Carcinogenesis vol.21 no.1 pp.107–111, 2000
Human DNA adducts of 1,3-butadiene, an important environmental carcinogen
Chunyan Zhao2, Pavel Vodicka1, Radim J.Sˇra´m1 and Kari Hemminki Center for Nutrition and Toxicology, Department of Biosciences, Karolinska Institute, NOVUM, 141 57 Huddinge, Sweden and 1Institute of Experimental Medicine, Academy of Sciences of Czech Republic, Videnska´ 1083, 142 20, Prague 4, Czech Republic 2To
whom correspondence should be addressed Email:
[email protected] Dedicated to the memory of Anthony Dipple.
The N-1-(2,3,4-trihydroxybutyl)adenine (N-1-THB-Ade) adducts induced by 1,3-butadiene (BD) were analysed from lymphocytes of 15 workers occupationally exposed to BD and 11 controls by 32P-post-labelling using HPLC with radioactivity detection. The difference in the adduct levels between the BD-exposed workers (4.5 ⍨ 7.7 adducts/ 109 nucleotides) and the controls (0.8 ⍨ 1.2 adducts/109 nucleotides) was statistically significant (Wilcoxon rank sum test, P ⍧ 0.038). This study shows for the first time BD-induced DNA adducts in humans and suggests that N-1-THB-Ade adducts may be used to biomonitor human exposure to BD.
Introduction 1,3-Butadiene (BD) is used principally as a monomer in the production of a wide range of polymers and co-polymers (1). It also occurs as an environmental contaminant present in automobile exhaust and cigarette smoke (2,3). The US EPA has estimated that BD is responsible for more cancer cases due to exposure to vehicle emissions than diesel particles, benzene and formaldehyde together (4). BD is a wellestablished carcinogen in rodents (5), while epidemiological studies have revealed associations between occupational exposure to BD and increased risk of leukaemia (6,7). In 1992, a working group of the IARC classified BD as ‘probably carcinogenic to humans’ (5). The formation of DNA adducts by the BD metabolites epoxybutene, diepoxybutane and epoxybutanediol (EBD) is likely to be critical in the carcinogenic process of BD. Thus, quantification of BD-induced DNA adducts in humans would be important for mechanistic considerations and risk assessment. In the past 10 years, a number of studies have reported on BD-induced DNA adducts in vitro and in mice and rats (8–11). However, the available analytical techniques have failed to detect DNA adducts of BD in humans. Using a revised 32P-post-labelling/HPLC-based method with sufficient sensitivity and specificity, we report here for the first time quantification of DNA adducts derived from BD in human samples. Abbreviations: BD, 1,3-butadiene; EBD, epoxybutanediol; N-1-THB-Ade, N-1-(2,3,4-trihydroxybutyl)adenine; N6-THB-Ade, N6-(2,3,4-trihydroxybutyl)adenine. © Oxford University Press
Materials and methods Chemicals Diepoxybutane was obtained from Aldrich Chemical Co. (Milwaukee, WI). [γ-32P]ATP and T4 polynucleotide kinase were from Amersham (Little Chalfont, UK). Salmon testis DNA (sodium salt), prostatic acid phosphatase and snake venom phosphodiesterase were acquired from Sigma Chemical Co. (St Louis, MO). Nuclease P1 was purchased from Boehringer Mannheim (Mannheim, Germany). Methanol of HPLC grade was from J.T. Baker (Deventer, The Netherlands). All other chemicals were of analytical grade and obtained from Sigma or Merck (Darmstadt, Germany). Subjects, lymphocyte preparation and DNA isolation The N-1-(2,3,4-trihydroxybutyl)adenine (N-1-THB-Ade) adducts were determined in 15 male exposed workers from a BD monomer unit in the Czech Republic (mean age ⫾ SD 43.5 ⫾ 11.5) and in 11 male control individuals (mean age 43.2 ⫾ 11.3) from the heat production unit. The mean employment time for exposed workers was 15.3 ⫾ 10.5 years. Cytogenetic biomarkers have been previously studied on the same subjects (individuals lacking enough DNA were not available in this study) (12). The data on BD exposure, age and smoking habits are summarized in Table I. The exposed group consisted of eight smokers and seven non-smokers, while in the control