Analysis of Oxidative DNA Damage 8-Hydroxy-2'-deoxyguanosine as

Feb 23, 2005 - An oxidative DNA biomarker, 8-hydroxy-2'-deoxyguanosine (8-OHdG), was determined in the livers and kidneys of stranded or by-caught cet...
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Environ. Sci. Technol. 2005, 39, 2455-2460

Analysis of Oxidative DNA Damage 8-Hydroxy-2′-deoxyguanosine as a Biomarker of Exposures to Persistent Pollutants for Marine Mammals CHI-SHAN LI,† KUEN-YUH WU,‡ GOU-PING CHANG-CHIEN,§ AND C H I N - C H E N G C H O U * ,† Department of Veterinary Medicine, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan, Division of Environmental Health and Occupational Medicine, National Health Research Institute, No. 100, Shihcyuan First Road, Kaohsiung 807, Taiwan, and Department of Chemical Engineering and Super Micro Mass Research and Technology Center, Cheng-Shiu Institute of Technology, 840 Cheng-Ching Road, Kaohsiung 833, Taiwan

An oxidative DNA biomarker, 8-hydroxy-2′-deoxyguanosine (8-OHdG), was determined in the livers and kidneys of stranded or by-caught cetaceans along the Taiwan coast through isotope-dilution liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to evaluate the feasibility of analyzing the DNA adduct in marine mammals and then to study the association between 8-OHdG and levels of polychlorinated biphenyls (PCBs) and dichlorodiphenyldichloroethylene (DDE) in the blubbers of the cetaceans. The mean values of the 8-OHdG from the liver and kidney samples were 19.83 ( 10.00 pmol/µmol deoxyguanosine (dG) (6.90-53.53 pmol/µmol dG) and 19.16 ( 7.48 pmol/µmol dG (5.36-39.36 pmol/µmol dG), respectively. In general, 8-OHdG was not related to the general health status of the by-caught and stranded animals and also was not related to species. However, the levels of 8-OHdG had a positive correlation with concentrations of PCBs, but not DDE, in female cetacean livers. In addition, when selected coplanar PCBs (dioxin-like congeners) were used to compare the 2,3,7,8-tetrachlorodibenzo-pdioxin equivalents (TEQs) with 8-OHdG of by-caught cetaceans, a high positive correlation (r ) 0.80, p < 0.01) was found in mature female animals. Thus, the detection of 8-OHdG in marine mammals with isotope-dilution LC/MS/ MS is possible, and the study of the relationship between oxidative DNA damage and environmental contaminants under natural exposure indicates that the level of 8-OHdG in female cetacean livers is associated with coplanar PCBs and the factor of sexual maturity.

Introduction Reactive oxygen species (ROS) through endogenous metabolic processes or exogenous sources such as carcinogens * Corresponding author phone: 886-2-2363-0495; fax: 886-2-23630495; e-mail: [email protected]. † National Taiwan University. ‡ National Health Research Institute. § Cheng-Shiu Institute of Technology. 10.1021/es0487123 CCC: $30.25 Published on Web 02/23/2005

 2005 American Chemical Society

and environmental pollutants may cause oxidative DNA damage in living cells (1). One of the many oxidative DNA adducts, 8-hydroxy-2′-deoxyguanosine (8-OHdG)sa nucleoside lesion, has been shown to cause G:C to T:A transversion (2), and 8-OHdG has been proposed to be an indicator of oxidative damage (3, 4). Production of 8-OHdG may be considered attributable to mutagenesis, carcinogenesis, and aging. The majority of the studies on 8-OHdG was in human and experimental animals (5-8), and the formation of 8-OHdG in mussels (9), fish (10, 11), and oysters (12) was reported, but not in marine mammals. Cetaceans, the top predators in marine food chains and having exceptional longevity, are suitable mammalian animals for the monitoring of contamination in marine ecosystems due to the distribution, transfer, and accumulation of environmental pollutants in the food chain (13). Therefore, studying 8-OHdG levels in cetaceans has the advantage of building the background levels of cetaceans and also revealing the impact of human activities on ocean biota. Exposure to certain contaminants can cause increased levels of oxidative DNA damage, like benzo[a]pyrene (9, 14) and dieldrin (10). Polychlorinated biphenyls (PCBs) and dichlorodiphenyldichloroethylene (DDE) have good stability and are easily bioaccumulated in marine food chains. Cetaceans, thus, are easily susceptible to the accumulation and toxicity of these organochlorines (15). Whether or not these environmental contaminants have genotoxicity and adversely affect these cetaceans, the detection of DNA adducts in organs can be used to determine the formation of DNA adducts resulting from many events and processes, such as the absorption of the agents, distribution to different tissues, activation of metabolism, and DNA repair systems (16-18). Thus, detection of DNA adducts in tissues represents the dynamic equilibrium between DNA damage and their repair (19) and also may provide an opportunity to evaluate the carcinogenesis potential. In this study, the levels of 8-OHdG in DNA isolated from livers and kidneys of cetaceans from the Taiwan waters were measured by isotope-dilution liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The influences of biological factors such as age, sex, and species differences between the stranded and accidentally caught individuals were analyzed to assess possible exposure to past oxidative agents, either endogenous or exogenous. In addition, according to our previous research of PCBs and DDE concentrations in the same cetaceans’ blubbers (20, 21), this study also evaluated the relationship between 8-OHdG and these organochlorines.

