Chapter 24
Immunoassays for Molecular Dosimetry Studies with Vinyl Chloride and Ethylene Oxide Michael J . Wraith, William P. Watson, and Alan S. Wright Shell Research, Ltd., Sittingbourne Research Centre, Sittingbourne, Kent, M E 9 8 A G , United Kingdom
Specific, sensitive radioimmunoassays have been developed for detecting 1,N-ethenodeoxyadenosine (EdA) and 3,N-ethenodeoxycytidine (EdC), two adducts reported to occur in liver DNA of rats chronically exposed to vinyl chloride. Application of these assays in the analysis of liver DNA from rats exposed orally to five daily doses of 50 mg/kg vinyl chloride failed to clearly detect EdA and EdC. The levels of detection were 1 adduct in 3x10 nucleotides and 1 adduct in 6x10 nucleotides respectively. A novel immunochemical procedure has also been developed for monitoring human exposures to ethylene oxide based on the reaction of ethylene oxide with the amino group of the N-terminal valine residue of the α-chain of human hemoglobin. The method is designed to measure the extent of this reaction by determining the product in the form of the aducted N-terminal tryptic heptapeptide. The method has been employed in monitoring exposures of workers to ethylene oxide and has been validated by comparison with a gas chromatography-mass spectrometry procedure. 6
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Despite the long-standing and diverse applications of immunochemistry in the clinical area, this technology is only now becoming widely exploited as a general tool in analytical and biochemistry, e.g. in environmental analysis. Initial studies in our laboratory were directed towards the analysis of the products of reactions between carcinogens and bio-macromolecules for applications in molecular dosimetry and biomonitoring programmes (1). Subsequently, the focus has shifted to applications in environmental monitoring where the major attributes of the methods, i.e. speed and simplicity combined with high sensitivity and specificity, were needed to cope with the increasingly heavy demands. Particular emphasis was placed on enzyme-linked immunosorbent assays (ELISA) with a view to establishing practical field assays (2). These developments have been greatly facilitated by the development of monoclonal antibody technology which has expanded the horizons of immunoassay (3). In particular this technology provides the potential for greater specificity and virtually unlimited supplies of antibodies which has allayed concerns about the long-term viability of the newly developed assays. The, so-called, non-traditional applications of immunoassay are therefore wellestablished (4) and have an important role in both applied and fundamental research. 0097-6156/91/0451-0272$06.00/0 © 1991 American Chemical Society
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Molecular dosimetry It is now widely accepted that the identification and quantitation of the products of interactions between D N A and electrophilic chemicals (or their metabolites) is of key importance in the understanding of the mutagenic and carcinogenic processes. In particular, qualitative and quantitative determinations of D N A adducts formed during low-level exposures to chemicals are essential for rational prospective risk assessments (5). Problems arise when extrapolations are made from high experimental doses or concentrations, designed to give measurable effects in experimental species, to low concentrations occurring in the environmental or occupational situation. For example, it is uncertain whether the extrapolation from high doses to low doses should be based on a linear regression to zero dose or on some alternative dose-response relationship. Furthermore, translation of the risk estimates from the test (experimental) species to man are subject to error, the assessments being complicated by species differences among factors that determine the response to a given exposure or dose of the carcinogen (6). These interpretative problems led to the development of the 'target-dose' approach (7), which seeks to improve the quality of risk assessment by providing a means of compensating for differences in the metabolism and metabolic disposition of carcinogens in the tissues of the test species and man. This approach, which is based on the estimation of the dose of ultimate carcinogen delivered to the critical cellular target (DNA), permits the investigation of the integrated operation of all the toxicokinetic and toxicodynamic factors that regulate tissue D N A dose. A further important consideration is that human tissue D N A is generally inaccessible for experimental purposes and this led to the development of indirect methods of assessment of D N A doses (8). Due to the electrophilic nature of mutagens and carcinogens they react with all types of nucleophilic centre, including those found in proteins. There are several examples where hemoglobin (Hb) is an appropriate dose monitor for DNA(9). Its advantages are that blood is readily and repeatedly obtainable from humans in useful quantities. Also the biological lifetime of the human erythrocyte (120 days) permits monitoring long after a particular exposure has ceased and provides high sensitivity by permitting adduct formation due to chronic, low-level exposure to accumulate. The estimation of target dose, either directly at D N A or indirectly in proteins, is based on measurements of the amounts of the adducts formed by chemical reactions occurring between the ultimate carcinogen and D N A bases or amino acid residues. These adducts occur in tissues at extremely low concentrations, typically 1 adduct per 1 0 bases or amino acids, and the technical demands on quantitative assays, without the use of radiolabelled carcinogens, are very high. In such instances, e.g. the monitoring of occupational exposure or conventional experimental carcinogenicity studies, the assay of adducts necessitates the development of extremely sensitive and specific procedures. In this respect immunochemical assays have made a significant contribution (10,11) and, because of their intrinsic selectivity, which often significantly reduces or obviates the need for preliminary purification steps, they are one of the principle methods of choice. 6 - 1 0