group seven were non-smokers and four smokers. The smoking history exceeded 1 year for all smokers. All subjects in this study were asked to complete a questionnaire which solicited information on smoking, diet, alcohol consumption, etc. as described (12). Any persons with medical treatment, radiography or vaccination within the previous 3 months were not included in the study. Air levels of BD were measured by personal and stationary monitoring for 8 h, one shift before blood sampling. Passive monitors of 3M type 3520 with a back-up section (3M, St Paul, MN) were used. Blood samples of 20 ml were collected in heparinized sterile tubes. Lymphocytes were obtained by centrifugal separation in a Ficol (Pharmacia Chemical Co.) gradient (13). DNA was isolated from the nuclei of lymphocytes by means of enzyme incubation and solvent extraction (14). In vitro reaction of EBD with DNA EBD was prepared by hydrolysis of 200 µl of diepoxybutane in 1.5 ml of water for 3 days at 37°C. Remaining diepoxybutane was extracted with toluene. Salmon testis DNA (2 mg/ml 50 mM Tris–HCl, pH 7.4) was reacted with 100 µl of EBD at 37°C for 24 h. The reaction mixture was extracted with ethylacetate followed by ethanol precipitation of the DNA. The purified DNA was dissolved in water and the concentration of the solution was measured. For determination of the N-1-THB-Ade adduct levels, 200 µg of the purified DNA was depurinated at pH 1 (0.1 M HCl, 70°C, 30 min). The released DNA bases were separated on a Beckman HPLC (Fullerton, CA) coupled with a diode array detector. A 5µ Kromasil 4.6⫻250 mm C18 column was used and run with a gradient starting isocratically from 100% 50 mM ammonium formate, pH 4.6, for 20 min, followed by a linear gradient to 40% methanol over 40 min. The adduct levels were calculated from standard curves obtained by injecting samples with known concentrations of N-1-THB-Ade, synthesized in our previous study (15). Concentrations of standards were determined by UV absorption using the published molar extinction coefficient for N-1-methyladenine (ε ⫽ 11900 at pH 8.8) (16). 32P-post-labelling
and separation N-1-THB-Ade adducts were analysed by a post-labelling procedure for dinucleotides as described by Randerath et al. (17). DNA (10 µg) was hydrolysed with a mixture of nuclease P1 (20 mU/µg DNA) and prostatic acid phosphatase (0.2 µg/µg DNA) at pH 5.2. After incubation at 37°C for 45 min, the reaction was terminated by adding 100 µl of cold ethanol. Proteins were precipitated for 20 min at –20°C. After centrifugation the supernatant was evaporated to dryness. Labelling of adducted dinucleotides was carried out in 2 µl containing 6 U T4 polynucleotide kinase and 14 µCi [32P]ATP. The reaction was carried out at pH 9.6, followed by adding snake venom phosphodiesterase (0.5 mU/µg DNA). The post-labelled sample containing 10 µg of DNA mixed with UV markers of one diastereomeric N-1-THB-5⬘dAMP pair, synthesized in our previous study (15), was injected into a
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Table I. N-1-THB-Ade adduct levels (adducts/109 nucleotides) in lymphocytes of BD-exposed workers Subject
Age
Smoking categorya
Cigarettes/day
BD exposure (mg/m3)b
N-1-THB-Ade
Exposed group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean ⫾ SD
57 50 28 59 23 49 49 24 45 48 38 44 43 44 57
S S S S S S S S NS NS NS NS NS NS NS
15 20 15 40 20 15 2 5
0.118 5.88 0.77 0.97 ⬍0.011 0.53 0.20 0.05 8.7 ⬍0.011 2.96 NRc 17.0 3.08 0.34
0.3 0.5 1.0 0.8 1.0 12.5 0.3 4.3 1.5 ⬍0.1 0.3 18.0 25.0 0.3 1.3 4.5 ⫾ 7.7d
Control group 1 2 3 4 5 6 7 8 9 10 11 Mean ⫾ SD
36 20 31 50 31 54 54 55 44 49 51
S S S S NS NS NS NS NS NS NS
10 20 10 25
0.036 0.038 0.038 0.15 ⬍0.005 0.03 ⬍0.005 ⬍0.006 ⬍0.005 ⬍0.006 ⬍0.006
⬍0.1 ⬍0.1 2.3 3.5 ⬍0.1 ⬍0.1 1.8 0.5 ⬍0.1 0.2 ⬍0.1 0.8 ⫾ 1.2
aS, smoker; NS, non-smoker. bData from S ˇ ra´ m et al. (12). cNR, no record. dSignificance determined by Wilcoxon rank sum test; P adducts/109 nucleotides) was used in the calculations.