Experimental Procedures Chemicals. All enzymes were from Roche, Mannheim, Germany. Solvents were of analytic grade (Merck, Darmstadt, Germany). Chemical reagents, salts, and 2′-deoxyguanosine (dG) were purchased from Sigma, St. Louis, MO. Unlabeled 8-hydroxy-2′-deoxyguanosine (8-OHdG) was from Calbiochem. Synthesis of 15N5-8-OHdG and 15N5-dG was according to Hu et al. (22). Field Sampling. Samples of cetaceans either accidentally entangled in fishing net or stranded (live or dead) were collected from the coasts of the Taiwan water areas with the help of the Taiwan Cetacean Society during 2000-2001. Carcasses were recorded carefully for the collection date, species, sex, length, and teeth wear. The developmental stage of each cetacean was classified into mature or immature according to the reference data for the body length in different VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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19.32 ( 8.86 (14)

16.51 (1) 35.40 (1) 24.98 (2) 17.89 (1) 15.58 (2) 13.45 (1) 19.17 ( 11.03 (3)

22.14 ( 10.82 (6) 19.44 (1) 14.09 (1) 15.74 ( 2.71 (3) 18.36 ( 6.85 (12) 23.22 (2) 24.98 (2) 17.50 ( 4.69 (19) 20.89 ( 13.75 (3) 18.68 ( 7.27 (23) 23.21 ( 10.70 (5) 25.35 (1) 19.16 ( 7.48 (78) 19.95 ( 10.26 (16)

17.48 ( 4.83 (18) 31.51.(1) 18.92 ( 7.35 (22) 29.26 (2) 25.35 (1) 19.13 ( 7.22 (64) a

Mean ( standard deviation.

b

Sample number.

17.46 ( 9.59 (18) 24.12 (1) 21.06 ( 10.62 (20) 16.72 (2) 24.48 (1) 19.80 ( 10.03 (62)

18.73 (

Bottlenose dolphin (Tursiops truncatus) Cuvier’s beaked whale (Ziphius cavirostris) Common dolphin (Delphinus delphis) Dwarf sperm whale (Kogia sinus) Fraser’s dolphin (Lagenodeiphis hosei) Finless porpoise (Neophocaenoides phocaenoides) Pygmy killer whale (Feresa attenuata) Pantropical spotted dolphin (Stenella attenuata) Pygmy sperm whale (Kogia breviceps) Risso’s dolphin (Grampus griseus) Rough-toothed dolphin (Steno bredanensis) Short-finned pilot whale (Globicephala macrorhynchus) total

43.91 (2) 18.39 ( 8.32 (12) 8.76 (1)

13.06 (1) 36.69 (2) 13.07 (1) 17.03 (1) 24.66 (1) 8.78 (1) 16.37 (1) 14.05 (1) 18.64 ( 3.20 (3) 13.04 (1) 23.27 ( 3.94 (3)

15.74 ( 2.71 (3) 18.21 ( 6.56 (11) 11.05 (1)

23.57 ( 11.44 (5)

17.78 ( 6.44 (6) 36.69 (2) 13.07 (1) 34.60 ( 15.26 (3) 18.87 ( 8.15 (13) 8.77 (2) 16.37 (1) 17.28 ( 9.35 (19) 20.01 ( 3.78 (4) 20.68 ( 10.50 (21) 20.65 ( 5.43 (5) 24.48 (1) 19.83 ( 10.00 (78)