Vinyl Chloride Dosimetry Studies Vinyl chloride (VC) has been known for many years to be a human carcinogen. It has therefore been the subject of intensive investigation and was chosen as a model for our molecular dosimetry studies in experimental animals. During 1980 work began on the development of radioimmunoassays (RIA) to quantitate VC-induced D N A adducts. The aim was to apply the RIAs in studies designed to investigate the quantitative relationships between exposures to low doses of V C , tissue D N A doses (including both target and non-target tissues) and hepatocarcinogenesis. A t the commencement of the study, three V C - D N A adducts had been described in rats exposed to V C in vivo (12,13,14). These comprised a major adduct,
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7
6
N -(2-oxoethyl)guanine (I), and two minor (cyclic) adducts, l,N -ethenodeoxyadenosine (II, EdA) and 3,N -ethenodeoxycytidine (III, EdC). During the course of the work evidence for a third cyclic adduct, ^,3-ethenoguanine (IV), was reported (15). (Figure 1). Part of the original objectives of the study was the development of a R I A for the major V C - D N A adduct. However, ^-alkylated deoxyguanosine adducts are chemically unstable and, although an immunogen was prepared, production of antibodies was not detected. Thus RIAs were developed for E d A and E d C . 4
Antibody Production. Immunogens were prepared by covalently linking l,N -ethenoadenosine and 3,N -ethenocytidine to bovine albumin (16). The use of the ribose forms of the adducts did not create a problem of specificity as the analytical samples were purified free of R N A . Ultimately, H P L C purification of E d A and E d C prior to analysis obviated all potential cross-reactivity problems. Rabbits were immunised by the multisite intradermal method (17) and high-titre antisera were generated. 6
4
Synthesis of Reference Standards and Radiolabeled Tracers. E d A and E d C were prepared (18) and purified by H P L C . In order to prepare [ I]-radiolabelled tracers a strategy was devised to introduce a group into the adduct molecules, at a point remote from the antigenic determinant, which could be subsequently iodinated. In summary, the acetal derived from the reaction of anisaldehyde with the ribose moieties of adenosine or cytidine was demethylated with lithium t-butyl mercaptide to give phenolic derivatives. These were then allowed to react with chloroacetaldehyde (18) to give the modified etheno adducts. Iodination was accomplished using sodium [ I]iodine and a solid-phase oxidising agent, 1,3,4,6-tetrachloro-3a-i6a-diphenylglycoluril (19). 125
125
Radioimmunoassay Parameters. The RIAs for E d A and E d C were developed by conventional approaches. The lower limit of detection for E d A was 1 pmol/ml which was equivalent to 0.05 pmol (3 χ 10 molecules) in the test sample volume. For E d C the lower limit of detection was 0.5 pmol/ml. Antibody affinities for both the E d A and E d C were estimated (20) to be in the order of 1 0 ~ M . Both antisera displayed high specificity for their respective antigen targets, with no cross-reactivity between these. Furthermore, cross-reactivity with normal deoxyribonucleosides, determined at the limits of solubility in the test system i.e. 3.6 ^mol/ml, was also very low and suggested that the RIAs would be capable of detecting one adducted deoxyribonucleoside in the presence of approximately 10 non-adducted deoxyribonucleosides. This level of crossreactivity indicated that the assays would be unable to meet the requirement of quantifying adducts in the presence of a molar excess of approximately 10 deoxyribonucleosides i.e. 1 adduct per cell. Assay sensitivity was therefore enhanced by concentrating and isolating the adducts by H P L C prior to R I A . Recoveries of 80% were typical. The removal of cross-reacting species maximised the assay sensitivities which were now limited only by the quantity of D N A available for analysis. The adopted procedure was based on a 5 mg D N A sample, equivalent to 9.25 χ 10 nucleotides. The lower limits of detection of the E d A and E d C RIAs were 3 χ 10 molecules and 1.5 χ 10 molecules respectively. Thus the E d A R I A was capable of detecting 1 adduct in 3 x 10 nucleotides and the E d C R I A 1 adduct in 6 x 10 nucleotides. 10
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Vinyl Chloride Exposure Study. Rats were exposed to V C for 5 days (daily doses of 50 mg/kg in corn oil by oral intubation). D N A was isolated from the livers and hydrolysed enzymatically prior to H P L C . Following collection of the appropriate fractions each was assayed by both RIAs. E d A was not detected. In the case of E d C values bordering on the lower limit of detection were obtained. Confirmation of this result would have required a 10-fold greater amount of D N A which was unavailable.
