32P-post-labelling of 7-(3-chloro-2-hydroxypropyl)guanine in white blood cells of workers occupationally exposed to epichlorohydrin

Kamila Plna2, Siv Osterman-Golkar1, Erika Nogradi and Dan Segerbäck

Center for Nutrition and Toxicology, Department of Biosciences, Karolinska Institute, Novum, S-141 57 Huddinge and
1 Department of Molecular Genome Research, Stockholm University, S-106 91 Stockholm, Sweden


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epichlorohydrin (ECH) is a simple 3-carbon epoxide of industrial importance. It has been shown to be genotoxic in several systems and carcinogenic in experimental animals. The aim of the present investigation was to study DNA adducts of ECH as a biomarker of occupational exposure to this chemical. 7-(3-Chloro-2-hydroxypropyl)guanine (7-CHP-guanine) was analysed in DNA from white blood cells using an anion exchange-based adduct enrichment protocol of the 32P-post-labelling/HPLC-based assay. Blood samples were collected from seven workers handling ECH (exposed), nine workers not handling ECH but normally present in the premises where this chemical is used (potentially exposed) and 13 office and factory workers from locations in the plant where ECH is not handled (controls). 7-CHP-guanine was detected in five of the seven workers exposed to ECH (1.6–7.1 mol/109 mol nucleotides) and in two of the nine workers potentially exposed to ECH (0.8–1.5 mol/109 mol nucleotides). This adduct was not detected in any of the 13 controls. The difference in adduct levels between exposed workers and controls was statistically significant (Mann–Whitney test, P < 0.001), as was the difference between exposed workers and potentially exposed workers (P = 0.017). The recovery of 7-CHP-guanine in the 32P-post-labelling assay was on average 48 ± 7%, which is considerably higher than previously reported for other 7-alkylguanines. The method used had a limit of detection of ~0.4 mol adduct/109 mol nucleotides using 20 µg DNA. This study shows for the first time ECH-induced DNA adducts in humans and suggests that 7-CHP-guanine may be used as a biomarker of occupational exposure to ECH.

Abbreviations: CHP, 3-chloro-2-hydroxypropyl; DHP, 2,3-dihydroxypropyl; ECH, epichlorohydrin; ESI-MS, electrospray ionization mass spectrometry.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epichlorohydrin (ECH) is extensively used in the manufacture of epoxy resins and for the production of synthetic glycerine and glycidyl ethers. There is no known environmental or endogenous source of ECH. The most likely route of exposure is by inhalation, although there is a possibility of dermal and oral absorption. Cells in blood samples of workers occupationally exposed to ECH were shown to have significantly higher numbers of chromosomal aberrations, in particular, there was an increase in chromatid and chromosomal breaks as compared with controls (1). In another study significant differences in sister chromatid exchanges and high frequency cells were found in workers occupationally exposed to ECH when compared with controls. In the same study the average haemoglobin adduct level was higher in the exposed than in the non-exposed donors, although the difference was not significant (2).

In experimental test systems ECH has been reported to induce mutagenic as well as clastogenic effects (37). ECH has been shown to be a carcinogen in animals (8,9) and it is classified by the IARC as probably carcinogenic to humans (10). Administration of ECH by gavage (11) or orally (12) to Wistar rats resulted in a high incidence of squamous cell carcinomas in the forestomach, while inhalation exposure to ECH produced nasal tumors (9).

ECH is a bifunctional directly acting alkylating agent, which besides the reactive epoxy group also contains a chlorine atom. The main products of reaction of ECH with macromolecules in cells are formed by attack on the epoxide ring resulting in 3-chloro-2-hydroxypropyl (CHP) adducts. Because of the OH group in the ß-position the electrophilic reactivity of the carbon attached to the chlorine is highly dependent on pH. Hydrolysis of CHP adducts results in formation of 2,3-dihydroxypropyl (DHP) adducts. The latter type of adduct is also formed by glycidol, another low molecular weight epoxide. Formation of DHP-haemoglobin adducts has been demonstrated in ECH-exposed rats and humans (2,13). In DNA, ECH reacts mainly with the 7-position of guanine (Figure 1Go), in analogy with other small alkylating agents (14). Upon treatment of calf thymus DNA in vitro with ECH only 7-CHP-guanine was detected, although other adducts were also formed in reactions with different deoxyribonucleosides (15). Formation of 7-CHP-guanine was studied in female Wistar rats exposed to ECH (16). Since alkylation at the 7-position of guanine adds a positive charge to the structure these products are relatively polar and labile to secondary modifications such as spontaneous depurination (17).



