Highly sensitive chemiluminescence immunoassay for benzo[a]pyrene-DNA adducts: validation by comparison with other methods, and use in human biomonitoring

Rao L. Divi1, Frederick A. Beland2, Peter P. Fu2, Linda S. Von Tungeln2, Bernadette Schoket3, Johanna Eltz Camara1, Monica Ghei1, Nathaniel Rothman1, Rashmi Sinha1 and Miriam C. Poirier1,4

1 National Cancer Institute, NIH, Bethesda, MD 20892-4255, USA,
2 National Center for Toxicological Research, Jefferson, AR 72079, USA and
3 Józef Fordor National Center for Public Health, Budapest H-1097, Hungary


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A chemiluminescence immunoassay (CIA) utilizing antiserum elicited against DNA modified with (±)-7ß, 8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]- pyrene (BPDE) has been developed and validated to study the formation of polycyclic aromatic hydrocarbon (PAH)–DNA adducts in human tissues. Advantages include a low limit of detection for 10b-(deoxyguanosin-N2-yl)-7ß,8{alpha},9{alpha}-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG, ~1.5 adducts/109 nucleotides using 20 µg DNA) and a high signal-to-noise ratio (>=100). The CIA BPDE–DNA standard curve gave 50% inhibition at 0.60 ± 0.08 fmol BPdG (mean ± SE, n = 30), which was a 10-fold increase in sensitivity compared with the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). Calf thymus DNA modified with [1,3-3H]BPDE was assayed by radiolabeling, 32P-postlabeling, DELFIA and CIA, and all assays gave similar values. Liver DNAs from mice exposed to 0.5 and 1.0 mg [7,8-3H]benzo[a]pyrene (BP) were assayed by the same four assays and a dose–response was obtained with all assays. The BPDE–DNA CIA was further validated in MCL-5 cells exposed to 4 µM BP for 24 h, where nuclear and mitochondrial DNA adduct levels were associated with an increase in DNA tail length measured by the Comet assay. Human peripheral blood cell (buffy coat) DNA samples (n = 43) obtained from 25 individuals who were either colorectal adenocarcinoma patients or controls were assayed by BPDE–DNA CIA. Three samples (7%) were non-detectable, and the remaining 40 samples had values between 0.71 and 2.21 PAH–DNA adducts/108 nucleotides. The intra-assay coefficient of variation (CV), for four wells on the same microtiter plate, was 1.85%. Sufficient DNA for two assays, on separate plates, was available for 38 of the 43 samples, and the PAH–DNA adduct values obtained were highly correlated (r2 = 0.95). Coded duplicate DNA samples from 15 individuals were assayed four times gave an inter-assay CV of 13.8%.

Abbreviations: BP, benzo[a]pyrene; BPDE, (±)-7ß,8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BPDE–DNA, DNA modified with BPDE having a single major adduct, BPdG; BPdG, 10b-(deoxyguanosin- N2-yl)-7ß,8{alpha},9{alpha}-trihydroxy 7,8,9,10-tetrahydrobenzo[a]pyrene; CIA, chemiluminescence immunoassay; CV, coefficient of variation; DELFIA, dissociation-enhanced lanthanide immunoassay; MCL-5, multi competent human lymphoblastoid cell line expressing the cytochrome P450s 1A1, 1A2, 2A6, 3A4, and 2E1, and epoxide hydrolase; PAH, polycyclic aromatic hydrocarbon; PBS, phosphate buffered saline; PBST, phosphate buffered saline with 0.05% Tween 20


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Benzo[a]pyrene (BP) is a human carcinogen and a major carcinogenic component of multiple human exposures involving polycyclic aromatic hydrocarbons (PAHs) (1,2). The PAHs escape into the ambient atmosphere as a result of partial combustion, sources of which include cigarette smoking, vehicle exhaust, indoor heating, and industrial processes (1,2). In addition to inhalation, exposure to PAHs can occur by consumption of charbroiled food (3,4) and, in some cases, even uncooked food (5). Attempts to monitor human genetic damage resulting from such exposures have met with increasing levels of success during the past ~20 years (6,7), but the risk of human cancer conferred by PAH–DNA adduct formation is still a subject of much interest and investigation (812). The ultimate success of such endeavors will depend, to a significant extent, upon the sensitivity and validity of the methodologies applied.