14.99 (1) 19.44 (1) 14.09 (1)

total stranded by-caught total stranded

(5)b 6.72a

by-caught

kidney 8-OHdG pmol/µmol dG liver

TABLE 1. Levels of 8-OHdG in Liver and Kidney Samples of Different Cetacean Species in Taiwan Waters

cetacean species and the status of reproductive organs and teeth wear (23, 24). The health status and nutritional state of cetaceans were determined by the gross pathological findings and blubber thickness. For each cetacean, a sample of liver and kidney was cut and wrapped with aluminum foil and frozen at -20 °C until analysis. In this study, a total of 156 samples (78 of liver and kidney each) from 12 cetacean species was analyzed, and the majority was from Risso’s dolphin, Pantropical spotted dolphin, and Fraser’s dolphin (Table 1). DNA Isolation. The method described by Helbock et al. (25) and Ravanat et al. (26) was used with modifications. Briefly, 200 mg of tissue was homogenized with 5 mL of buffer A (320 mM sucrose, 5 mM MgCl2, 10 mM Tris, 0.1 mM desferrioxamine, 1% Triton X-100, pH 7.5). After being centrifuged at 2000g for 10 min at 4 °C, the pellets were washed twice with 3 mL of buffer A. A total of 600 µL of buffer B (10 mM Tris, 5 mM EDTA-Na2, 0.15 mM desferrioxamine, pH 8) and 35 µL of 10% sodium dodecyl sulfate was added, and the sample was vigorously agitated. After 30 µL of RNase A (1 mg/mL in RNase buffers10 mM Tris, 1 mM EDTA, 2.5 mM desferrioxamine, pH 7.4) and 8 µL of RNase T1 (1 U/µL in RNase buffer) was added, the sample was incubated at 50 °C for 20 min to remove contaminated RNA from DNA samples, and then 30 µL of Proteinase K (20 mg/mL) was added to incubate at 37 °C for 2.5 h. Thereafter, 1.2 mL of NaI solution (7.6 M NaI, 40 mM Tris, 20 mM EDTA-Na2, 0.3 mM desferrioxamine, pH 8.0) and 2 mL of 2-propanol was added, and the sample was inverted gently to precipitate DNA. After centrifugation at 5000g for 15 min at 4 °C, the DNA was washed with 1 mL of 40% 2-propanol and 1 mL of 70% ethanol. Last, DNA was collected by centrifugation and dissoloved in 1 mL of 0.1 mM desferrioxamine overnight. The concentration of DNA was determined by UV absorption spectroscopy; the ratio of A260/A280 between 1.4 and 1.9 was acceptable. Then, 20 µg of DNA was added to 40 µL of 15N5-8-OHdG (40 ng/mL) and 15N5-dG (120 ng/mL) solution as an internal standard. After the addition of 10 µL of 1 U/µL nuclease P1 (dissolved in 300 mM sodium acetate and 1 mM ZnSO4 aqueous solution, pH 5.3), the sample was incubated at 37 °C for 2 h. Then, 10 µL of 10X alkaline phosphatase buffer and 0.2 µL of alkaline phosphatase was added, and the aliquot underwent 2 h incubation. Finally, 10 µL of 0.1 M HCl was added to neutralize the solution, and the DNA sample was diluted with 1 mL of deionized water. Analysis of 8-OHdG using High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry (HPLC/MS/MS). The method of solid-phase extraction and HPLC/MS/MS used by Hu et al. (22) was followed and described next. After the addition of 75 µL of 1 M ammonium acetate buffer (pH 5.25), the diluted DNA solution was applied to Sep-Pak C18 cartridge (100 mg/1 mL; Waters, Milford, MA) preconditioned with 1 mL of 100% methanol and 1 mL of deionized water. Subsequently, the column was washed with 1 mL of deionized water, and then the fraction containing 8-OHdG was eluted by 1.5 mL of 40% methanol (v/v). The eluent was dried under vacuum for 3 h at 40 °C, and then the residue was redissolved in 200 µL of 5% acetonitrile containing 0.1% formic acid. Finally, 180 µL of aliquot was used for 8-OHdG analysis, and the remaining 20 µL sample was diluted 30 times with 580 µL of 5% acetonitrile containing 0.1% formic acid for dG analysis. Of the previous sample solution, 20 µL of each were injected into the HPLC/MS/MS instrument. This included a PE 200 autosampler and two PE 200 micropumps (Perkin-Elmer, Norwalk, CT), equipped with a Polyamine-II endcapped HPLC column (150 × 2.0 mm, 5 µm, YMC) and an identical guard column (10 × 2 mm, YMC); the previous HPLC was connected to a triple-quadrupole mass spectrometer (API 3000, Applied Biosystems, Foster

15.86 ( 7.06d (5.36-31.42) 25.54 ( 8.96 (14.06-35.40) d

Range. c p < 0.01 (stranded male group vs stranded female group). b

Mean ( standard deviation.

