Carcinogenesis vol.22 no.7 pp.1015–1018, 2001
ACCELERATED PAPER
Myeloperoxidase – 463A variant reduces benzo[a]pyrene diol epoxide DNA adducts in skin of coal tar treated patients
Margarita Rojas, Roger Godschalk, Kroum Alexandrov, Ingolf Cascorbi1, Erik Kriek2, Judith Ostertag3, Frederik-Jan Van Schooten4 and Helmut Bartsch5 German Cancer Research Center (DKFZ), Division of Toxicology and Cancer Risk Factors, PO Box 101949, D-69009 Heidelberg, Germany, 1Ernst Moritz Arndt, University Institute of Pharmacology, Medical Faculty, Friedrich-Loeffler-Strasse 23d, D-17487 Greifswald, Germany, 2Straat van Gibraltar 32, NL-1183 GW Amstelveen, The Netherlands, 3University Hospital Maastricht (AzM), Department of Dermatology, PO Box 5800, NL6202 AZ Maastricht, The Netherlands and 4Maastricht University, Department of Health Risk Analysis and Toxicology, PO Box 616, NL-6200 MD Maastricht, The Netherlands 5To
whom correspondence should be addressed Email:
[email protected]
The skin of atopic dermatitis patients provides an excellent model to study the role of inflammation in benzo[a]pyrene (BaP) activation, since these individuals are often topically treated with ointments containing high concentrations of BaP. In this study we have determined, by HPLC with fluorescence detection, the BaP diol epoxide (BPDE)–DNA adduct levels in human skin after topical treatment with coal tar and their modulation by the –463G→A myeloperoxidase (MPO) polymorphism, which reduces MPO mRNA expression. BPDE–DNA adduct levels were 2.2 and 14.2 adducts/108 nt for MPO–463AA/AG and –463GG, respectively. The predominant BaP tetrol observed was tetrol I-1, which is derived after hydrolysis of the antiBPDE–DNA adduct. The tetrol I-1/II-2 ratio, corresponding to the anti/syn ratio, was 6.7. The 32P-post-labeling assay was also performed and thin layer chromatograms showed a major spot with a chromatographic location corresponding to BPDE–DNA. The mean values of the BPDE–DNA adduct spots were 3.8 ⍨ 2.4 per 108 nt for MPO–463AA/ AG (n ⍧ 3) and 18.4 ⍨ 11.0 per 108 nt for MPO–463GG (n ⍧ 7), respectively (P ⍧ 0.03). One individual with the homozygous mutant genotype (–463AA) even had a 13-fold lower adduct level (1.4 per 108 nt) as compared to MPO– 463GG subjects. In conclusion, these data show for the first time: (i) the in vivo formation of BPDE–DNA adducts in human skin treated with coal tar; (ii) that the MPO– 463AA/AG genotype reduced BPDE–DNA adduct levels in human skin. Introduction There is increasing evidence that the formation of tumorigenic benzo[a]pyrene (BaP) 7,8-diol-9,10-epoxide (BPDE)–DNA adducts is modulated by polymorphisms in genes that encode for carcinogen metabolizing enzymes (1–4). The same genes were found to modulate the risk for developing lung and head and neck cancer in smokers (5). Four recent studies showed Abbreviations: BaP, benzo[a]pyrene; BPDE, benzo[a]pyrene diol epoxide; HPLC–FD, high performance liquid chromatography with fluorescence detection; MPO, myeloperoxidase; PAH, polycyclic aromatic hydrocarbon; ROS, reactive oxygen species. © Oxford University Press
that the mutant myeloperoxidase (MPO) genotype plays a protective role against lung cancer (6–9). In the case of a frequent MPO–463G→A polymorphism (allelic frequency in Caucasians 21.5%; (7)), the expression of MPO mRNA is strongly reduced in carriers of the mutated allele (MPO– 463AA/AG), leading to reduced MPO activity (10). MPO is an enzyme primarily found in the lysosomes of neutrophils. It generates hypochlorous acid providing microbicidal activity (11) and recruitment of neutrophils into human tissues due to inflammatory stimuli results in the release of free radicals. BaP activation occurs mainly via the sequential oxidation to BaP 7,8-oxide, BaP 7,8-diol and BPDE, the ultimate carcinogenic metabolite. The drug metabolizing enzymes CYP1A1, CYP2C9 and CYP3A4 (12,13) are known to mediate these processes, but the epoxidation of BaP 7,8-diol to BPDE is also affected by peroxyl free radicals, generated by prostaglandin H synthase or MPO (14,15). Thus, MPO activity may be implicated in the activation of BaP to BPDE, in particular in organs that contain relatively low levels of cytochrome P450s. Consequently, reduced MPO activity in mutant MPO (MPO–463AA/AG) subjects would decrease the formation of BPDE upon exposure to BaP and therefore BPDE–DNA adduct levels are expected to be less in MPO–463AA/AG individuals as compared with those with the wild-type (MPO–463GG) genotype. To confirm this hypothesis, we investigated skin samples from patients with atopic dermatitis, in which a strong inflammatory response in the skin is involved. During therapeutic treatment with coal tar ointments these individuals are highly exposed to polycyclic aromatic hydrocarbons (PAHs), particularly BaP. Indeed, high levels of PAH-related DNA adducts were previously detected in the skin of these treated patients using the 