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Conclusions. Specific and sensitive RIAs have been developed for the two minor adducts reported to occur in the liver D N A of rats exposed to V C . The results obtained in the experiment reported here were inconclusive and subsequent acute exposure studies performed in this laboratory and elsewhere (14) with C-labelled V C failed to confirm the earlier findings (13). However, a recent study (21) in which rats were exposed to 2000 ppm of V C for 10 days has reported measurements of the minor V C adducts by R I A . The results observed in this range of V C exposure conditions supports the idea that measurable levels of the minor cyclic adducts are only found after long-term exposures or following exposures during rapid growth e.g. in neonates. Current views (22) are that the minor cyclic adducts are criticial to the promutagenic, and carcinogenic, activity of V C . However, there still remains a need to develop a simple quantitative method for the major N^oxoethylguanine adduct as this will provide a more sensitive monitor, especially at low exposure doses. 14
Ethylene Oxide Exposure Monitoring The utility of H b as a D N A dose monitor was investigated, using ethylene oxide (EO) as the model carcinogen. Prior to this study, inhalation experiments in rats exposed to C E O had indicated a rapid absorption and equilibration of E O throughout the tissues (23). The results of these studies were consistent with the view that, in the case of E O , H b would be an effective tissue D N A dose monitor. 1 4
Peptide approach. A R I A was developed (1) for use in biomedical monitoring of E O exposure. The principal adducted amino acids formed in human H b by reaction with E O are N (2-hydroxyethyl)valine (a- and 0-chain), N (2-hydroxyethyl)histidine, N -(2-hydroxyethyl)histidine and S-(2-hydroxyethyl)cysteine. The possibility of developing antibodies against the adducted amino acids was judged to be low as their small molecular size offers only limited antigenicity. A n alternative approach was to raise antibodies against a peptide from human H b which contained an EO-adducted amino acid. The peptide selected was the N terminal heptapeptide released from the α-chains of human H b by the action of trypsin (24) (Figure 2). The adducted heptapeptide and the unmodified analogue were synthesized chemically. Both peptides were analysed by high-performance liquid chromatography and were homogeneous on two different stationary phases. In addition, fast atom bombardment-mass spectrometry of both peptides gave ( M + H)+ ions consistent with the required molecular weights. r
3
Radioimmunoassay development. The hydroxyethylated (HOEt) peptide was radioiodinated at the amino group of the C terminal lysine using a conjugation procedure (25). This radioactive tracer was used in the optimized R I A and to monitor incorporation of the H O E t peptide during preparation of the immunogen. The H O E t peptide was coupled to horse albumin using l-ethyl-3-(3-dimethylaminopropyl)carbodiimide, resulting in approximately 16 mol adducted peptide per mol immunogen. Four rabbits were immunized, and antisera from one animal (R103B9) demonstrated sufficiently low cross-reactivity when tested against the nonH O E t peptide, native human H b and the peptides from trypsin-hydrolysed H b , to be useful for the development of a R I A (see Figure 3). The cross-reactivity results indicated that it was possible to quantify the H O E t peptide in the presence of a lC^-fold excess of the nonH O E t peptide. During development of the assay, attempts were made to analyse native H b treated with E O ; however, very low recoveries indicated that the antibody was capable of binding the H O E t peptide only after its release by trypsin hydrolysis. Additional assessments of specificity indicated that the antibody bound equally well to the equivalent H O E t peptide from rat H b . This was probably due to the sequence homology of the first three amino acids of the α-chains of human and rat H b (also rabbit, mouse and chimpanzee Hb). In contrast, the antibody did not bind the analogous propylene oxide adducted peptide, indicating high
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Ο
CHXHO
(IV) Figure 1. Vinyl chloride-DNA adducts
CH
3
CH
3
CH \ 3 / 3 Ο CH,OH Ο CH Ο CH II I II I II I N-C-CH-NH-C-CH-NH-C-CH-NH-CHjr-CH^OH N
C
C
H
2
0 =C
I NH I CH-CH, I 3