31 31 mature immature

a

17.65 ( 6.39 (8.87-31.51) 20.52 ( 7.76 (9.95-39.36) 31 33 11 5 17.72 ( 8.51 (7.77-36.68) 21.87 ( 11.11 (6.90-39.36)

Age 21.81 ( 11.59 (13.04-53.53) 15.86 ( 4.94 (8.78-20.59)

19.34 ( 7.88 (9.22-39.36) 18.92 ( 6.62 (8.87-36.07) 32 32 Gender 17.07 ( 4.72 (13.04-26.67) 22.82 ( 13.50 (8.78-53.53) 8 8 21.91 ( 10.82a (8.89-51.58)b 17.69 ( 8.86 (6.90-44.48) 31 31 male female

p ) 0.04 (stranded mature group vs stranded immature group).

9 5

13.35 ( 4.52c (5.36-19.44) 25.28 ( 8.17 (16.51-35.40) 7 7

stranded n kidney by-caught n stranded n liver by-caught n

Analysis of 8-OHdG by Isotope-Dilution LC/MS/MS. Selected reaction monitoring (SRM) for 8-OHdG, 15N5-labled 8-OHdG, dG, and 15N5-labled-dG was obtained from LC/ MS/MS chromatograms. The positive ESI mass spectrum of 8-OHdG contained a precursor ion at m/z 284.1 and a product ion at m/z 168.0; a precursor ion at m/z 289.1 and product ion at m/z 173.0 were characterized as 15N5-labled 8-OHdG. The precursor ions at m/z 268.1 and 273.1, and the corresponding product ions at m/z 152.0 and 157.0, were each for dG and 15N5-dG used for SRM. A linear calibration curve ranging from 0.04 to 20 ng/mL was obtained using aqueous standard solutions (r2 ) 0.9999), each calibration solution contained 1.64 ng/mL 15N5-labled 8-OHdG, and two linear calibration curves ranging from 0.08 to 3.2 ng/mL (r2 ) 0.9988) and 4-16 ng/mL (r2 ) 0.9998) were of dG containing 0.4 ng/mL 15N5-labled-dG. Limit of detection (LOD) of the instrument for 8-OHdG was 0.024 ng/mL determined as the concentration that yielded a signal-to-noise (S/N) ratio of 3.9 using diluted standard solutions. The coefficients of variation were 4.3 and 7.1% for within-run and betweenrun, respectively. This study showed that it is feasible to use isotope-dilution LC/MS/MS to detect the 8-OHdG levels in cetacean livers and kidneys. The chaotrophic sodium iodide method in the current analysis was chosen to minimize artificial oxidation through experimental procedures (25, 27). The advantages of the isotope-dilution method were to correct for losses during the sample preparation and variations in the mass spectrometric response by calibration with isotopically labeled internal standards, 15N5-labled 8-OHdG and 15N5dG. Moreover, the tandem mass spectrometry had high sensitivity by reducing the background levels (18, 28, 29). Thus, through the previous processes, detection of 8-OHdG in tissues of cetaceans was possible and promising. However, during the DNA isolation procedure in some decomposed or age-rotten samples, we were unable to purify enough DNA (the ratio of A260/A280 below 1.4 or above 1.9), and these samples were excluded for further analysis. This resulted in about 5.5% of samples lost and also produced several unpaired numbers of liver and kidney samples for 8-OHdG analysis. Thus, improvement of the method to recover the DNA as much as possible is necessary for a better understanding of such decomposed biological materials. Concentrations of 8-OHdG in Cetacean Liver and Kidney. The average 8-OHdG levels in liver and kidney

8-OHdG pmol/µmol dG (range)

Results and Discussion

TABLE 2. Levels of 8-OHdG in Liver and Kidney Samples of By-Caught and Stranded Cetaceans in Taiwan Waters Classified by Gender and Age

