Influence of Genotypic Polymorphisms of the CYP1A1 and GSTM1 Genes on DNA-Protein Crosslinks (DPC) among Incinerator Workers in Hospitals in Taiwan

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(1)11. ORIGINAL ARTICLE. Influence of Genotypic Polymorphisms of the CYP1A1 and GSTM1 Genes on DNA-Protein Crosslinks (DPC) Among Incinerator Workers in Hospitals in Taiwan 1. Fang-Yang Wu, Chinq-Huey Tsai, Hsien-Wen Kuo, Kwang Victor Lo , Ching-Tsan Tsai Institute of Environmental Health, China Medical University, Taichung, Taiwan; 1. Civil Engineering Department, University of British Columbia, Vancouver, British Columbia, Canada.. Objectives. The purpose of this study was to explore the association between polymorphisms of the Cytochrome P450 (CYP1A1) and Glutathione S-transferase M1 genes and DNA-protein crosslinks (DPC) among medical waste incinerator (MWI) workers in Taiwan. Methods. The exposure group comprised 31 MWI workers from eight hospitals. We selected 31 healthy, age- and gender-matched subjects with no history of malignant tumor, medication that might induce mutation, or exposure to carcinogens to serve as the control group. No significant differences in demographic or behavioral characteristics, such as age, gender, cigarette smoking, betel nut chewing, and alcohol consumption were found between the exposure and control groups. CYP1A1 and GSTM1 polymorphisms were analyzed by PCR. DPC were determined by a sodium dodecyl sulfate (SDS) precipitation assay. Results. The frequency distributions of the homozygous variant of CYP1A1 Msp I (v/v) in the exposure and control groups were 51.6% and 58.1%, respectively; however, the difference was not significant. Yet, there was a significant difference between the exposure and control groups in the GSTM1 null genotype (41.9% vs 25.8%; OR = 1.4, p = 0.01). There was also a significant difference in DPC levels between both groups (1.5% vs 0.9%, p < 0.01). Furthermore, no significant relationship was found between CYP1A1 and GSTM1 genotypes and DPC levels in either group. Multiple regression analysis revealed that exposure to medical waste incineration was the only significant factor which predicted the variance in DPC levels (R 2 = 0.85, p < 0.01). Conclusions. This study suggests that DPC is a sensitive biomarker of DNA damage in workers exposed to MWI hazards. No association between the CYP1A1 and GSTM1 genes and DPC was observed. ( Mid Taiwan J Med 2004;9:11-8). Key words CYP1A1, DPC, GSTM1, incinerator workers, medical waste. INTRODUCTION. In Taiwan, there are 170 hospitals with more than 100 beds, and most of them are located Received : 3 November 2003. Revised : 15 December 2003. Accepted : 22 December 2003. Address reprint requests to : Ching-Tsan Tsai, Institute of Environmental Health, China Medical University, 91 Hsueh-Shih Road, Taichung 404, Taiwan.. in metropolitan areas. Approximately 95% of the medical waste generated in hospitals is treated by incineration. Of the 170 hospitals, 26 of them use small-sized incinerators to process medical waste containing chemotherapeutic medications for tumors. Many vials and intravenous bottles are incinerated in the process, and sometimes the.