32P-post-labeling assay (16). This patient group thus offers a unique opportunity to study the impact of inflammatory conditions and the role of the MPO genetic polymorphism on the in vivo metabolic activation of BaP. Materials and methods Study population and treatment Three male and seven female patients diagnosed as suffering from atopic eczema (age 34 ⫾ 5 years, range 18–52 years) were treated with coal tar ointments for 21 ⫾ 9 days (range 7–33 days), covering 20–86% of their body surface (16). Treatment started twice a day with 3% coal tar (coal tar type, pix lithantracis) in petrolatum or in 10% zinc oxide plus 90% petrolatum for the first 2–3 days. In all cases this treatment was well tolerated and was intensified by applying 5% coal tar in petrolatum/zinc oxide-petrolatum twice a day for another 2–3 days. Subsequently a 10% coal tar ointment was applied. The BaP content of these ointments was 52 mg/kg for each percent of coal tar (e.g. the 10% ointment contained 520 mg/kg BaP). The dose of BaP per kg body wt varied according to the percentage of the body surface treated with coal tar, but the concentration per cm2 surface was similar for all subjects. The numbers of treatments were equal for all subjects. After clearance by the ethical committee and informed consent, punch biopsies (diameter 4 mm) were obtained under local anesthesia after 1 week of continuous application of coal tar ointments (2 applications/day). As skin biopsies are difficult to obtain, the number of patients in this study was relatively small. Nevertheless,
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M.Rojas et al. these unique samples offered the possibility of studying the impact of MPO genetic variants on the formation of DNA damage in human skin. Biopsy samples were stored at –20°C until DNA isolation. 32P-post-labeling DNA was isolated using phenol extraction and 32P post-labeling analysis was performed to assess the levels of bulky DNA adducts as described previously (16). Briefly, DNA was digested with micrococcal endonuclease and spleen phosphodiesterase and subsequently treated with nuclease P1 to dephosphorylate unmodified nucleotides. Labeling was carried out with excess of [γ-32P]ATP and T4 polynucleotide kinase. 5⬘-Labeled adducts were resolved on polyethyleneimine–cellulose TLC sheets (Merck, Darmstadt, Germany) using the solvents as described (16). In each experiment three standards of [3H]BPDE-modified DNA with known modification levels (1 per 107, 108 and 109 nt) were run in parallel for quantification purposes. Quantification was performed using a phosphorimager (Molecular Dynamics, Sunnyvale, CA). A detection limit for BPDE–DNA adducts of 艋0.1 adducts/108 nt was achieved. Inter-assay variation was ⬍20%.
Analysis of BaP adducts by HPLC with fluorescence detection (HPLC–FD) BPDE–DNA adduct levels were measured using HPLC–FD of BaP tetrols (17,18), which enables characterization of the diastereoisomers that formed BPDE–DNA adducts. Because of low amounts of DNA, samples were pooled according to their MPO genotype [one sample pooled the wild-type MPO– 463GG subjects (n ⫽ 7) and the other the MPO–463AA/AG (n ⫽ 3)]. Human skin DNA samples from individuals with the MPO–463GG (96 µg) and MPO–463AA/AG (67 µg) genotypes were hydrolyzed with fluorescence-free 0.1 N HCl. The hydrolysate was loaded onto a guard column equilibrated with 10% MeOH and washed for 20 min with 12 ml of 10% MeOH. Subsequently, the pre-column was switched to the analytical C18 column and the hydrolysate products were eluted (see ref. 17 for details). Fluorescence was assessed at an excitation wavelength of 344 nm and emission wavelength of 398 nm. The detection limit was 0.5 pg BaP tetrol I-1 (derived from antiBPDE) and BaP tetrol II-2 (derived from syn-BPDE). The level of each BaP tetrol was determined using a standard curve generated from the fluorescence peak area of authentic BaP tetrol standards analyzed just before the analysis of skin samples. Because in all our previous analyses of human DNA samples (17–20) we did not detect the formation of BaP tetrol II-1 (trans-syn-BaP tetrol) we used it as internal standard (2 pg added to each HPLC run) for verification of the relative retention time. MPO genotyping The MPO–463G→A polymorphism was genotyped by a PCR–RFLP based method as described before (7) using the restriction enzyme AciI. Three fragments were produced for the homozygous wild-type MPO–463GG (168, 121 and 61 bp) and two fragments for MPO–463AA (289 and 61 bp). Fragments were separated on a 2.5% agarose gel stained with ethidium bromide. Statistical analysis Data are presented as means ⫾ standard deviations. Non-parametric tests were applied to assess the differences between carriers of the MPO–463GG and carriers of the MPO–463AG/AA genotype. A gene dose effect (MPO– 463GG ⬎ MPO–463AG ⬎ MPO–463AA) was studied by the Jonckheere– Terpstra test. P ⬍ 0.05 was considered significant.