o=c
I NH CI H - C H C 0 H I 0 =C 2
2
NH I CH-(CH ) NH I 2
4
2
c/\>H Figure 2. The N-terminal heptapeptide released from the α-chain of ethylene oxide-treated haemoglobin by trypsin hydrolysis
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1 0
-12
1 0
-11
1 0
-10
1
0
-9
1
0
Heptapeptide m o l . m l "
-8
1
0
-7
1
0
- β
1
Figure 3. Examples of cross-reactivity of three antisera • RIA
Δ GC-MS
Δ Δ CD
Ο CD Ô
Ε
1.5
c
& * Δ ·. Δ
Non-exposed
Γ Δ • Δ - Δ Δ
Exposed
Figure 4. Levels of N-(2-hydroxyethyl)valine in the α-chain of H b
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specificity for the H O E t modification. The lower limit of detection of the optimized R I A was 25 fmol/50 μ\ sample, which in conjunction with the cross-reactivity data gave an overall sensitivity of 0.14 pmol H O E t peptide/g globin. Subsequent to the R I A development an E L I S A method for E O exposure monitoring, was developed. The E L I S A method was more rapid and convenient than the R I A , retaining the advantages of immunochemical analysis but without the hazards of radioactivity. Biomonitoring study. The R I A was validated in a study of hospital workers potentially exposed to E O . Samples of blood were obtained from a group of operatives employed in E O sterilization of medical equipment and supplies. Blood samples were also obtained from a group not involved in sterilization work. Test and control samples were analysed using the R I A procedure and were also analysed independently using a G C - M S method for N (2-hydroxyethyl)valine (26) (Figure 4). Significant differences were found between potentially exposed workers and the control group. Background levels of hydroxyethylation were also found in the unexposed group, in agreement with earlier findings (27). In the RIA, background levels of α-chain N-(2-hydroxyethyl) valine ranged from 0.14-0.44 nmol/g globin (mean 0.25; SD, 0.09; η = 14) in samples from the unexposed group. Samples from the potentially exposed operatives gave corresponding values ranging from 0.11 -1.51 nmol/g globin (mean 0.58; SD, 0.37; n = 17). Corresponding data obtained by the G C - M S method were as follows: unexposed group 0.05 - 0.67 nmol/g globin (mean 0.27; SD, 0.2; η =13), exposed group 0.21-2.11 nmol/g globin (mean 0.83; SD 0.61; n = 15). The independent G C - M S analysis thus gave results that were in very good agreement with the R I A data (I). Conclusions. A novel, sensitive and specific immunochemical biomonitoring method for E O exposure was developed. The R I A was validated against an existing G C - M S method, and the two widely differing analytical methods showed excellent agreement. A n assumption made at the outset of this study was that the N-terminal valine residues of the a- and 0-chains of H b would display similar reactivities towards E O . This remains to be proven experimentally. The observation of background levels of hydroxyethylation of H b suggested the possible occurrence of the corresponding adducts as a 'background' in D N A . Potential sources of hydroxyethylating agents included cigarette smoke, engine exhausts, intestinal bacteria and lipid peroxidation. The origins and significance of background alkylations are under investigation (28).
Final Remarks In recent years the demands for trace analysis in toxicology and environmental monitoring have been steadily increasing. A t the same time the costs of conventional methods of analysis have been escalating with continued advances in the sophistication of instruments. This has led to a search for more economic alternatives with performances comparable to existing technology. Immunoassay techniques match these requirements and have additional benefits which can fulfil the changing needs of the analyst. For example, although antibodies are usually very specific, it is possible, by accident or design, to produce antibodies of general specificity. This allows the development of generic immunoassays or immunoaffinity chromatography methods e.g. to quantify polyaromatic hydrocarbon adducts of D N A (29). Applications of immunoassay outside the medical field are now widespread and fully established.
Acknowledgment We thank our colleagues R. Davies, A . E . Crane, D . Potter, R . L . Ball, E . Akerman and D . W . Britton for their assistance and Mrs. B . Whitehead for typing the manuscript.
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