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Fig. 1. 7-(3-Chloro-2-hydroxypropyl)deoxyguanosine 5'-monophosphate (7-CHP-5'-dGMP). R, deoxyribose 5'-monophosphate.

 
7-Substituted guanines are the predominant DNA adducts formed from most alkylating agents and are therefore usually a first choice as markers of exposure to such chemicals. 7-Substituted guanines are not expected to be genotoxic per se, but the apurinic sites formed can be miscoding lesions, inducing mainly G->T transversions (18). Other adducts might be biologically more significant, but if present at low levels difficult to analyse. We report here the quantification of 7-CHP-guanine in white blood cells of workers occupationally exposed to ECH using an anion exchange-based adduct enrichment protocol of the 32P-post-labelling/HPLC-based assay (1922).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
ECH (>99.5%) and glycidol, used for preparation of adduct standards, were obtained from Fluka Chemie (Buchs, Switzerland). [{gamma}-32P]ATP (sp. act. >5000 Ci/mmol) and T4 polynucleotide kinase were from Amersham (Little Chalfont, UK). All nucleotides used for preparation of standards, salmon testes DNA, alkaline phosphatase and micrococcal nuclease were from Sigma Chemical Co. (St Louis, MO). Spleen phosphodiesterase and nuclease P1 were obtained from Boehringer Mannheim (Mannheim, Germany). Methanol and 1 ml Bakerbond anion exchange cartridges were from J.T. Baker (Deventer, The Netherlands). All other chemicals were of analytical grade and obtained from Sigma or Merck Chemical Co. (Darmstadt, Germany).

Reaction with nucleotides
3'-dGMP or 5'-dGMP (1–3 mg) was reacted with 200 mM ECH at pH 8.3 and room temperature for 20 h. The modified nucleotides were isolated by HPLC and were characterized by UV and mass spectrometry and the ring-opening reaction. 7-DHP-dGMP was prepared by reaction of 5'-dGMP with glycidol.

Mass spectrometric analysis
Electrospray ionization mass spectrometry (ESI-MS) was performed using a Finnigan LCQ LC/MSn system equipped with an ion trap mass analyser. The spectra were obtained in the negative ion mode. The samples, dissolved in an eluent of water/acetonitrile/NH3 (49:50:1%), were applied by loop injection (10 µl) into the running solvent from the syringe pump, at a flow rate of 0.1 ml/min. For MS/MS experiments precursor ions were selected in the ion trap analyser and fragmented. The full scan data were acquired for m/z 70–1000 and for MS/MS data m/z 70–500. Electrospray voltage was 5.0 kV and capillary temperature 250°C.

The ESI-MS spectrum of dGMP 7-substituted with ECH showed a deprotonated molecular ion [M-H] at m/z 438, confirming formation of a mono-alkylated nucleotide with an alkyl group, -CH2-CH(OH)-CH2Cl. The MS/MS experiment using m/z 438 as the parental ion showed characteristic fragments at m/z 402, 241.9, 206.2 and 195. The fragment m/z 402 was interpreted as a fragment formed after loss of the chlorine atom ([M-H-Cl]). The fragments m/z 241.9 and 206.2 corresponded to the CHP-guanine part of the molecule and the same fragment after loss of the chlorine atom, respectively. The fragment m/z 195 corresponds to deoxyribose with a phosphate group.

In vitro reaction with DNA
Salmon testes DNA (2 mg/ml 20 mM Tris–HCl, pH 7.4) was incubated with 40 mM ECH at 37°C for 19 h. The reaction mixture was extracted twice with ethyl acetate and precipitated with ethanol. 7-CHP-guanine was released from DNA by selective depurination (pH 6.0, 100°C, 30 min). Residual DNA was precipitated by ethanol and after centrifugation the supernatant was transferred to a new tube. The solvent was evaporated in a Savant Speed Vac centrifuge and the redissolved residue analysed by HPLC. The eluate was monitored by UV (280 nm) and the amount of 7-CHP-guanine quantified from a standard curve, obtained by injection of dilutions of a 7-(2-hydroxypropyl)guanine standard of known concentration (21).