(2) 12. leftover fluids in these vials and bottles are not cleaned out prior to incineration. Although the incinerating process of chemotherapeutic waste reduces its mass and volume effectively, toxic compounds can concentrate in the fly ashes or be vaporized [1]. Incinerators produce numerous carcinogenic compounds, such as polycyclic aromatic hydrocarbons (PAHs) [1]. Once these toxic compounds pass through the pollution control equipment, they accumulate in the environment and even in human tissue [2]. Cytochrome P450 (CYP1A1) enzyme metabolizes PAH compounds into electrophilic intermediates, and its polymorphic gene is known to be susceptible to particular carcinogens [3,4]. Greater enzyme activity in some individuals might augment the metabolism of PAHs, thereby increasing the risk of lung cancer [3,4]. Glutathione S-transferase (GSTM1) is an important detoxification enzyme responsible for the conjunction of glutathione and electrophilic compounds, which prevents them from reacting with DNA or proteins [5]. Individuals with the GSTM1 null genotype tend to detoxify PAHs less effectively. They therefore have higher levels of DNA lesions, which put them at greater risk of developing some types of cancer. Although this mechanism has been discussed in numerous papers [6-8], no association between the GSTM1 null type and DNA damage or cancer has been found [9,10]. The relationship between individuals' exposure to PAHs at work or in the environment and DNA-adducts has been explored by a few researchers [11,12]. For example, the formation of PAH-DNA adducts in white blood cells was found to be associated with the level of exposure to PAHs [11]. Whyatt and his colleagues examined Polish mothers and their neonates and found that DNA damage was related to local air pollution levels, and that DNA-adduct levels were drastically higher in certain heavily polluted areas [12]. In addition, a number of studies have shown that exposure to environmental contaminants or use of chemotherapeutic agents induced the formation of DNA-protein crosslinks (DPC) [13-. DPC and Incinerator Workers in Hospital. 16]. It has also been shown that levels of DPCs are associated with DNA strand breakage [16], sister chromatid exchanges (SCE) [17], cell transformation [18], augmentation of cell toxicity, and alteration of cell growth [19]. The formation of DPC reflects the net effects of competing metabolic and detoxification pathways and DNA repair. The purpose of this study was to measure the level of DNA damage in MWI workers according to the levels of DPC. We then examined the association between DPC levels and polymorphisms of the CYP1A1 and GSTM1 genes. MATERIALS AND METHODS. Study Subjects. This research was a case-control study. Thirty-one MWI workers were selected from 8 hospitals. Participants completed a questionnaire, and informed consent was obtained. The contents of the questionnaire included demographic data (such as gender and age), lifestyle behavior (such as cigarette smoking and alcohol consumption), and work history. Thirty-one healthy subjects matched by gender, age, and cigarette smoking behavior living within an uncontaminated area were selected as the control group. Furthermore, 6 mL of whole blood were drawn from each subject, of which 3 mL were stored in EDTA tubes for DNA extraction and genotype analyses. The remainder was kept in heparin tubes for DPC analyses. Measurement of Genotyping. Genomic DNA was prepared from human blood lymphocytes by standardized procedures. The CYP1A1 MspI polymorphism from each subject was analyzed by PCR-based methods, as described by Kawajiri et al [20]. The MspI polymorphism was assessed by amplifying a 295bp region of DNA containing an MspI restriction site followed by digestion with MspI. CYP1A1 (w/w) lacked the MspI cleavage site, CYP1A1 (v/v) was homozygous for the allele with the MspI site, and CYP1A1 (w/v) was heterozygous for the MspI site. The GSTM1 genotypes were determined by.

(3) Fang-Yang Wu, et al.. 13. Table 1. Demographic characteristics of the medical waste incinerator (MWI) and control groups. Variable Age (yr) Gender (%) Male Female Smoking (%) Betel nut consumption (%) Alcohol consumption (%) Work duration (yr) ≥5 ≤5. Controls (n = 31). MWI workers (n = 31) 35.8. 10.0. 36.1. 24 (77.4) 7 (22.6) 16 (51.6) 4 (12.9) 12 (38.7). p 0.91. 10.3. 1.00 1.00 1.00 0.81 0.72. 24 (77.4) 7 (22.6) 16 (51.6) 3 (9.7) 8 (25.8). 9 (29.1) 22 (70.9). Table 2. Odd ratios (OR) for both groups based on CYP1A1 and GSTM1 genotypes. Genotype CYP1A1 Msp I w/w + w/v v/v GSTM1 GSTM1 ( + ) GSTM1 ( – ). MWI workers n (% ). Controls n (% ). OR. 15 (48.4) 16 (51.6). 13 (41.9) 18 (58.1). 1.0 0.8. 18 (58.1) 13 (41.9). 23 (74.2) 8 (25.8). 1.0 1.4. p 0.45. 0.01. a modified method proposed by Comstock et al [21]. The β-globin primers described by Bell et al were included in the polymerase chain reaction (PCR) to confirm the presence of amplifiable DNA in the samples [7]. The commercial PCR kit containing Taq DNA polymerase (Blossom, Taiwan) required 1 µg DNA. Reactions were heated for 2 min at 94˚C, 2 min at 53˚C, and 3 min at 72˚C, with the cycle repeated 35 times in a Thermal Cycler. PCR products were electrophoresed on a 3% agarose gel. Individuals with an intact GSTM1 gene exhibited amplification of the 273bp GSTM1 fragments. A 100bp β-globin fragment served as a positive internal control. Measurement of DPC. DPC was detected by methods described previously [22]. Briefly, white blood cells (2 106) were lysed in 0.5% sodium dodecyl sulfate (SDS), as well as 20 mM Tris-HCl (pH 7.5) solution, and stored at 70 C before analyses. Meanwhile, fluorescence was measured at 450 nm during excitation at 360 nm by a Horfer Model Fluorescence Spectrophotometer.. Statistical Analysis. All data analyses were done by the SPSS 8.0 statistical software. χ 2 -tests and t tests compared the basic characteristics and distributions of CYP1A1, GSTM1 and DPC values in the research and control groups. To examine the association between polymorphic genes and MWI workers, odds ratio (OR) was calculated by unconditional logistic regression. Finally, factors affecting DPC levels were analyzed by the ordinary multiple regression model. RESULTS. Frequency distributions of personal characteristics for both groups are shown in Table 1. The mean ages of the MWI workers and those in the control group were 35.8 and 36.1 years, respectively (p = 0.9; non-significant). The number of men and women in both groups were equal (24 males and 7 females). There were no significant differences between the groups in cigarette smoking, betel nut chewing or alcohol.