⫽ 0.038 compared with control group. For non-detectable adduct levels, the detection limit (0.1
Analysis of results Adduct levels were calculated from the radioactivity of the samples. Recovery of N-1-THB-Ade adducts in the post-labelling assay was estimated from analysis of in vitro EBD-treated DNA with a known concentration of N-1THB-Ade adducts and was used to correct the adduct levels determined in human samples. The Wilcoxon rank sum test was applied for comparison of two samples. Linear multiple regression analysis was used to assess the influence of BD exposure and smoking on the adduct levels. Fig. 1. Base-catalysed rearrangement of N-1-(2,3,4-trihydroxybutyl)deoxyadenosine 5⬘-monophosphate to N6-(2,3,4-trihydroxybutyl)deoxyadenosine 5⬘-monophosphate. R, deoxyribose 5⬘-monophosphate.
Beckman HPLC coupled with a UV and a Beckman 171 radioisotope detector. A 5µ Prodigy 2.0⫻250 mm C18 reversed phase column was used. HPLC fractions containing post-labelled N-1-THB-Ade adducts were collected from 2–5 HPLC runs of parallel samples. Therefore, between 20 and 50 µg DNA were analysed for each sample. The pooled samples were frozen and lyophilized. The dry residue was treated with 0.1 M NaOH at pH 13 (80°C, 30 min), representing conditions under which N-1-THB-Ade is completely converted to N6-(2,3,4-trihydroxybutyl)adenine (N6-THB-Ade) adducts (Figure 1). The corresponding N6-THB-Ade adducts formed by the Dimroth rearrangement were further analysed by HPLC. For collection of N-1-THB-Ade adducts, the column was run isocratically with 100% 50 mM ammonium formate, pH 4.6, for 20 min, followed by a linear gradient of 100% 0.2 M ammonium formate containing 20 mM phosphoric acid, pH 4.6, to 100% methanol over 55 min. For separation of N6-THB-Ade adducts, the column was run with a linear gradient of 99% 0.2 M ammonium formate containing 20 mM phosphoric acid, pH 4.6, to 10% methanol over 45 min and then maintained for 15 min, at which time the N6-THB-Ade adducts eluted. The flow rate was 0.2 ml/min.
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Results HPLC analysis of the post-labelled EBD-treated DNA showed two adduct fractions which were assigned to diastereomeric N-1-THB-Ade adducts, identified by their co-migration with synthesized UV markers of N-1-THB-5⬘-dAMP (Figure 2A). Upon treatment of the collected N-1-THB-Ade adducts with base they were quantitatively converted to N6-THB-Ade adducts (diastereomers co-migrated) (Figure 2B), which were identified by their co-migration with an N6-THB-5⬘-dAMP standard synthesized previously (15). This DNA standard was labelled in parallel with each set of DNA from human lymphocyte samples and used as an external standard for correction of determined adduct level. Based on known adduct levels in EBD-treated DNA, the recovery of N-1-THB-Ade adducts from DNA hydrolysis through all steps of the postlabelling procedure was 40 ⫾ 5% and the recovery of N-1THB-Ade adducts rearranged to N6-THB-Ade adducts was close to 100%.
DNA adducts of 1,3-butadiene
Fig. 2. HPLC chromatograms of 32P-post-labelled EBD-treated DNA (10 µg) analysed with radioisotope and UV detectors. (A) N-1-THB-Ade adducts in EBD-treated DNA, (B) N6-THB-Ade adducts formed by rearrangement of the collected N-1-THB-Ade adducts from (A). The positions of the adducts are indicated with arrows. –, radioactivity; ····, UV. Note that the UV detector was installed 0.6 min after the radioactivity detector.
