s believed to be the c r i t i c a l target for chemical carcinogens, such agents also bind to RNA and proteins. Quantitation of adducts on either hemoglobin or albumin has been used as an alternate marker of exposure to environmental carcinogens. In contrast to DNA adduct measurements, large amounts of protein can be obtained from blood samples and no repair occurs suggesting that chronic, low levels of exposure may be measurable. Red blood c e l l s have an average lifespan of 4 months while albumin has a h a l f l i f e of 21 days. Thus, only recent exposure w i l l be quantifiable with protein adduct measurement. We have concentrated on the development of immunologic methods for the measurement of carcinogen-DNA and protein adducts as well as measurement of the carcinogen i t s e l f i n body f l u i d s such as urine and sera. These antibodies can be used in highly s e n s i t i v e competitive enzyme-linked immunosorbent assays (ELISA) with c o l o r or fluorescence-endpoint detection to give femto ( 1 0 - ) mole sensitivities. For DNA adduct measurement, with t h i s l e v e l of s e n s i t i v i t y , and the a b i l i t y to assay 50ug of DNA per well adduct levels i n the range of 1/10 nucleotides can be measured. Table I l i s t s the monoclonal antibodies recognizing carcinogen-DNA adducts that we have developed to date. Several of these antibodies, including those recognizing a f l a t o x i n , benzo(a)pyrene d i o l epoxide and 8-methoxypsoralen-DNA adducts, have been applied to adduct detection in humans. A major advantage of immunologic methods i s 15

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Table I. Monoclonal Antibodies Recognizing CarcinogenDNA and Protein Adducts Acetylaminofluorene-DNA Aflatoxin-DNA 4-Aminobiphenyl-guanos ine Aminopyrene-DNA Benzo(a)pyrene d i o l epoxide-DNA Benzo(a)pyrene d i o l epoxide-guanosine Ethenoadenine Ethenocytidine 7- (Hydroxyethy1)guanos ine 8-Methoxyps ora1en-DNA 8-Oxoguanos ine Tr imethy1ange1icine-DNA Aflatoxin Bi-albumin Benzo(a)pyrene d i o l epoxide-protein Ethylene oxide-hemoglobin

(3) (4) unpublished (5)

(6) (6) (7) (7)

Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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that once a s e n s i t i v e and s p e c i f i c method has been developed i t can e a s i l y be applied to the large number of samples that are c o l l e c t e d in epidemiologic studies. However, before an immunoassay can be developed the structure of the adduct of interest must be known and i t must be possible to synthesize an appropriate immunogen. Also shown in Table I are the antibodies we have recently developed for measurement of carcinogen-protein adducts. Only the antibody recognizing benzo(a)pyrene d i o l epoxide-protein adducts has been applied to human samples (see below).