To study the stability of 7-CHP-guanine in DNA, ECH-treated salmon testes DNA (1.0 mg/ml) was incubated at 37°C in 20 mM Tris–HCl, pH 7.4. Aliquots of 0.1 ml were removed after 0, 8, 24, 48, 72, 96, 120 and 144 h and DNA precipitated with ethanol. The washed and dried precipitates were analysed for 7-CHP-guanine (and 7-DHP-guanine) as described above.

Samples collection and preparation
Blood samples of 3–5 ml were collected in November 1997 from 29 employees of an ECH plant in Sweden. In this factory ECH is primarily used for production of resins and adhesives for industrial applications. ECH arrives at the plant in railway wagons and is transferred to storage containers. From these it is pumped into a closed reactor system where the resin is prepared. The final product contains <0.001% ECH. Potential exposures are primarily during transfer from the railway wagons, during reactor operations and during servicing of the plumbing, etc. (exposure category A). Since these operations are performed on an irregular basis most exposures are likely to be intermittent. Among the workers seven were handling ECH (exposed; mean age ± SD 46 ± 12 years), nine were not handling ECH but were normally present in the premises where this chemical is used (potentially exposed; mean age ± SD 43 ± 11 years) and 13 were office and factory workers from locations in the plant where ECH is not handled (controls; mean age ± SD 46 ± 8 years). Smokers were represented in all groups and 25% of the workers were women. Occasional personal and stationary monitoring of ECH over several years had indicated air levels from non-detectable (<0.01 p.p.m.) to 0.5 p.p.m. When transferring ECH from the railway wagons to storage tanks air levels of up to 2.1 p.p.m. were recorded, but workers involved in those operations wear protective clothing and gas masks. The occupational exposure limit for ECH in Sweden is 0.5 p.p.m. (time-weighted average).

Nuclei were prepared from blood samples and DNA isolated by means of enzyme incubation and solvent extraction (23). In order to minimize spontaneous depurination during DNA preparation, the pH of all solutions was 8.0 and all steps except the enzymatic incubations were performed on ice and a refrigerated centrifuge was used. By taking these precautions the expected adduct loss was <1%. DNA concentration was measured by UV absorption at 260 nm using 20 A = 1 mg/ml. RNA contamination was checked by HPLC after digestion to deoxyribonucleosides (24). Following digestion samples were separated by HPLC with UV detection at 254 nm. The ratio adenosine: deoxyadenosine was used as a measure of RNA contamination and was considered to be sufficiently low if it was <1%. All samples were coded prior to analysis and each sample was analysed at least twice.

32P-post-labelling
Analysis of 7-CHP-guanine in ECH-treated DNA by the 32P-post-labelling assay was carried out as previously described for the 7-substituted guanine of propylene oxide (21) with some modifications. Briefly, 10 µg of DNA was dissolved in 6 µl of 0.5 mM Bicine, 3 µM CaCl2, pH 9.0, containing 400 mU micrococcal nuclease and incubated for 2 h at 37°C. Spleen phosphodiesterase (48 mU in 6 µl of water) and 2 µl of 20 mM ammonium acetate, pH 5.0, were added and the incubation continued for another 40 min. The digest was evaporated to dryness in a vacuum centrifuge, redissolved in 200 µl of 5 mM ammonium formate buffer, pH 5.2, and purified from normal nucleotides by anion exchange chromatography (21).

Adduct standards and column-enriched adduct-containing fractions from digested DNA samples were 32P-labelled by 1 h incubation at 37°C with 14 µCi of [32P]ATP and 6 U polynucleotide kinase in a total volume of 2 µl of labelling buffer (20 mM CHES, pH 9.6, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM spermidine). To this was added 0.5 µl of 1 M ammonium formate, pH 4.6, and labelled bisphosphates were dephosphorylated by incubation with nuclease P1 (2.5 µg in 0.5 µl of 1.5 mM ZnCl2) for 15 min at 37°C. 32P-labelled 7-CHP-5'-dGMP was separated from residual ATP, residual normal nucleotides and background products by HPLC with an on-line radioisotope detector. A synthesized UV marker (7-CHP-5'-dGMP) was added to all samples prior to HPLC analysis and monitored by UV absorption at 260 nm.