(4) 14. DPC and Incinerator Workers in Hospital. Table 3. DPC values in the MWI and control groups. n. Mean. 31 31. MWI workers Control group SD = standard deviation.. 1.5 0.9. p. SD. < 0.01. 0.1 0.2. Table 4. Effects of the CYP1A1 and GSTM1 genotypes on DPC values. Genotype CYP1A1 Msp I w/w + w/v v/v GSTM1 GSTM1 ( + ) GSTM1 ( – ). n. MWI workers (n = 31) Mean SD. p. n. Controls (n = 31) Mean SD. p 0.17. 15 16. 1.6 1.5. 0.1 0.1. 0.30. 18 13. 0.9 1.0. 0.9 0.2. 18 13. 1.5 1.5. 0.1 0.1. 0.40. 8 23. 0.9 1.1. 0.2 0.8. 0.27. Table 5. Factors affecting levels of DPC by multiple regression analysis. Variable MWI workers/control group Smoking (yes/no) Betel nut consumption (yes/no) Alcohol consumption (yes/no) CYP1A1 Msp I (v/v vs w/w+w/v) GSTM1 (null/non-null) R 2 = 0.85.. Regression coefficient 0.93 0.04 – 0.05 – 0.02 – 0.06 0.04. consumption. As for the MWI workers' work duration, 71% of the subjects (n = 22) had worked at a hospital incinerator for less than 5 years, and 29% (n = 9) for more than 5 years. Table 2 shows the frequency distributions and comparisons of CYP1A1 and GSTM1 genotypes. The frequencies of the CYP1A1 Msp I wild type (w/w + w/v) and variant type (v/v) in the exposure group were 48.4% and 51.6%, respectively (41.9% and 58.1%, respectively in the control group). There were no significant differences between the groups. In the exposure group, 58.1% had the GSTM1 non-null genotype (+) and 41.9% had the GSTM1 null genotype (–) (74.2% and 25.8% in the control group); there was a significant difference between the groups (OR = 1.4, p = 0.01). Comparison of DPC levels between the groups is shown in Table 3. The DPC levels for the exposure and control groups were 1.5% and 0.9%, respectively which was significant (p <. p < 0.01 0.49 0.36 0.67 0.29 0.52. 0.01). Table 4 shows the DPC level in CYP1A1 and GSTM1 genotypes. For the exposure group, the DPC levels in CYP1A1 Msp I wild type (w/w + w/v), variant type (v/v), GSTM1 non-null genotype, and null genotype were 1.6%, 1.5%, 1.5%, and 1.5%, respectively. In the control group, the percentages were 0.9%, 1.0%, 0.9%, and 1.1%, respectively. No significant differences were found in either group. Table 5 shows the factors affecting DPC levels according to multiple regression analysis. Only exposure to medical waste combustion at the worksite (MWI workers compared to controls) was found to be a significant risk factor, after adjusting for the other factors (R2 = 0.85, p < 0.01). DISCUSSION. The combustion of medical waste in hospital incinerators produces known human carcinogens such as polycyclic aromatic.