Measurement of Exposure to P o l y c y c l i c Aromatic Hydrocarbons: Antibodies to Benzo(a)pyrene d i o l epoxide-DNA Benzo(a)pyrene (BP), a p o l y c y c l i c aromatic hydrocarbon (PAH), i s a ubiquitous environmental pollutant r e s u l t i n g from the incomplete combustion of organic material, including f o s s i l fuels. It i s found in urban a i r , sidestream and mainstream cigarette smoke and the food supply. BP is generally used as a representative indicator of t o t a l PAH concentration. Extensive studies from several laboratories have determined that i t is metabolized in vivo to 7,8-dihydroxy-9,10epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-I), the major e l e c t r o p h i l i e , mutagenic and carcinogenic metabolite involved in DNA binding (Figure 1, reviewed in (11)). The complete structure and conformation of the adduct formed between BPDE-I and DNA in v i t r o i s known and results from binding of the C-10 position of BPDE-I to the 2-amino group of guanosine (Figure 1). This adduct has been detected as the major adduct formed when a variety of human, bovine and rodent c e l l s are exposed to BP in culture. We have developed polyclonal and monoclonal antibodies recognizing t h i s adduct. Two types of antigens were used, the modified DNA e l e c t r o s t a t i c a l l y complexed to bovine serum albumin (BSA) or the ribose form of the nucleoside monoadduct covalently coupled through the adjacent hydroxides on the ribose r i n g to BSA (6,12). When f i r s t characterized, the antisera developed against BPDE-I-DNA were found to be highly s p e c i f i c for the modified DNA not recognizing BP i t s e l f or nonmodified DNA. In addition, there was no c r o s s r e a c t i v i t y with several other carcinogen modified DNAs, including acetylaminofluorene and 8-methoxypsoralen-DNA. More recently, both the polyclonal and monoclonal antibodies were found to crossreact with s t r u c t u r a l l y related d i o l epoxide adducts of several other PAHs including chrysene and benz(a)anthracene (13). For example, polyclonal antibody #29,(12) obtained from animals immunized with BPDE-I-DNA, recognizes DNA modified by chrysene-1,2diol-3,4-epoxide more e f f i c i e n t l y (50* i n h i b i t i o n at 18 fmol) than i t recognizes BPDE-I-DNA (50* i n h i b i t i o n at 30 fmol). This antibody also binds to DNA modified by benz(a)anthracene-8,9-diol-10,11epoxide (50* i n h i b i t i o n at 42 fmol) and 3 , 4 - d i o l - l , 2 - e p o x i d e (50* i n h i b i t i o n at 114 fmol). These results indicate that multiple adducts may be detected by the ELISA. Since humans are exposed to BP in complex mixtures containing a number of other PAHs, a number of different adducts may be present. The i d e n t i t y of the adducts cannot be determined and thus absolute quantitation of adducts i s not possible. However, since a number of PAHs in addition to BP are




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Figure 1. Structure of benzo(a)pyrene ( 1 ) , BPDE-I ( 2 ) and the adduct of BPDE-I with guanine ( 3 ) .


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carcinogenic, the ELISA provides a b i o l o g i c a l l y relevant general index o f DNA binding by t h i s class o f compounds. Measured values are expressed as femtomole equivalents o f BP adducts which would cause a s i m i l a r i n h i b i t i o n i n the assay. We have also recently determined that antibody #29 detects adducts more e f f i c i e n t l y i n highly modified DNA (1.2 adducts/100 nucleotides) than i n DNA modified to a lower l e v e l (1.5/10 ) (14). This e f f i c i e n c y also varied with the type o f ELISA used. With the color endpoint ELISA there was a 2.5 f o l d difference between the high and low modified DNA samples but with the fluorescence endpoint ELISA the difference was 10 f o l d . These two assays d i f f e r i n antibody d i l u t i o n and concentration o f antigen used for plate coating. The antigen used f o r antibody development was highly modified DNA. C l u s t e r i n g o f adducts or some unique determinents present on highly modified DNA may be responsible f o r the higher s e n s i t i v i t y with these samples. U t i l i z a t i o n o f highly modified DNA i n the standard curve i n our o r i g i n a l studies resulted i n an underestimation o f adduct l e v e l s . In contrast, antibodies recognizing 8-MOP-DNA have s i m i l a r c r o s s r e a c t i v i t y with adducts i n high and low modified DNA (15). These results demonstrate the importance o f thorough characterization o f a n t i s e r a before application to human samples and the u t i l i z a t i o n o f appropriate standards f o r analyzing b i o l o g i c a l samples. Currently, we use polyclonal antibody #29 i n a competitive ELISA with a low modified BPDE-I-DNA standard and fluorescence endpoint detection. This assay has a 50% i n h i b i t i o n of antibody binding with low modified DNA o f about 44 fmol. I f 20% i n h i b i t i o n i s taken as the lower l i m i t o f d e t e c t a b i l i t y , adduct l e v e l s o f about 1/10 nucleotides can be determined.