HPLC separations
The previously described Beckman HPLC system (21) was used for all separations. A 4µ Genesis 4.6x250 mm C18 reversed phase column (Jones Chromatography, Mid Glamorgan, UK) was run with a linear gradient of 100% 50 mM ammonium formate, pH 4.65, to 15% methanol over 30 min (system A) for separation of dGMP standards or depurinated DNA.

For separation of 32P-labelled samples a Beckman 171 radioisotope detector was connected to the HPLC system (21). A pre-column filter and a 5µ Kromasil 2.0x100 mm C18 pre-column (Phenomenex, Torrace, CA) were installed in front of the analytical column (5µ Luna 2.0x250 mm C18 reversed phase column; Phenomenex). Inorganic phosphate, residual ATP as well as labelled residual nucleotides were separated from adducts on the pre-column by diverting the first 3.8 ml (19 min) to the waste using a four-port switching valve (Valco Instruments, Houston, TX). The HPLC system was first run isocratically with 99% 0.5 M ammonium formate containing 20 mM phosphoric acid (pH 4.6) and 1% methanol for 21 min, followed by 98% 0.5 M ammonium formate containing 20 mM phosphoric acid and 1.3% ion-pairing agent triethylamine (pH 8.2) for 45 min and finally a linear gradient to 100% methanol for 15 min (system B).

Analysis of results
The efficiency of the HPLC on-line radioisotope detection was determined by injecting known amounts (measured by scintillation counting) of [32P]ATP. Recovery in the post-labelling assay was estimated from analysis of an ECH-modified salmon testes DNA sample with a known level of 7-CHP-guanine (see above). This DNA standard (diluted 1300-fold with unmodified DNA) was labelled in parallel with each set of DNA from human blood samples and used as an external standard for correction of determined adduct levels. The Mann–Whitney two sample test was used for comparison of the ECH exposure categories.


    Results
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 Materials and methods
 Results
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 References
 
Reaction with nucleotides
Following HPLC separation of the reaction products between ECH and 5'- or 3'-dGMP two major peaks of the same amplitude and identical UV spectra were observed (not shown). When these, presumably diastereomeric, products were depurinated (pH 6.0, 100°C, 30 min) and separated by HPLC a single peak was detected, which displayed a UV spectrum typical for 7-alkylated guanine. Furthermore, the dGMP products exhibited UV spectra at different pH values characteristic of 7-alkylated dGMP and treatment with base indicated formation of ring-opened derivatives (17,25). ESI-MS data confirmed the expected molecular weight of the major products formed in the reaction of ECH with 5'-dGMP. It was therefore concluded that the isolated products were indeed the expected 7-CHP-5'- and 7-CHP-3'-dGMP. 7-DHP-5'-dGMP, prepared by reacting glycidol with 5'-dGMP, was purified and characterized in the same way. This product was not detected after reaction of 5'-dGMP with ECH.

In vitro reaction with DNA
When ECH-reacted DNA was depurinated at pH 6 and analysed by HPLC, one major peak not present in the untreated DNA was observed (Figure 2Go). This product and synthetically produced 7-CHP-guanine had identical UV spectra and HPLC retention times. The level of 7-CHP-guanine in the ECH-treated DNA (8.5 mol/103 mol nucleotides) was determined from HPLC analysis of depurinated duplicate samples of 100 µg. One additional peak not present in control DNA was observed. This peak had the same retention time and UV spectrum as synthetic 7-DHP-guanine (Figure 2Go). The level of this adduct amounted to a few percent of 7-CHP-guanine.



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Fig. 2. HPLC separation (system A) of bases released from partly depurinated epichlorohydrin-treated DNA.

 
The rate of spontaneous depurination of 7-CHP-guanine from ECH-treated DNA was tested by incubation at pH 7.4 (20 mM Tris–HCl) and 37°C. The found half-life for depurination was 72 h. In this experiment, no additional formation of the secondary product 7-DHP-guanine was observed during incubation for up to 144 h.

32P-post-labelling
32P-post-labelling and HPLC separation of ECH-treated DNA after adduct enrichment on anion exchange cartridges revealed two major peaks not present in the untreated DNA (not shown). These peaks co-eluted with the UV traces of each of the two diastereomers of 7-CHP-5'-dGMP and they were not detected when the enriched adduct fraction was heated (pH 5.2, 100°C, 30 min) prior to labelling. The sensitivity of the HPLC analysis with on-line radioisotope detection was improved if normal nucleotides were separated from 7-CHP-5'-dGMP on the pre-column and diverted to the waste. This resulted in a decrease in the baseline radioactivity. However, using this method the first eluting diastereomer of 7-CHP-5'-dGMP went to the waste and only the last eluting diastereomer could be analysed (Figure 3AGo).