The classic methods for PAH–DNA adduct determination have included radiolabeling, 32P-postlabeling, and immunoassays utilizing antisera elicited against DNA samples modified with PAHs (7,13). Each of these approaches has advantages and disadvantages, but only occasionally have they been compared in the same study for DNA samples modified in vitro and in vivo (14,15). All of the aforementioned assays, with the exception of radiolabeling, are commonly used with human tissues, but the resulting data have been plagued with variability and difficulties in achieving specificity. The current work describes development and validation of an immunoassay for PAH–DNA adducts that is sufficiently sensitive to obtain measurable values for most human samples, and also has minimal variability and a good signal-to-noise ratio compared with similar immunoassays (16,17).

The highly sensitive BPDE–DNA chemiluminescence immunoassay (CIA) detects BPDE–DNA adducts in experimental samples exposed to BP, and PAH–DNA adducts in human samples, where typical exposures include multiple hydrocarbons. In this study the assay has been validated using a standard calf thymus DNA modified in vitro with [3H]-BPDE, by comparing the CIA data with results obtained using several other methods for BPdG adduct determination. In addition, we have examined DNA adducts in livers of BP-exposed mice, in BP-exposed cultured MCL-5 cells, and in human peripheral blood DNA samples. For the human samples, we have expressed the BPDE–DNA CIA measurements as PAH–DNA adducts, as the antiserum is specific for DNA samples modified with several carcinogenic PAHs (18).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Culture of MCL-5 cells and isolation of nuclear and mitochondrial DNA
MCL-5 cells (19) were purchased from Gentest Corporation (Woburn, MA) and maintained as suspension cultures in RPMI 1640 medium, without histidine but containing 2 mM 1-histidinol, supplemented with 9% horse serum, 30 µg/ml 5-aminolevulinic acid, and 200 µg/ml hygromycin B. Cells were passaged every 2–3 days to maintain a concentration of 2.5 x 105 cells/ml. Cells were exposed to 0 or 4.0 µM BP (in 2% dimethylsulfoxide) for 24 h.

Cells were suspended in three packed cell volumes of PBS containing 2 mM mercaptoethanol and 0.2 mM phenylmethylsulfonyl fluoride, and centrifuged at 1000 g for 5 min at 22°C. Cell pellets were homogenized in two packed cell volumes of the above buffer using a tight-fitting Dounce homogenizer (12 strokes). Mitochondria were stabilized by adding two packed cell volumes of buffer containing 210 mM mannitol, 70 mM sucrose, 20 mM HEPES, pH 8.0, 2 mM EDTA, 2 µg/ml leupeptin, and 2 mM dithiothreitol. The nuclei were pelleted by centrifugation at 1600 g for 8 min at 22°C and the supernatant was subsequently centrifuged at 20 000 g at 4°C for 15 min to collect the mitochondrial pellet. DNA was isolated separately from nuclear and mitochondrial pellets using QIAamp DNA Mini Kits (Qiagen, USA), as per the manufacturer's protocol. The DNA concentration was determined by UV spectrometry.

In vitro modification of calf thymus standard DNA with [1,3-3H]BPDE
Calf thymus DNA (472 mg) in 400 ml 5 mM Bis–Tris, 0.1 mM EDTA, pH 7.1 was treated for 2 h with 4.2 µg [1,3-3H]BPDE (1744 mCi/mmol) that had been dissolved in 5.9 ml dimethylsulfoxide. The mixture was extracted three times with n-butanol and three times with isoamyl alcohol. These solvents had previously been saturated with 50 mM Bis–Tris, 1 mM EDTA, pH 7.1. The DNA was precipitated by the addition of ethanol and NaCl, washed with 70% ethanol, and redissolved in 5 mM Bis–Tris, 1 mM EDTA, pH 7.1 (15). The DNA concentration was determined by UV spectrometry and the extent of radiolabeled BPdG modification was established by liquid scintillation counting in sextuplicate.