(5) Fang-Yang Wu, et al.. hydrocarbons (PAHs), aromatic amine, and dioxin [23]. PAH compounds enter the body via inhalation, and then accumulate in the lungs. They are distributed to other organ tissues through the bloodstream [23]. DNA damage, observed as sister chromatid exchanges (SCE), DNA adducts and DPC, is a suitable endpoint for monitoring the biological effect of exposure to potential carcinogens. These biomarkers are extensively used to evaluate occupational and environmental exposure. It has been shown previously that people living adjacent to chemical waste disposal sites have increased levels of SCEs [24]. That study indicated that PAH-DNA adduct levels vary according to the type of pollutant, and that smokers have higher adduct values than non-smokers [25]. Studies have also indicated that chemotherapeutic agents, such as cyclophosphamide, are capable of introducing cross-links in DNA [15,16]. Chemical carcinogens are metabolized by a wide variety of enzymes. Multiple forms of human cytochrome P-450 are involved in the oxidative metabolism of chemical carcinogens, such as PAHs [26]. Most studies have focused on the CYP1A1 enzyme and glutathione Stransferase M1 (GST M1) when studying PAHDNA adducts [3-8]. To our knowledge, no study has yet examined the effects of GSTM1 and CYP1A1 MspI genotypes on DPC formation among MWI workers in hospitals. In this study, no positive association between the MspI polymorphism and DPC was found, which was similar to the findings of our previous study of 60 breast cancer patients [27]. The proportion of the variant genotype (v/v) of CYP 1A1 was slightly lower in the incinerator workers than in the controls (51.6% vs 58.1%). The GST Ml gene is responsible for detoxification of reactive intermediates of PAH, by conjugating to glutathione. The GST Ml gene is polymorphic in humans in that the gene is either present or absent (null genotype) [21]. This study showed that the prevalence of the null genotype among the control group was 25.8%, but the prevalence among workers was 41.9%. In. 15. our study of breast cancer patients, the GST Ml gene (–) was found in 56.7% of cancer patients and in 41.7% of controls [27]. In addition, this study investigated the association between the GSTM1 null genotype and DPC levels. We hypothesized that subjects with this genotype would have higher levels of DPC because of their decreased ability to detoxify PAH metabolites. However, DPC levels were not significantly lower in subjects with the GSTM1 null genotype. DNA tends to be more sensitive than proteins to chemicals, although the effects of chemicals on both proteins and DNA are complex. Many chemicals enhance the formation of DPC [13,14,16,28]. DPC formation in treated cells may persist since its presence is easily detectable following the removal of the genotoxic agent [29,30]. Normal cells are able to repair these lesions with fidelity or by introducing errors. However, genotoxic compounds may reduce a cells ability to repair, possibly resulting in the production of DPC during DNA replication [31]. This study showed that DPC values in MWI workers were significantly higher than those in the control group (1.5% and 0.9%, respectively). Our previous studies have indicated an association between DPC and breast cancer patients [27] and between DPC and lead workers [32]. Hence, DPC formation may be associated with exposure to chemical compounds. In conclusion, this study suggests that DPC is a sensitive biomarker of DNA damage in workers exposed to MWI hazards. However, exposure to specific chemicals was not directly measured, which limited interpretation of this study. No significant differences in polymorphic genes were found between the worker and control groups. Further research is required to investigate the precise relationship between the various genotypes and MWI workers. ACKNOWLEDGMENT. This study was supported by the China Medical University Hospital (DMR-90-132) in Taichung, Taiwan..

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(8) 18. CYP1A1 GSTM1 DNA-Protein Crosslinks 1. 1. (CYPlA1. GSTM1). DNA-protein crosslinks (DPC). 8. 31. CYP1A1 (wt/vt) 41.9%. (wt/wt). 48.4%. (vt/vt). 58.1%. 51.6%. GSTM1. 58.1%. 41.9%. 74.2% 1.5%. 25.8%. 0.9%. ( p < 0.01) CYP1A1. GSTM1. DPC. DPC. DPC 2004;9:11-8. CYP1A1. DPC. GSTM1. 404. 19. 2003. 11. 3. 2003. 12. 22. 2003. 12. 15. DPC.

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