City, CA) equipped with a TurboionSpray source. The mobile phase chosen was 80% acetonitrile with 0.1% formic acid and delivered at a flow rate of 300 µL/min. Eluent from the HPLC system was electrospray-ionized in the positive mode. For all the samples, the [M + H]+ ion was selected by the first mass filter, and then the [M + H-116]+ ions, corresponding to BH2+, were selected by the last mass filter. Nitrogen was used as the nebulizing, curtain, heater (6 L/min), and collision gases, and the TurboionSpray probe temperature was set at 300 °C. Statistical Analysis. Data were analyzed using SAS-PC System Version 8.2 for Windows (SAS Institute Inc., Cary, NC). The results were presented as mean ( standard deviation; a p-value < 0.05 was considered a significant difference. The PCBs and DDE concentrations in the same cetaceans’ blubber were obtained from our previous research (20, 21), and the correlations between the levels of 8-OHdG with PCBs and DDE were calculated using Pearson’s correlation analysis. For comparing paired groups, a Student’s t-test was used to determine whether the differences were statistically significant. Duncan’s multiple-range test was also applied to differentiate 8-OHdG concentrations among the species.

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TABLE 3. Correlations between DDE, PCBs, and 8-OHdG of Liver and Kidney in Female and Male Cetaceans DDE in blubber

PCBs in blubber

8-OHdG in liver

8-OHdG in kidney

Female DDE PCBs 8-OHdG in liver 8-OHdG in kidney

1.000a (25)b 0.691 (25)c 0.411 (23) -0.133 (24)

DDE PCBs 8-OHdG in liver 8-OHdG in kidney

1.000 (30) 0.480 (30)c 0.039 (28) 0.087 (29)

1.000 (25) 0.575 (23)c -0.148 (24)

1.000 (39) 0.061 (36)

1.000 (39)

1.000 (39) -0.008 (37)

1.000 (39)

Male

a

Pearson’s correlation coefficients.

b

1.000 (30) 0.130 (28) 0.028 (29)

Sample number. c p < 0.01.

FIGURE 1. Correlations between the 2,3,7,8-TCDD Toxic Equivalents (TEQs) and 8-OHdG Levels in By-Caught Cetaceans of Taiwan Waters. samples of cetaceans in Taiwan waters were 19.83 ( 10.00 pmol /µmol dG and 19.16 ( 7.48 pmol /µmol dG, respectively. Generally, the background levels of 8-OHdG in cetacean liver were within the range of those previously determined by high-performance liquid chromatography-electrochemical detection (HPLC-ECD) and reported for the control group of fish (2-4/105 dG) (11), rat (∼0.13-6/105 dG) (30-33), and human liver (0.8-3.1/105 dG) (34). The levels of 8-OHdG in cetacean kidney were also similar to the levels previous found in rat kidney (1.8 ( 0.3/105 dG) (35). Results of 8-OHdG in different cetacean species were given in Table 1, and data of the different gender and maturity in by-caught and stranded animals were shown in Table 2. For species, the highest mean level of 8-OHdG in liver and kidney was from Cuvier’s beaked whale (36.69 pmol /µmol dG) and Short-finned pilot whale (25.35 pmol /µmol dG), respectively; the lowest mean level in liver and kidney was from Finless porpoise (8.77 pmol /µmol dG) and Common dolphin (14.09 pmol /µmol dG), respectively (Table 1). Overall, no significant differences were observed in the total level of 8-OHdG between by-caught and stranded cetaceans in liver and kidney and also between species. However, most of the sample numbers of different species was very limited; thus, more sample data were necessary for a better representative to the levels of 8-OHdG in some species (Table 1). The 8-OHdG values in liver and kidney of the by-caught and stranded group did not significantly differ between maturity status and genders, 2458