In vivo modification of mouse liver DNA with [7,8-3H]BP
Eight-week-old male B6C3F1 mice (four/group) were injected intraperitoneally with 0, 0.5, or 1.0 mg [7,8-3H]benzo[a]pyrene (125 mCi/mmol) in 100 µl trioctanoin. After 24 h, the mice were euthanized, livers were pooled by group, nuclei were prepared (20), and DNA was isolated by enzymatic digestion, extraction with organic solvents and precipitation (21). The DNA concentration was determined by UV spectrometry and the extent of radiolabeled BP-DNA modification was established by scintillation counting in triplicate.

HPLC analysis of DNA modified in vitro with [1,3-3H]BPDE
DNA reacted in vitro with [1,3-3H]BPDE was hydrolyzed to nucleosides by treatment with DNase I, followed by alkaline phosphatase and snake venom phosphodiesterase as described (22). The nucleoside adducts were extracted three times with water-saturated n-butanol. The n-butanol extracts were combined, back-extracted once with n-butanol-saturated water, evaporated, and the residue was redissolved in methanol for HPLC analysis. The n-butanol extracts contained 98% of the radioactivity associated with the DNA. Non-radioactive BPdG was added to the samples to serve as a UV marker and the samples were analyzed by reversed-phase HPLC using a Zorbax ODS C18 column (9.4 x 250 mm) with an HPLC system consisting of two Waters Model 510 pumps, a Waters U6K injector, and a Waters Model 680 automated gradient controller. The peaks were monitored at 254 nm with a Hewlett-Packard 1040M diode array spectrophotometric detector. Samples were eluted with a 50 min linear gradient of 20–80% methanol, with a flow rate of 3 mL/min. Fractions were collected at 1 min intervals for measurement of radioactivity.

32P-Postlabeling analyses of DNA adducts
The 32P-postlabeling protocol used for the analysis of DNA adducts employed nuclease P1 enhancement as described by Phillips and Castegnaro (23), and was based on the approaches reported previously (2426). Adducts were resolved on 10 x 10 cm polyethylenimine-cellulose thin layer chromatography plates by multi-directional chromatography and visualized by PhosphorImager. The quantitation has been reported (23).

Comet assay
The Comet assay, a single cell electrophoresis, was performed using the Trevigen Comet Assay kit (Trevigen, Gaithersburg, MD). Briefly, cell suspensions were harvested by centrifugation and resuspended at 1 x 105 cells/ml in Ca2+ and Mg2+ free phosphate buffered saline (PBS). The cell suspension was mixed with low melting agarose at 42°C, transferred to chilled microscopic slides and incubated at 4°C for 10 min. The slides were immersed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% sodium lauryl sarcosinate and 0.01% Triton X-100) and placed in the dark at 4°C for 1 h. The slides were then transferred to alkali buffer (0.3 M NaOH, 1 mM EDTA) and incubated at room temperature for 1 h. The slides were washed twice with 0.09 M Tris–borate, 2 mM EDTA, pH 8.3, subjected to horizontal electrophoresis using 0.09 M Tris–borate, 2 mM EDTA, pH 8.3 at 1 V/cm for 15 min, and then stained with SYBR Green. The cells were visualized by fluorescence microscopy and digitized using Image Pro Plus 3.0. Nuclear diameter, and tail length from the center of the nucleus, were expressed as µm, after measurements were taken by NIH image analysis software (http://rsb.info.nih.gov/nih-image/index.html') using the macros of Herbert M.Geller (http://www2.umdnj.edu/%7Egeller/lab/comet.htm).