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Detection o f PAH-DNA Adducts i n Humans Occupational Exposure. Antibodies recognizing PAH-DNA have been used i n several studies to quantitate adduct levels i n humans. Lung, the target tissue f o r PAH carcinogenesis, i s not a v a i l a b l e on a routine basis from healthy individuals. Therefore, white blood c e l l s have been u t i l i z e d i n a number o f biomonitoring studies as a surrogate source of DNA since i t i s r e a d i l y and repeatedly available. A 30ml blood sample provides the 500ug o f DNA required f o r duplicate assays i n t r i p l i c a t e wells as normally c a r r i e d out. In collaboration with K. Hemminki, Institute o f Occupational Health, Finland, adducts were measured i n white blood c e l l DNA from iron foundry workers and controls (16). Foundry workers are known to be at elevated r i s k o f lung cancer (17) and constitute a model population for purposes o f v a l i d a t i n g PAH-DNA adducts as a marker o f b i o l o g i c a l l y e f f e c t i v e dose. Workers were c l a s s i f i e d into high, medium, or low exposure to BP based on a i r monitoring data and an i n d u s t r i a l hygienist's evaluation o f the job description (Table I I ) . A dose response relationship was seen between estimated exposure to BP and adduct l e v e l s . Adducts i n these samples were also analyzed by two other laboratories by [ P ] postlabeling (18). In t h i s method, the DNA i s enzymatically digested to 3'monophosphates followed by labeling with T4 polynucleotide kinase 32


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and gamma [ P ] ATP. Normal nucleotides are separated from adducted ones by thin layer chromatography and adducts v i s u a l i z e d by autoradiography (19-20). While adduct levels were lower i n the postlabeling assay, there was a good c o r r e l a t i o n between the immunoassay and postlabeling data. From a subset of 9 foundry workers, we recently c o l l e c t e d blood samples immediately after a one month vacation and then again after the individuals had been working for 3 months. Mean adduct levels increased 10 f o l d after return to the work environment (Table I I ) . Although the sample number i s small, these results suggest rapid repair of adducts in white blood c e l l s .


Table I I . PAH-DNA adducts in white blood c e l l s of foundry oven workers determined by ELISA. Exposure l e v e l ug BP/m