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except those in the kidney of the stranded female group (25.28 ( 8.17 pmol /µmol dG) versus the stranded male group (13.35 ( 4.52 pmol /µmol dG) (p < 0.01) and of the stranded mature group (15.86 ( 7.06 pmol /µmol dG) versus the stranded immature group (25.54 ( 8.96 pmol /µmol dG) (p ) 0.04) (Table 2). Stranded cetaceans had poor health status due to diseases, injury, and/or starvation when they were found. A variety of ROS is continuously generated in response to the inflammatory and pathological processes; thus, the most abundant oxidative DNA adduct, 8-OHdG, may result from the continuous ROS generation associated with chronic inflammation (36, 37). Therefore, we compared the stranded cetaceans to the accidentally caught ones to support the hypothesis through a natural exposure but not from an experimental control study. However, the levels of 8-OHdG in liver and kidney in our data did not comply with the health status of individuals. Fraga et al. (6) demonstrated that the levels of 8-OHdG in the liver, kidney, and intestine of rat increased during aging might be due to decreased repair efficiency or increased rate of oxidative DNA damage. In this study, there is no agedependent increase of 8-OHdG, and the corresponding level in the stranded immature group was even higher than the mature group (p ) 0.04), contrary to Fraga’s study (Table 2). Since factors of stranding were difficult to determine and due to the limitations of acquiring the precise age of individual

animals in this study, age-dependent associations of 8-OHdG in cetaceans were difficult to examine in the current nature study. As to stranded female cetaceans’ kidneys having more oxidative DNA damage than males’ (p < 0.01) (Table 2), gender might be a factor. However, many spurious factors in stranded animals are still mysterious, and additional research is necessary to disclose the relationship. Correlation between 8-OHdG and PCBs, DDE. The concentrations of 19 PCBs and DDE in 73 bubblers from our previous investigation (20, 21) were retrieved and were compared with those same origin animals’ liver or kidney paired samples for the 8-OHdG studies. In brief, the PCBs and DDE concentrations were determined by gas chromatography/mass spectrometry (GC/MS) after sample homogenization, saponification, liquid-liquid extraction, and silica gel solid-phase extraction. Among all sample results, no significant correlation was found between DDE and 8-OHdG. The same findings were also found between paired correlations of PCBs and 8-OHdG, DDE and 8-OHdG, for the bycaught group, stranded group, different maturities group, and different species groups. However, there was significant positive correlation demonstrated between PCBs and 8-OHdG in female liver samples (r ) 0.58, p < 0.01) but not in male (Table 3). These results indicated that oxidative DNA damage may be associated with sex-specific exposure to PCBs. The 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs) using mammal-specific 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent factors (TEFs) reported by the World Health Organization were applied to assess the toxic potential of coplanar PCBs (38). In this study, non-ortho coplanar PCB77, -126, and -169 and mono-ortho coplanar PCB105, -118, -156, -157, and -189 from our previous detected PCB congeners were selected for DNA oxidative damage assessment. To minimize the confounding factors, livers only from healthy by-caught cetaceans were chosen to assess the correlation of 8-OHdG and TEQs. A significant correlation (r ) 0.31, p < 0.05) was found between 8-OHdG and TEQs in total bycaught cetaceans, and a better correlation was shown for female animals (r ) 0.62, p < 0.01). A highly positive correlation was found when considering mature female cetaceans only (r ) 0.80, p < 0.01) (Figure 1). This indicates a relationship that becomes stronger with aging and sexual maturity in 8-OHdG and TEQs in female animals. Wyde et al. (39) reported that the induction of 8-oxo-dG by TCDD occurred through a chronic, sex-specific, estrogendependent mechanism. Tritscher et al. (40) also detected higher levels of 8-OHdG from liver of TCDD-treated intact rats than from the liver of TCDD-treated ovariectomized rats. A possible mechanism is through increasing the metabolism of endogenous estrogens by TCDD-induced enzymes, and the process of metabolism of estrogens also can lead to redox cycling and formation of ROS and thus lead to increased oxidative damage in the cell (40-43). In this study, these results suggested that high concentrations of PCBs in cetaceans and increased estrogen metabolism by coplanar PCBs in the liver could be part of the factors contributing to the formation of 8-OHdG in mature female cetaceans. To our knowledge, this was the first report on the levels of 8-OHdG in cetaceans. Most results of the paired groups in this study had no statistical significance, which might be due to the fact that background levels of 8-OHdG were affected by many factors. Thus, increasing the investigation population is necessary. However, this study was investigated by using a natural exposure model, not in the laboratory, where cetaceans were continuously exposed to low-dose environmental contaminants. The result of this study indicated a strong potential risk of formation of 8-OHdG in the liver of mature female cetaceans from continuous exposure to coplanar PCBs.

Acknowledgments We thank the Taiwan Cetacean Society for providing the samples and Dr. Chiung-Wen Hu’s help to process these samples.

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Received for review August 18, 2004. Revised manuscript received January 15, 2005. Accepted January 18, 2005. ES0487123