BPDE–DNA adducts by DELFIA
DNA samples were subjected to sonication and denaturation before conducting the DELFIA and the CIA. Specifically, aliquots of sample DNA in PBS were sonicated for 15 s using a Sonifier Cell Disruptor (Heat Systems-Ultrasonics, Plainview, NY), heat-denatured at 90°C for 2 min, and cooled immediately on ice.

The BPDE–DNA DELFIA was carried out as previously described (16). Essentially, 0.1 ng of DNA, modified to 35.9 pmol BPdG/µg DNA (1.2% modification), was used to coat microtiter plates. Non-specific binding was blocked with 1% fetal calf serum in PBS containing 0.05% Tween-20 (PBST). All washes were carried out with an automated plate washer (Ultra Wash Plus; Dynatech Laboratories, Gaithersburg, MD) using PBST. Diluted (1 : 1 x 106) rabbit BPDE–DNA antiserum was mixed with equal volumes of either the standard BPDE–DNA (modified to 0.8 BPdG/106 nucleotides (16)) or the sample DNA, and incubated on the plate. The DNA content in the standard curve wells was maintained equivalent to the amount of DNA used in the sample wells by adding appropriate amounts of sonicated and denatured carrier calf thymus DNA to normalize the matrix effect.

After washing, plates were sequentially incubated with biotinylated rabbit anti-IgG (1 : 2500) and europium-labeled streptavidin (1 : 2000), and the final fluorescent signal was generated by the addition of Enhancement solution (Wallac, Gaithersburg, MD) before reading on the Wallac 1234 Research Fluorometer. The standard curve 50% inhibition (mean ± SD) was 6.3 ± 0.24 fmol BPdG per well (n = 25) and the lower limit of detection, using 20 µg of DNA per well, was ~1.3 adducts/108 nucleotides (16).

BPDE–DNA determination by CIA
Microtiter plates for the CIA were 96-well high-binding LIA plates (Greiner Labortechnik, FRG). The CIA-specific reagents, including biotinylated antirabbit IgG, streptavidin–alkaline phosphatase, I block (casein), and CDP-Star with Emerald II, were obtained from Tropix (Bedford, MA).

DNA adducts were measured by BPDE–DNA CIA using an approach similar to that described previously for tamoxifen–DNA adducts (27). Briefly, 40 pg of sonicated BPDE–DNA (modified to 35.9 pmol BPdG/µg DNA, 1.2% modification (16)) or calf thymus DNA were coated on microtitration plates in 0.1 ml of Reactibind (Pierce) solution at room temperature for 48 h. Plates were washed with three cycles of PBST containing 0.05% of NaN3 (see the BPDE–DNA DELFIA, above), and stored frozen until use. For assay, non-specific binding was blocked by incubating the wells with 310 µl casein (0.25%) in PBST for 1 h at 37°C. Equal volumes (50 µl) of anti-BPDE–DNA antibody (diluted to 1 : 3 x 106 in 0.25% casein) and serially diluted BPDE–DNA standard (0.03–27 fmol of BPdG in DNA modified to 1.0 BPdG per 106 nucleotides (16)) or test sample, in PBS, were mixed and incubated at 37°C for 15 min prior to adding to the microtiter plate wells. The DNA content in the standard curve wells was maintained equivalent to the amount of DNA used in the sample wells by adding appropriate amounts of sonicated and denatured carrier calf thymus DNA to normalize the matrix effect. The final mixture of DNA and antibody (final dilution 1 : 6 x 106 in 0.25% casein) was incubated for 90 min on the microtiter plate. After washing three times with PBST, biotinylated anti-rabbit IgG (100 µl of 1 : 5000 dilution in 0.25% casein in PBST) was transferred to wells and incubated for an additional 90 min at room temperature. After washing again, streptavidin-alkaline phosphatase (100 µl of 1 : 6000 in 0.25% casein in PBST) was added and incubated at room temperature for 60 min, followed by washing with three cycles of PBST and two cycles of Tris buffer (20 mM Tris–1 mM MgCl2, pH 9.5). Finally, CDP-Star containing Emerald II enhancer (100 µl) was transferred to wells, plates were incubated at room temperature for 30 min and at 4°C for 15 h, and luminescence was read at both times using a TR717 Microplate Luminometer (PE Applied Biosystems, Foster City, CA) at 542 nm. The standard curve 50% inhibition (mean ± SE) was 0.60 ± 0.08 fmol BPdG per well (n = 30) and the lower limit of detection, using 20 µg of DNA per well, was ~1.5 adducts/109 nucleotides.