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Fig. 3. HPLC separation (system B) of 32P-post-labelled DNA: (A) treated in vitro with epichlorohydrin; (B) from a control rat liver; (C) from a rat exposed to epichlorohydrin. The position of 7-CHP-5'-dGMP is indicated by an arrow. —, radioactivity; ––-, UV.

 
The recovery of 7-CHP-guanine from ECH-modified salmon testes DNA (external standard) was 48 ± 7% and the labelling efficiency of the standard 7-CHP-3'-dGMP was 75 ± 10%. The same DNA was also diluted stepwise with unmodified DNA to investigate the sensitivity of the method, which was estimated to be 0.4 mol adduct/109 mol nucleotides using 20 µg DNA.

Analysis of liver DNA from an ECH-exposed rat (i.p. injection, 90 mg ECH/kg body wt; ref. 16) showed a radioactive peak co-eluting with the UV trace of 7-CHP-5'-dGMP (Figure 3CGo). The radioactivity present in this peak corresponded to an adduct level of 1.5 mol/107 mol nucleotides. 7-CHP-guanine was not detected in liver DNA of an unexposed rat (Figure 3BGo).

Data on 7-CHP-5'-dGMP levels in the human samples are presented in Table IGo together with ECH exposure categories, smoking habits and the time of sample collection (in relation to the last work shift). A radioactive peak co-eluting with the UV marker of 7-CHP-5'-dGMP was detected in ECH-exposed workers (Figure 4A and BGo), but not in controls (Figure 4DGo). The identity of the adduct was further confirmed by heating the sample prior to labelling, which resulted in loss of the radioactive peak, consistent with depurination of 7-substituted dGMP (Figure 4CGo).


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Table I. Levels of 7-(3-chloro-2-hydroxypropyl)guanine (mol/109 mol nucleotides) in workers occupationally exposed to epichlorohydrin
 


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Fig. 4. HPLC separation (system B) of 32P-post-labelled DNA from white blood cells of: (A) subject 3, an epichlorohydrin-exposed worker; (B) subject 2, an epichlorohydrin-exposed worker; (C) subject 2, an epichlorohydrin-exposed worker (sample depurinated before labelling); (D) subject 25, a control person. The position of 7-CHP-5'-dGMP is indicated by an arrow. —, radioactivity; ––-, UV.

 

    Discussion
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 Materials and methods
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This is the first report on detection of ECH-induced DNA adducts in humans. 7-Alkylguanine is the major type of adduct found after reaction of ECH, as well as of most other simple alkylating agents, with DNA in vitro (15) and it was therefore a logical choice for analysis of occupational exposure to ECH. We have previously used the post-labelling assay for analysis of 7-alkylguanines of allylglycidyl ether (20) and propylene oxide (21). The specificity of the method was achieved by using structurally characterized 7-CHP-5'-dGMP as an internal UV standard.

7-Alkylguanines are prone to depurination which could cause problems in analysis of such adducts at the nucleoside or nucleotide level (26,27). In DNA, under physiological conditions, 7-alkylguanines are relatively stable and the half-life for 7-CHP-guanine in DNA (72 h) is in the same range as has been determined for other adducts at guanine N-7 (21,2831).

At high pH, because of the hydroxy group, -CH2-CH(OH)-CH2Cl is expected to undergo hydrolysis, although under physiological conditions the process is slow. During incubation for 1 h at 37°C and pH 9.3–9.6 (conditions used for the T4 polynucleotide kinase reaction step in the 32P-post-labelling assay) we observed an ~7% loss of the standard 7-CHP-3'-dGMP due to conversion to 7-DHP-3'-dGMP. However, this secondary product was not observed after incubation of 5'-dGMP with ECH at pH 8.3 and room temperature for 20 h. In a previous study, a half-life of 213 h was estimated for hydrolysis of 3-CHP-deoxyuridine to yield 3-DHP-deoxyuridine under physiological conditions (15). The depurination of 7-CHP-guanine in DNA under physiological conditions is a much faster process than formation of 7-DHP-guanine. Therefore, 7-CHP-guanine is expected to be the predominant adduct in vivo.