Results The 32P-post-labeling assay was performed on DNA samples from each individual and high levels of total hydrophobic DNA adducts were observed (100.3 ⫾ 93.3 adducts/108 nt) obtained from the total amount of radioactivity in the diagonal radioactive zone. All thin layer chromatograms showed a major spot with the chromatographic location corresponding to the BPDE–DNA adduct (Figure 1). The mean level of this adduct in skin samples was 14.0 ⫾ 11.5 adducts/108 nt and was found to be 5-fold lower in skin of patients with at least one mutant MPO–463A allele (3.8 ⫾ 2.4 adducts/108 nt, n ⫽ 3) as compared with individuals carrying homozygously the high activity –463G variant (18.4 ⫾ 11.0 adducts/108 nt, n ⫽ 7), this difference being statistically significant (P ⫽ 0.03, Mann–Whitney U-test). One individual who was homozygous for the mutant allele (MPO–463AA) even had a 13-fold lower adduct level (1.4 adducts/108 nt) (Table I), which is an indication of a gene dose effect (P ⬍ 0.01, Jonckheere– 1016
Fig. 1. (A) Representative [32P]DNA adduct maps from human skin samples obtained from patients with atopic dermatitis following topical treatment with coal tar for 1 week. Thin layer chromatograms of the MPO–463GG and MPO–463AA genotypes are shown. (B) Typical reverse phase HPLC fluorometric profile of coal tar treated human skin after acid hydrolysis of pooled DNA samples from individuals with the MPO–463GG and MPO–463AA/AG genotypes. I-1, BaP tetrol I-1; II-2, BaP tetrol II-2; I.S., internal standard.
Terpstra test). Genetic polymorphisms were also determined for GSTM1 (prevalence of GSTM1 null 40%) and CYP1A1 (all except one were CYP1A1 wild-type), respectively, but these did not affect DNA adduct levels as determined by 32Ppost-labeling (data not shown). BPDE–DNA adduct levels in human skin after 1 week of topical treatment with coal tar were then determined by our HPLC–FD assay (17,18). This HPLC–FD method has the advantage over 32P-post-labeling that it can identify specific BPDE–DNA adducts. Representative profiles of the hydrolysis products from skin samples are shown in Figure 1. A fluorescent product was found in all HPLC runs which corresponded in its retention time to the standard BaP tetrol I-1 (trans-antiBaP tetrol) derived from anti-BPDE–DNA (17). The presence of product II-2 was observed in MPO–463GG samples only and had the same retention time on HPLC as the BaP tetrol II-2 (cis-syn-BaP tetrol) standard, derived from hydrolysis of syn-BPDE–DNA adducts (17). Background fluorescence of a hydrolysate of calf thymus DNA did not show any fluorescence peaks. These results indicate the formation of both anti- as well as syn-BPDE–DNA adducts in human skin (Figure 1) with an anti/syn adduct ratio of 7:1. The DNA adduct level in MPO–463GG carriers represents the sum of anti- and syn-
Myeloperoxidase –463A variant
Table I. MPO genotype and BPDE–DNA adduct levels in human skin from patients with atopic dermatitis treated topically with coal tar ointments Detection method
MPO genotype
No. of subjects (n)
Adduct level per 108 nt
32P
–463 GGb –463 AGb
7 2
18.4 ⫾ 11.0 5.0 ⫾ 1.7
–463 AAb
1
1.4 (P ⬍ 0.01)d
–463 GGb –463AA/AGb
7 3
14.2f 2.2f
post-labelinga
HPLC–FDe
}
Ratio anti/syn BaP tetrol
3.8 ⫾ 2.4 (P ⫽ 0.03)c 6.7 ⬎11g
aAdduct
level determined by 32P post-labeling using the BPDE–DNA spot only. were equal for all individuals. MPO-463GG by the Mann–Whitney U-test. dP value for trend (Jonckheere–Terpstra test). eAdduct level determined by HPLC–FD as the sum of tetrol I-1 and tetrol II-2 (Figure 1B) derived from anti- and syn-BPDE–DNA. fPooled samples. gBaP tetrol II-2 was below the detection limit (艋0.2 adducts/108 nt). bThe number of treatments cP value as compared with
BPDE–DNA adducts, while in MPO–463AA/AG individuals only anti-BPDE-DNA adducts were found (Table I). DNA adduct levels determined by HPLC–FD were 2.2 and 14.2 adducts/108 nt for MPO–463AA/AG and MPO–463GG individuals, respectively. Discussion BPDE–DNA adducts were previously detected in human lung, colon, atherosclerotic lesions and white blood cells (17–20), but no information was available on the occurrence of BPDEDNA adducts in human skin (reviewed in ref. 4). In the 1930s BaP was identified as a strongly carcinogenic component of coal tar (21) and it has been one of the most extensively studied carcinogens in the past century (22). High levels of hydrophobic bulky DNA adducts were observed in skin of patients treated with coal tars containing high concentrations