Human lymphocyte samples, source and DNA preparation
A series of 43 peripheral blood lymphocyte DNA samples, from 25 individuals who were either colorectal adenocarcinoma patients or controls, was obtained from the Navy Colon Adenoma study. This case–control study of colorectal adenomas in active and retired military officers and their families was designed to evaluate meat cooking practices and tumor susceptibility. The study was approved by the institutional review boards of both the National Cancer Institute and the National Naval Medical Center and has been described in detail elsewhere (28). These 43 samples comprised a small subset of a much larger study, and were assayed for the purpose of obtaining quality control parameters. The sample set consisted of 36 samples (labeled 1–36) from 18 patients that were replicates or coded duplicates, two from each of the 18 patients. In addition, there were seven single samples from each of seven patients. The total number of samples was therefore 43. The samples assayed remain coded with respect to the original purpose of the study, so the numbers of cases and controls are currently unknown. Our intention here was to explore the usefulness of the BPDE–DNA CIA for human biomonitoring and to address specific quality control issues including sensitivity, background level, assay reproducibility, and fraction of samples measurable by the assay.

Blood was obtained, allowed to clot at room temperature and centrifuged to obtain serum (top), buffy coat (nucleated cells – middle), and hemoglobin (bottom). Buffy coat samples were retrieved and stored frozen at –80°C until DNA isolation was performed. DNA was prepared as described by Daly et al. (29). Briefly, nuclei were collected by centrifugation of buffy coat samples lysed in sucrose buffer. The nuclear pellet was resuspended in neutral Tris–HCl buffer, incubated at 65°C for 30 min, and extracted with 2 ml chloroform. Following centrifugation, the aqueous DNA-containing upper phase was transferred to a fresh tube, and the DNA was precipitated in ethanol and spooled. The spooled DNA was washed in 70% ethanol and allowed to dry at room temperature for 20 min. In the absence of visible spooled material, DNA was recovered by centrifugation. DNA was resuspended in 50 to 400 µl 10 mM Tris–HCl, 1 mM EDTA, pH 7.4 by incubation at 60°C for 8–16 h. DNA was stored, following aliquoting, at –80°C. DNA purity was checked by electrophoresis on ethidium bromide-containing agarose minigels using Tris–Borate-EDTA buffer (30). Molecular weight markers (either 100 bp ladder from Gibco–BRL or lambda HindIII digest) were also included on each gel. Bands were visualized on a UV transilluminator, and gels photographed using a Kodak DC40 digital camera.


    Results
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 References
 
In vitro modified calf thymus [1,3-3H]BPDE–DNA
A large quantity of calf thymus DNA was modified with [1,3-3H]BPDE (23) with the intention of preparing a standard that could be utilized by many laboratories for assay validation. Digestion of the DNA and subsequent analysis by HPLC demonstrated a single major peak of radioactivity at 40 min that co-chromatographed with an authentic BPdG adduct standard (Figure 1Go). The modification level determined by radiolabeling (Table IGo) was 110.7 ± 2.1 adducts/108 nucleotides (mean ± SD, six measurements). When the same DNA was measured by 32P-postlabeling (Figure 2AGo), BPDE–DNA DELFIA, and BPDE–DNA CIA, the values obtained (Table IGo) were very similar to the radiolabeling value.