The recovery of 7-alkylguanines in the post-labelling assay is in the range 10–20% using the original protocol for DNA digestion (20,21,32,33). A similar recovery was initially found for ECH and since the labelling efficiency of the standard 7-CHP-3'-dGMP was high (75%), most losses during the post-labelling procedure probably occurred as a consequence of spontaneous depurination during DNA digestion and cartridge enrichment. Therefore, a modified digestion protocol was introduced, with shorter incubation time and increased concentration of spleen phosphodiesterase. With this new protocol complete hydrolysis of DNA was obtained and the recovery of 7-CHP-guanine was increased ~2-fold (to 48%). The new protocol was used to analyse the concentration of 7-CHP-guanine in liver DNA of a rat exposed to ECH. The adduct level found (1.5 mol/107 mol nucleotides) corresponded well with the concentration (1.8 mol/107 mol nucleotides) found in a previous study, where 7-CHP-guanine in a DNA sample from pooled tissues (mainly liver) was analysed using 3H-radiochromatography (16).

7-CHP-guanine was detected in DNA from white blood cells in five of seven workers handling ECH (exposed), in levels ranging from 1.6 to 7.1 mol/109 mol nucleotides. The adduct was not found in any of the 13 controls. However, 7-CHP-guanine was detected in two of nine workers not handling ECH (potentially exposed), but at somewhat lower levels than in the exposed group (0.8–1.5 mol/109 mol nucleotides). Since these workers are normally present at the premises where this chemical is used, exposure to ECH cannot be excluded. Smoking habit had no effect on adduct formation. The difference in adduct levels between exposed workers and controls was statistically significant (Mann–Whitney test, P < 0.001) as well as the difference between exposed workers and potentially exposed workers (P = 0.017). The fact that this adduct was not detected among the controls supports the hypothesis that there are no other sources of ECH than occupational. In a previous study, N-DHP-valine in haemoglobin was used as a biomarker of ECH in workers (2). The possibility of detecting exposure by means of this adduct was limited by a relatively high background of unknown origin (2).

Most blood samples from workers of exposure category A and about half of the samples from category B were collected during or directly following the last work shift (Table IGo). With just a single sample from each worker and not knowing if they were exposed or not during the last shift it is difficult to judge if the adducts analysed originate from past or recent exposures. However, 7-CHP-guanine is expected to be lost due to spontaneous depurination (t1/2 72 h) and, possibly, repair and therefore the measured levels of 7-CHP-guanine probably reflect relatively recent exposures (probably within the last 2 weeks).

The only other 7-alkylguanines measured so far in humans are 7-methyl- and 7-(2-hydroxyethyl)guanine. Using the 32P-post-labelling assay, levels of 7-methyl and 7-(2-hydroxyethyl)guanine were determined in white blood cells of non-smokers and smokers (33,34). 7-Methylguanine ranged between 180 and 350 and 190 and 420 mol/109 mol nucleotides in non-smokers and smokers, respectively, and 7-(2-hydroxyethyl)guanine ranged between 21 and 81 and 28 and 97 mol/109 mol nucleotides in non-smokers and in smokers, respectively. In comparison with these values, the levels of 7-CHP-guanine detected in this study (0.8–7.1 mol/109 mol nucleotides) are low. However, due to the high recovery in the assay and the lack of detectable background levels it was possible to analyse the very low concentrations of this adduct.

In conclusion, this study demonstrates for the first time the presence of 7-CHP-guanine in ECH-exposed workers. 7-CHP-guanine is one of a few specific DNA adducts detected in human tissues and this study is one of a few where increased adduct levels have been reported in an occupational environment, if not counting exposure to polycyclic aromatic hydrocarbons. Because of the high assay sensitivity and the absence of background levels, 7-CHP-guanine could be a suitable marker for monitoring occupational exposure to low levels of ECH.


    Acknowledgments
 
Dr Mikko Koskinen is acknowledged for performing the mass spectrometric analyses. This work was financially supported by the Swedish Council for Work Life Research.


    Notes
 
2 To whom correspondence should be addressed Email: kamila.plna{at}cnt.ki.se

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    References
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 Abstract
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 Materials and methods
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 References
 

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Received July 12, 1999; revised September 29, 1999; accepted October 1, 1999.





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