The interference of background peaks close to the early eluting N-1-THB-Ade adducts makes it difficult to directly detect low levels of adducts in human samples. The sensitivity of the assay could be considerably increased when the adduct fraction was rearranged to the N6 position; the rearranged adduct eluted much later and was free of background radioactivity. Consequently, the quantification of N-1-THB-Ade adducts in human samples was based on the level of the rearranged N6-THB-Ade adducts. Radioactivity peaks, cochromatographying with the UV marker of N6-THB-5⬘-dAMP, were detected in lymphocyte DNA from exposed workers (Figure 3A and B) and some controls (Figure 3C). The identity of the fraction as the N6-THB-Ade adduct was further confirmed by using three different chromatographic systems and by methylation of the adducts by dimethylsulphate. In these tests, the radioactivity peak always followed the N6THB-5⬘-dAMP standard (data not shown). Table I lists BD exposure, smoking habits and N-1-THBAde adduct levels of the subjects. For subjects with nondetectable adducts, a value of 1 adduct/1010 nucleotides (the detection limit) was assigned. It should be noted that BD exposure reflects an 8 h exposure, one shift before blood sampling, and is thus subject to large chance fluctuations. The presence of N-1-THB-Ade adducts was detected in 14 of 15 samples from BD-exposed workers and in five of 11 controls. The difference in the adduct levels between the BD-exposed workers (4.5 ⫾ 7.7 adducts/109 nucleotides) and the controls (0.8 ⫾ 1.2 adducts/109 nucleotides) was statistically significant (Wilcoxon rank sum test, P ⫽ 0.038). Linear multiple regression analysis was used to assess the influence of individual BD exposure and smoking on the levels of N-1-THB-Ade adducts in the 14 exposed subjects (one individual without an exposure record was excluded). The dependent variable was DNA adduct level; individual BD exposure and daily consumption of cigarettes (for non-smokers, 0 was used in the calculation) were independent variables. The analysis showed that
Fig. 3. HPLC chromatograms of 32P-post-labelled human lymphocyte DNA of: (A) exposed worker 13 (20 µg DNA analysed) with an adduct level of 25.0/109 nucleotides; (B) exposed worker 8 (50 µg DNA) with an adduct level of 4.3/109 nucleotides; (C) control 11 (30 µg DNA) with nondetectable adducts. The positions of N6-THB-Ade adducts are indicated with arrows. –, radioactivity, ····, UV. Note that the UV detector was installed 0.6 min after the radioactivity detector.
only BD exposure was significantly related to an increase in N-1-THB-Ade adduct levels (F for slope ⫽ 5.51, P ⬍ 0.05; t ⫽ 3.2, P ⬍ 0.01) rather than to daily consumption of cigarettes, explaining 50% of the total variation. When this analysis was carried out on the whole study population, the results showed that only BD exposure was significantly associated with an increase in N-1-THB-Ade adduct levels (F for slope ⫽ 12.23, P ⬍ 0.001; t ⫽ 4.9, P ⬍ 0.001), explaining 53% of the total variation. When the controls were subdivided into smokers and non-smokers, the adduct levels in four smokers (1.5 ⫾ 1.7) were ~5 times higher than those in seven non-smokers (0.3 ⫾ 0.6). However, the difference was not significant by Wilcoxon rank sum test. Discussion This is the first report identifying BD-induced DNA adducts in humans. Many other end-points have been described elsewhere from the same population (12), but the comparisons between DNA adducts and other parameters are beyond the scope of the present publication. The usefulness of a method in human biomonitoring requires high sensitivity combined with high specificity because the adduct levels are low. The analysis of N-1-THB-Ade adducts has been difficult due to the background products during HPLC separation. However, when the N-1THB-Ade adducts were analysed by collecting the radioactive peaks and converting them to the corresponding N6-THB-Ade adducts by Dimroth rearrangement, the products were well separated. Since HPLC analysis was carried out in two steps, 109