Ν

High Medium Low Control

>0.2 0.05-0.2 lxl0 _ D imethy lbenz ( a ) anthracene Competitors

6



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protein. This low s e n s i t i v i t y i s probably due to burying of the adduct i n hydrophobic regions of the protein. Others have suggested that release of t e t r o l s by acid hydrolysis, followed by quantitation, i s a s e n s i t i v e method for determination of protein adducts (33-34). While t h i s method worked well on protein treated i n v i t r o with BPDEI, our i n i t i a l studies i n mice treated with radiolabeled BP indicated that only low levels of r a d i o a c t i v i t y could be released from globin by a c i d treatment (35). For this reason, we used an alternate approach for measurement of protein adducts. When protein modified i n v i t r o with BDPE-I was enzymatically digested to peptides and amino acids before ELISA s e n s i t i v i t y was increased 3-4 f o l d (Table I I I ) . This assay was validated using globin isolated from animals treated with radiolabeled BP. The ELISA was able to detect 90-100% of the adducts measured by r a d i o a c t i v i t y (36). These animal studies also indicated that adduct levels were about 10 f o l d higher in albumin than i n globin. For t h i s reason, our i n i t i a l work on human samples has been with albumin isolated from workers o c c u p â t i o n a l l y exposed to PAHs. Albumin was isolated by Reactive blue 2-Sepharose CL-4B a f f i n i t y chromatography and enzymatically digested with insoluble protease coupled to carboxymethyl c e l l u l o s e , which was subsequently removed by centrifugation. Samples were then analyzed by competitive ELISA with antibody 8E11. I n i t i a l studies have been c a r r i e d out on a small number of roofers o c c u p â t i o n a l l y exposed during the removal of an old p i t c h roof and application of new hot asphalt. These studies were c a r r i e d out in collaboration with R. Herbert, Mt. Sinai Medical Center, NY. Seventy percent of the roofers samples were p o s i t i v e with a mean l e v e l of 5.4fmol/ug while 62% of the controls had detectable adduct levels (mean of 4.0fmol/ug). In t h i s small number of subjects there was a trend but no s i g n i f i c a n t difference between roofers and controls. However, we are continuing studies of PAH-albumin adducts using a larger sample. Monitoring Exposure to Aflatoxin Bi Detection of Aflatoxin-DNA Adducts. Aflatoxin Bi (AFBi), i s a fungal metabolite produced by s p e c i f i c strains of Aspergillus flavus. This mycotoxin contaminates the food supply in many regions e s p e c i a l l y i n Asia and A f r i c a . Epidemiological studies have demonstrated a strong association between exposure to this potent l i v e r carcinogen and human l i v e r cancer incidence (37). AFBi i s metabolized in vivo by microsomal mixed-function oxidases to produce a reactive e l e c t r o p h i l i c epoxide which binds to the N7 position of guanine (Figure 3). This adduct i s unstable having an apparent h a l f l i f e of 7.5hr and either depurinates or imidazole ring-opens (Figure 3) (38). The imidazole ring opened (iro) AFBi-FAPy adducts are stable, have been shown to accumulate in treated animals and may play an important role in hepatocarcinogenesis (39). We have developed monoclonal antibodies recognize AFBi-FAPy adducts from animals immunized with DNA modified in v i t r o with AFBi followed by base treatment to convert adducts to the FAPy form (4). An immunogen was prepared by complexing t h i s DNA with methylated BSA. Competitive ELISAs using antibody 6A10 are shown i n Figure 4. This antibody has higher r e a c t i v i t y with denatured highly



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DNA

Figure 3. Structure of a f l a t o x i n Bi-guanine (1) and imidazole r i n g opened aflatoxin Bi-guanine (AFBi-FAPy), (2).

100

10 r 0I 10

.

.

.

100

1000

10000

I 100000

fmole Figure 4. Competitive ELISA of antibody 6A10 binding to i r o A F B i DNA. The competitors were highly modified denatured i r o AFBi-DNA (2.5 adducts/100 nucleotides) (A), highly modified denatured A F B i DNA (7 adducts/100 nucleotides) ( ψ ) , and low modified denatured i r o AFBi-DNA (4 adducts/10 nucleotides) ( Δ ) . Reproduced with permission from Ref. 4, Copyright 1988, Cancer Research. 5


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modified i r o AFBi-DNA (2.5 adducts/100 nucleotides, 50% i n h i b i t i o n at 160fmol) than with i r o AFBi-DNA modified to a lower l e v e l (4 adducts/10 nucleotide, 50% i n h i b i t i o n 500fmol) or with denatured highly modified AFBi-DNA (50% i n h i b i t i o n at 1300fmol). Since AFBi-DNA contains both ring-opened and ring-closed adducts, these results suggest that the antibody binds more strongly with the r i n g opened adduct against which i t was generated. Adducts are detected about 4 - f o l d more e f f i c i e n t l y in highly modified DNA than in DNA with a lower modification l e v e l . While there i s no c r o s s r e a c t i v i t y with unmodified native c a l f thymus DNA, there i s minor c r o s s r e a c t i v i t y with unmodified denatured DNA. There i s no c r o s s r e a c t i v i t y with free aflatoxins, a f l a t o x i n - p r o t e i n conjugates, AFBi-guanine or several other carcinogen modified DNAs including DNAs modified by acetylaminofluorene, psoralen or BPDE-I. The l i m i t of s e n s i t i v i t y , based on assaying 50ug of DNA/well and >20% i n h i b i t i o n , i s 5 adducts/10 nucleotides. To validate the immunoassay, antibody 6A10 was used to detect adducts in l i v e r and kidney of mice and rats treated with radiolabeled AFBi. The ELISA detected 55-65% of t o t a l adducts measured by r a d i o a c t i v i t y in both rat and mouse l i v e r (4). R e l a t i v e l y high adduct levels (>1/10 ) were also detectable i n 2 of 8 tumor adjacent-normal tissues and 7 of 7 tumor tissues obtained from l i v e r cancer patients from Taiwan (Table IV).