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Fig. 1. HPLC analysis of the enzymatic hydrolysate from calf thymus DNA reacted with [3H]BPDE. Profile was obtained from 2 mg of DNA reacted at a molar ratio of 9.43 x 10-6 : 1 of BPDE:DNA nucleotides. The peak at 40 min shows co-chromatography of radiolabeling and UV absorbance.

 

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Table I. DNA adduct levels (per 108 nucleotides) induced in calf thymus DNA modified with [1,3-3H]BPDEa and measured by different methods
 


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Fig. 2. 32P-Postlabeling analysis of: (A) calf thymus DNA modified in vitro with [3H]BPDE as described in Figure 1Go; (B, C, D) liver DNA from mice treated with 0.0, 0.5 or 1.0 mg of [7,8-3H]BP, respectively. The adducts were visualized with a phosphorimager using attenuation factors of 500, 250 and 400, respectively.

 
In vivo modification of mouse liver DNA with [7, 8-3H]BP
A dose–response for binding of BP to mouse liver DNA was obtained with the intraperitoneal injection of 0.5 and 1.0 mg [7,8 -3H]BP into mice (Table IIGo); radiolabeling values were 86.4 and 137.6 BP molecules/108 nucleotides, respectively (23). These numbers reflect the total quantity of radiolabeled BP bound to mouse liver DNA. Both the BPDE–DNA DELFIA and BPDE–DNA CIA determinations showed dose-responses, however, both underestimated the total amount of BP bound to DNA. The DELFIA and CIA detected 5–6% and 12–13% of the total signal, respectively, suggesting that most of the DNA-bound BP radioactivity was not in a form structurally similar to BPdG, or at least recognizable by the BPDE–DNA antiserum. The 32P-postlabeling assay (23) detected 13–16% of the total hepatic DNA-bound radioactivity in the form of the BPdG adduct, which was visualized by thin layer chromatography (Figure 2B, C, D, by comparison with AGo). Overall, the values obtained by the immunoassays and the 32P-postlabeling method were very comparable, and data from all of these assays indicate that much of the DNA-bound radioactivity was in a form structurally different from the BPdG adduct.


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Table II. DNA adduct levels (per 108 nucleotides)a in liver DNA from mice given 0.5 or 1.0 mg [7,8-3H]BP by intraperitoneal injection: comparison of different methods
 
Validation using the MCL-5 cells and Comet assay
MCL-5 cells are a stable multi-competent human lymphoblastoid cell line expressing cytochrome P450s 1A1, 1A2, 2A6, 3A4 and 2E1, as well as epoxide hydrolase (19). The MCL-5 cells were cultured for 24 h in the presence of 4 µM BP and the samples assayed for genotoxicity by two different methodologies. From one set of flasks, DNA was isolated and used for determination of BPdG adducts by the CIA. The data showed values of 5.8 BPdG adducts/108 nucleotides for nuclear DNA and 63.5 BPdG adducts/108 nucleotides for mitochondrial DNA (Table IIIGo). Cells in a second set of flasks were examined by Comet assay, and there was a substantial increase in comet tail length (Figure 3Go and Table IIIGo), accompanied by a decrease in nuclear size, in the BP-exposed cells, compared with the unexposed cells (Table IIIGo). Thus, DNA damage observed as tail lengthening in the Comet assay associated with DNA adduct formation.


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Table III. Determination of BPdG values and Comet assay parameters in MCL-5 cells exposed to 4 µM BP for 24 h
 


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Fig. 3. Comet assay of MCL-5 cells. (A) Unexposed cells and (B) cells exposed for 24 h to 4 µM BP. Values for the decrease in nuclear size and the increase in Comet tail length are presented in Table IIIGo.