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the adduct fractions from several HPLC runs could be combined and therefore the effective level of detection was boosted to ~1 adduct/1010 nucleotides. The specificity of the method was achieved by using fully structurally characterized standards of the adducts as internal UV markers. Thus, the 32P-postlabelling/HPLC methodology provided a robust and sensitive means of quantifying the N-1-THB-Ade adducts in humans. The N-1-THB-Ade adducts in lymphocyte DNA samples obtained from BD-exposed workers were detected in 14 of 15 exposed workers at levels of 0.3–25.0 adducts/109 nucleotides. There was an apparent positive correlation between individual exposure to BD and adduct level but it was mainly due to one individual (no. 13). Occupational exposure to BD is characterized by transient peak exposures and a detailed assessment of exposure would require personal monitoring over an extended period of time (1). Although the determination of the BD exposure covers only the last shift prior to blood sampling and virtually no data are available on exposure history, the significant correlation between DNA adducts and workplace concentrations of BD indicated a predominant role of BD exposure in DNA adduct formation. Because of uncertainties concerning BD exposure assessment and halflives of the N-1-THB-Ade adducts in lymphocytes, the observed correlation is probably an underestimate. In previous studies, trihydroxybutyl haemoglobin adducts have been found in BD-exposed rats and humans and the levels exceeded those of epoxybutene adducts (18). Trihydroxybutyl DNA adducts are also the major adducts in mice and rats (11). These results suggest that EBD may be an important proximal metabolite of BD-induced carcinogenesis. An alternative pathway is via direct attack by diepoxybutane and subsequent hydrolysis of the remaining epoxide. So far, data on the half-life and repair of N-1-THB-Ade adducts in vivo are not available. In this study, we could not directly analyse N6-THB-Ade adducts because of the presence of interfering background peaks close to the retention time of this product. According to a recent study on propylene oxide, the half-life of N-1-alkyladenine disappearance from doublestranded DNA was 9.2 days in vitro at neutral pH (19). However, in an in vivo experiment, no decrease in the N-1 adduct of propylene oxide was observed in rat liver 3 days after cessation of exposure, suggesting that the adduct is reasonably stable in vivo (19). As a corollary, these findings suggest that N-1-THB-Ade adducts may be the major initial products in vivo. Dimroth rearrangement of this product could be the source of N6-THB-Ade adducts previously detected in vivo (11). Further studies on the persistence of N-1-THBAde adducts in humans are required to estimate the important determinant of BD-mediated carcinogenesis. In this study, N-1-THB-Ade adducts were detected in five of 11 controls at levels of 0.2–3.5 adducts/109 nucleotides. The presence of haemoglobin adducts of EBD and epoxybutene was also observed in control workers (19,20). Since the control cohort was recruited in the same plant, occasional visits to BD-contaminated areas of the plant cannot be excluded. In our previous work N-1-THB-Ade adducts were detected in the liver of rats exposed to 300 p.p.m. [conversion factor (mg/m3) ⫽ 2.21⫻p.p.m.] BD for 5 days (6 h/day) by inhalation (15). The adduct level was found to be 11 adducts/109 nucleotides (corrected by recovery obtained in the present study). In comparison, the present subjects, who were exposed to an ~200-fold lower concentration, had adduct levels only 50% less that those found in rat liver. However, such crude 110
comparisons do not take into account non-linear dose–response relationships, such as metabolic saturation processes, nor do they consider half-lives of adducts, duration of exposure and steady-state levels of adducts, which are reached in chronic human exposure but probably not in the short-term animal experiment. Smoking is another important source of exposure to BD. It has been estimated that the amount of BD in the mainstream smoke of 30 cigarettes corresponds to an occupational exposure of 0.22 mg/m3 for 8 h at work (20). Our results on the control subjects were in line with this estimation: smokers had higher adduct levels than non-smokers, but the difference was not statistically significant. The effect of smoking could not be measured among the exposed because of the confounding effect of occupational exposure. In conclusion, this study demonstrated the presence of N-1THB-Ade adducts in humans. N-1-THB-Ade adducts are chemically stable and efficiently labelled in the post-labelling assay, which recommends it as a biomarker of BD exposure. Acknowledgements We thank Dr Chuanhui Dong for help in statistical analysis. This study was supported by the Swedish Council for Work Life Research.
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