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Table IV. Levels of AFBi-FAPY Adducts in Human Liver Patient

Tumor Adj acen t -norma 1 adducts/10 nucleotides 3.5 ND* 3.2 ND 1.2 ND 3.4 1.7 1.2 1.2 1.8 ND ND ND L3 NA 6

1 2 3 4 5 6 7 8 9 *ND, non detectable NA, not assayed

One potential a r t i f a c t of competitive ELISAs i s nonspecific i n h i b i t i o n of antibody binding by factors such as high or low s a l t concentration and pH. Thus, to further support these r e s u l t s , samples were tested i n a competitive ELISA using a monoclonal antibody with s p e c i f i t y for unrelated 8-methoxypsoralen-DNA adducts (8). Since these patients had not been treated with psoralen and there i s l i t t l e environmental exposure no psoralen adducts should be present. The l e v e l of i n h i b i t i o n of a l l samples was less than 20% i n d i c a t i n g there was no nonspecific i n h i b i t i o n of antibody binding by the matrix of the DNA samples. The difference between tumor and nontumor tissue may not be s i g n i f i c a n t because of the small number of samples but w i l l be further investigated in ongoing studies. In addition, because of the r e l a t i v e l y high adduct


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levels present in some samples, we have recently begun to u t i l i z e fluorescence methods to confirm adduct levels quantitated by ELISA. I n i t i a l studies with i n v i t r o modified DNA indicate that fluorescence spectroscopy can r e a d i l y detect adducts down to a l e v e l of 1/10 . We are currently developing immunoaffinity techniques to i s o l a t e AFBi modified DNA fragments from p a r t i a l l y digested human DNA samples. By combining the s p e c i f i c i t y of the immunoaffinity technique with the s e n s i t i v i t y of fluorescence spectroscopy, we hope to obtain supportive data for the ELISA r e s u l t s . A s i m i l a r approach has been used by others to confirm the presence of BPDE-I-DNA adducts in human placental DNA (34) and excised aflatoxin-DNA adducts i n urine (see Groopman et a l , t h i s p u b l i c a t i o n ) .


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Immunohistochemical Detection of Aflatoxin Bi-DNA Adducts. The antibodies recognizing AFBi-DNA are also being applied i n immunohistochemical studies to l o c a l i z e adducts. I n i t i a l studies have u t i l i z e d hepatocytes treated in v i t r o with 40uM AFBi. Six hours after exposure, c e l l s were fixed and treated with bicarbonate solution to convert the adducts to the stable r i n g opened form recognized by antibody 6A10. Staining with adduct s p e c i f i c antibody was followed by FITC labeled secondary antibodies. Figure 5 shows s p e c i f i c nuclear s t a i n i n g in treated c e l l s but not i n controls. This method i s now being applied to the detection of adducts i n human l i v e r biopsies. Multiple Adduct Analysis. Human exposure to environmental carcinogens usually occurs in the form of complex mixtures. To monitor exposure to these mixtures, we would i d e a l l y l i k e to perform multiple ELISAs on DNA samples from the same individual to determine a l l adducts present. To circumvent the l i m i t e d a v a i l a b i l i t y of white blood c e l l DNA, we have recently developed methods in which two different DNA adducts can be measured i n a DNA sample with s p e c i f i c antibodies recognizing the individual adducts. Initial studies have been on DNA modified in v i t r o with BPDE-I and subsequently modified with 8-methoxypsoralen and UVA l i g h t . A mixture of the antibodies recognizing both adducts, each at the appropriate f i n a l d i l u t i o n for the ELISA, was used. The competitive ELISA was c a r r i e d out on s e r i a l d i l u t i o n s of the modified DNA and mixed antibodies. This competitive mixture was f i r s t added to plates coated with BPDE-I-DNA and after a 90 min incubation, transferred to plates coated with 8-methoxypsoralen-DNA. Each plate was then incubated with a l k a l i n e phosphatase secondary antibody conjugate as in the standard assay. For both BPDE-I and 8-MOP-DNA, s i m i l a r 50* i n h i b i t i o n s were found when only one adduct or both were present. These results suggest that i t may be possible to make a c o c k t a i l of antisera to s p e c i f i c DNA adducts and, by sequential transfer to plates coated with the appropriate antigen, quantitate a number of different DNA adducts in a s i n g l e sample. Since the adducts are not destroyed by incubation with antibodies in the ELISA, DNA can also be recovered from the competitive mixture on the microwell plate and r e p u r i f i e d . The DNA can then be u t i l i z e d for additional analysis by alternate methods such as postlabeling or fluorescence.