 
Determination of BPdG in human lymphocyte DNA
A series of 43 peripheral blood cell (buffy coat) DNA samples, from 25 individuals who were either colorectal adenocarcinoma patients or controls, was obtained from the Navy Colon Adenoma study (28). The sample set (Table IVGo) consisted of 18 duplicates that were received as 36 separate coded samples, and seven single samples from each of seven patients that were submitted without replicates. Each sample was analyzed by BPDE–DNA CIA, using 10 µg of DNA per microtiter plate well and three experimental wells plus one control well per plate. Of the total 43 samples, 38 were assayed twice, as there was insufficient DNA to assay the remaining five samples a second time. For the 38 samples assayed twice, the two assays were performed on different days using separate plates and the correlation between the two assays (Figure 4Go) was excellent (r2 = 0.948). The intra-assay coefficient of variation (CV) for microtiter wells on the same plate was 1.85%. Three samples out of the 43 (7%) were below the limit of detection, and for the 40 measurable samples, values ranged from 0.71 to 2.21 adducts/108 nucleotides.


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Table IV. Human blood cell DNA samples from the Navy Colon Adenoma Study
 


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Fig. 4. Comparison between two different BPDE–DNA CIA values (Analysis 1 and Analysis 2) performed on different microtiter plates and on different days but using DNA from the same tube. The figure shows PAH–DNA adduct values for 38 samples, where the inter-assay correlation coefficient (r2) is 0.948. The units for the ordinate and abscissa are PAH–DNA adducts/1010 nucleotides.

 
Among the total of 43 samples, 36 samples were coded duplicates from 18 individuals provided to us with 36 different numbers as if they were separate samples (Table IVGo). Fifteen pairs of coded duplicates (30 samples) were assayed twice. When the pairs were identified, there were 15 samples each assayed a total of four different times (two coded duplicates with two assays each) on different microtiter plates; the coefficient of variation for these assays was 13.8%. As this is much larger than the intra (three wells on one plate)- or inter (two assays of DNA from the same tube)-assay variation (Figure 4Go), it would appear that the error associated with handling and aliquotting the DNA samples comprises a large source of variability. For three pairs of coded duplicate samples, provided to us as six samples, there was unacceptable variation in the values for the separate-sample duplicates (but not for the replicate assays from the same sample tube). For each of the three pairs, one sample had a measurable value and the second sample was undetectable. When third and fourth assays were performed on one pair of samples, the discrepancy disappeared.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The purpose of this study was two-fold: first to develop a highly sensitive and stable immunoassay (CIA) for the determination of PAH–DNA adducts in human samples, and second to validate the CIA by comparison with previously established DNA adduct detection methods using modified DNA standards and biological samples obtained in vitro and in vivo.

The newly-developed BPDE–DNA CIA appears to be superior to similar immunoassays (16,17), with a level of sensitivity (1.5 BPdG adducts/109 nucleotides) that is usually seen with 32P-postlabeling. The high signal-to-noise ratio gives a consistent assay with a stability that provides a considerable advantage compared with the BPDE–DNA DELFIA. In the DELFIA and previous immunoassays using this antiserum, high backgrounds resulted in variability that was constantly a problem. In contrast, in this assay the standard curves are more reproducible, largely because the problem of high background has been solved. One general disadvantage of the immunoassays, the fact that they require large quantities of DNA, has not been completely eliminated. However, using 10 µg of DNA per microtiter plate well in the BPDE–DNA CIA we found only three out of 43 human samples that gave values below the limit of detection. This constitutes a substantial improvement over the earlier immunoassays that used 35 µg of DNA per microtiter plate well (17), as the total DNA requirement has been reduced from ~250 µg to ~80 µg. The fact that most human samples were in the detectable range for this assay suggests that we may have eliminated the problem of performing studies where a substantial fraction of samples assayed have non-detectable adduct levels. In this set of samples, most of the individuals are not considered to have consistently received high PAH exposures, such as those encountered in some polluted occupational environments, and yet PAH–DNA adducts were measurable in most samples.