of PAH, especially BaP (16,23). Similar levels and patterns of these DNA adducts were found in mouse skin (23), which is known to be susceptible to the carcinogenic activity of PAHs, thus implying a potential hazard for man after dermal exposure to such mixtures (24). In this study we could show for the first time that BaP, which is present in coal tar, is metabolically activated in human skin in vivo and forms the anti-BPDE–DNA adduct, thought to initiate the carcinogenic process. Previously it was shown from our work and others that metabolic activation of BaP in human tissues and cells is modulated by polymorphisms in genes that encode for enzymes like CYP1A1 and GSTM1 (1–5). Also, MPO has been shown to transform environmental procarcinogens such as BaP and aromatic amines to highly reactive intermediates by the generation of reactive oxygen species (ROS) (15,25–29). The relative contributions of cytochrome P450s occurring in skin and ROS to the formation of BPDE–DNA adducts in human tissues is still the subject of investigation (27,29). Patients with atopic dermatitis have a strong dermal inflammatory response in which MPO is involved and, therefore, these subjects have allowed us to examine the impact of inflammatory conditions on BPDE–DNA adduct formation in their skin. BPDE–DNA adduct levels were found to be significantly increased in lung epithelium cells after incubation with BaP and stimulated neutrophils known to generate peroxyl free radicals (30). Furthermore, MPO was found to enhance the formation of BPDE–DNA adducts in lung tissue of experi-
mental animals (15,25). Therefore, it could be expected that the target dose of BaP in coal tar treated skin would be modified by the MPO polymorphism. Indeed, the MPO– 463AA/AG genotype has been linked to a markedly reduced risk for lung cancer (6–9), suggesting that lower MPO activity in carriers of the MPO–463AA/AG genotype represents a protecting factor. Our results in skin samples of atopic dermatitis patients confirmed this hypothesis and provided mechanistic insights: we found strongly reduced BPDE–DNA adduct levels (5- to 6-fold) in MPO–463AA/AG individuals as compared with the MPO–463GG genotype (wild-type). This implies that coal tar treated patients with the MPO–463AA/ AG genotype form less BaP-derived DNA damage in their target organs and thus appear to be at lower risk for developing PAH-associated tumors. Analysis of the ratio between anti- and syn-derived BaP tetrols has proven to be useful in distinguishing the activation pathways of BaP 7,8-diol by epoxidation in vitro and in vivo (15,25,28). An anti/syn ratio of 艌2.5 was found to be characteristic for peroxide-dependent oxidation of BaP 7,8-diol (29). In the case of skin of MPO–463GG subjects the presence of antiand syn-BPDE–DNA adducts was observed with an anti/syn ratio of 6.7. Although this ratio may suggest the involvement of peroxyl free radicals or MPO in the metabolic activation of BaP in the skin of dermatitis patients, it is still difficult to determine the exact relative contribution of cytochrome P450s and ROS in our study, because the skin was exposed to BaP and not BaP 7,8-diol, but only the latter would allow discrimination between cytochrome P450- and ROS-related pathways (see refs 15,25,27–29 for details). Further studies are necessary to determine the relative contribution of MPO genotypes to the formation of BPDE–DNA adducts in human skin. In conclusion, this study provides an example of a gene– environment interaction in which the individual genetic background determines the DNA damaging effect of an external exposure to carcinogens. Our results show for the first time: (i) the in vivo formation of BPDE–DNA adducts in human skin after therapeutic dermal exposure to coal tar; (ii) that BPDE–DNA adduct levels in human skin are modulated by the MPO genotype, with carriers of the MPO–463AA/ AG genotype having reduced BPDE–DNA adduct levels in their skin. 1017
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Acknowledgements This work is dedicated to the late James A.Miller, McArdle Laboratory for Cancer Research, Madison, Wisconsin, USA. The authors gratefully acknowledge Dr C.Ittrich (German Cancer Research Center, Division of Statistics) for her help in the statistical analysis and Drs J. Nair and A. Risch (German Cancer Research Center, Division of Cancer Risk Factors and Toxicology) for critically reading the manuscript.
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