Figure 5. Indirect inrnmofluorescence s t a i n i n g of hepatocytes treated with 40uM aflatoxin Bi (A); and control untreated c e l l s ( Β ) . Slides were treated with 50mM sodium carbonate pH 9.5 after f i x a t i o n , then with RNase, proteinase Κ and 50mM NaOH. Antibody 6A10 was used at a 1:2 d i l u t i o n and goat anti-mouse IgG conjugated with fluorescein at 1:40.


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Discussion These studies demonstrate that immunologic methods have s u f f i c i e n t s e n s i t i v i t y to monitor human exposure to environmental carcinogens. Immunoassays also have the advantage of ease of application to large number of samples making them ideal for epidemiologic studies. However, human samples frequently have adducts l e v e l s near the l i m i t s of s e n s i t i v i t y of these assays. In addition, because competitive ELISAs measure general i n h i b i t i o n of antibody binding to plates, a r t i f a c t s can sometimes interfere with quantitation. For these reasons, i t i s desirable to use other methods to confirm adduct levels determined by ELISA. unfortunately, alternate methods, with the required s e n s i t i v i t y , are not always a v a i l a b l e . The studies discussed above on PAH adducts i n foundry workers are examples of t h i s approach. While the postlabeling assay may not be measuring the i d e n t i c a l adducts as the ELISA, the c o r r e l a t i o n of both assays with exposure l e v e l and with each other provides support for the relevance of the determinations. S i m i l a r l y , for a f l a t o x i n DNA adducts we are attempting to u t i l i z e fluorescence measurements to confirm immunoassay data. The current immunofluorescence method i s l i m i t e d to the detection of adduct levels around 1/10 nucleotides. Computerassisted video microscopy systems or the use of b i o t i n - s t r e p t a v i d i n s t a i n i n g should further increase s e n s i t i v i t y . It may then be possible to u t i l i z e these methods for adduct detection i n human samples from occupational or environmental exposures. Since adducts can in theory be v i s u a l i z e d i n single c e l l s , the small amount of material obtained at biopsy could be u t i l i z e d . While methods for the determination of DNA adducts in humans provides information about the b i o l o g i c a l l y e f f e c t i v e dose of a carcinogen, and can therefore be used as a marker of exposure, information about the relationship of these measurements to r i s k i s unknown. Future epidemiologic studies are needed to provide t h i s information. 6

Acknowledgments Studies on placental DNA adducts in smokers and nonsmokers were c a r r i e d out i n collaboration with R. Everson. Studies on coal tar treated p s o r i a s i s patients were c a r r i e d out i n collaboration with D. Warburton, M. Toor and V. DeLeo. The cooperation of S.W. Hsu and D.S. Chen in the c o l l e c t i o n of samples from cancer patients i n Taiwan i s also g r e a t f u l l y acknowledged. This work was supported by grants from the National I n s t i t u t e of Health CA21111, OH02622 and a g i f t from the L u c i l l e P. Markey Charitable Trust

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