The radiolabeled BPDE–DNA modified to 1.1 BPdG adducts/106 nucleotides was prepared as part of an inter-laboratory effort to synthesize DNA standards modified with chemical carcinogens in the range of human biological samples (23). The idea was that many laboratories could use such modified DNA samples as standardization reagents when assaying human DNA by multiple different methods. A previous publication by Beland et al. (15) has described the synthesis and validation of a DNA modified with 4-aminobiphenyl designed to be made available to many laboratories. Other efforts to use standards for 32P-postlabeling assays have been published previously (14,23,31) and attest to the difficulties of such endeavors. For the radiolabeled in vitro-modified BPDE–DNA standard described here we compared the modification levels by radiolabeling, 32P-postlabeling (23), and the two immunoassays, the BPDE–DNA DELFIA and the BPDE–DNA CIA. The radiolabeling value was similar to those obtained using all the other methods, suggesting that good inter-laboratory standardization for PAH–DNA adduct formation is potentially achievable using this calf thymus DNA modified in vitro with BPdG.

To investigate the usefulness of shared experimental model standards, mice were exposed to 0.5 and 1.0 mg of [7,8-3H]-BP, and the DNA modification determined by radiolabeling, BPDE–DNA DELFIA, and BPDE–DNA CIA. These were the same samples that had been shared earlier in the postlabeling trial (23), the data for which are shown in Table IIGo. This experiment was complicated by the fact that much of the radioactivity bound to liver DNA was not in the form of the BPdG adduct and therefore not recognizable by the BPDE–DNA antiserum. In addition much of the radiolabeling was not measurable by 32P-postlabeling, confirming the previous supposition. Unlike mouse skin DNA, which contains mainly different enantiomeric forms of BPdG after topical BP exposure (32,33), the liver appears to metabolize BP to multiple different DNA-adducted forms, only a small fraction of which comprise the BPdG adduct (34,35). Nonetheless, a comparable dose–response was observed for the two different doses by 32P-postlabeling and both immunoassays.

The use of MCL-5 cells in this study has provided an opportunity to compare multiple different genotoxic end points in this unique cell line. MCL-5 cells are human B-lymphoblastoid cells transfected with cDNAs encoding for carcinogen activating enzymes (19). In several previous studies (3638) these cells have been exposed to BP and monitored for genotoxicity using end points that include DNA fragmentation by Comet assay, DNA adducts by 32P-postlabeling, and mutagenesis. In this study the Comet assay data provide a further validation of the BPDE–DNA CIA, as the Comet tail lengths were similar to those previously reported (38). In addition, by comparison with nuclear BPdG adduct levels, 10-fold higher BPdG adduct levels were found in mitochondrial DNA. These data reproduce literature findings (39,40) and give further validation to the BPDE–DNA CIA.

Overall, this study describes the validation of a new, highly-sensitive immunoassay for the determination of PAH–DNA adducts. The major changes, compared with the BPDE–DNA DELFIA, include the use of different microtiter plates, a different blocking agent, more highly-diluted antiserum, and a chemiluminescent end signal. It should be noted that for any immunoassay, the sensitivity depends upon a particular combination of plate, coating, blocking, antibody, and standard, and the sensitivity of this CIA may change if the conditions vary. In this study we have applied and validated the BPDE–DNA CIA using a DNA sample modified with BPDE in vitro, and in vivo-modified samples from mouse liver, cultured human cells and human blood cell DNA. Because the BPDE–DNA CIA is highly sensitive (detecting measurable values in >90% of the samples assayed), and reproducible, it is a promising method for human biomonitoring in molecular epidemiology studies.


    Notes
 
4 To whom correspondence should be addressed at: Carcinogen–DNA Interactions Section, National Cancer Institute, Building 37, Room 4032, NIH, 37 Convent Dr., MSC-4255, Bethesda, MD 20892-4255, USA Email: poirierm{at}exchange.nih.gov Back


    Acknowledgments
 
We greatly appreciate the editorial assistance of Mrs Bettie Sugar.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 24, 2002; revised August 21, 2002; accepted August 27, 2002.