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Institute of Public Health, University of Copenhagen, Denmark
1Correspondence: Institute of Public Health, c/o Department of Pharmacology, The Panum Institute, Bldg. 18.5, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. E-mail: s.loft{at}pubhealth.ku.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ethinylestradiol (EE) exposure in vivo and in vitro after incubation with EE with or without an antiestrogen. In vivo, preadult (30–35 days) and adult (110–120 days) Wistar rats received 0, 2.8, or 56 mg EE/kg body weight as intraperitoneal injections (n=6). After 1 or 4 h, the 8-oxodG/106 dG ratio was measured in the liver, kidneys, and testes. Testes DNA analysis revealed an age-related effect (adult animals had a higher ratio than the young animals) and a concentration effect in preadult rats (increased EE-concentration caused increased ratio), but no time effect. No differences were found in the liver or kidneys. In vitro, testicular cells were isolated and incubated with EE concentrations ranging from 0.1 to 1000 nM. The results indicated an increase in 8-oxodG/106 dG from 0 to 10 nM estrogen. At 1000 nM, the level was close to control level. Coincubation of 10 nM EE (maximum damage) with an estrogen antagonist, ICI 182.780, abolished the effect at 10 nM, indicating that the damaging effect is estrogen receptor mediated.—Wellejus, A., Loft, S. Receptor-mediated ethinylestradiol-induced oxidative DNA damage in rat testicular cells.
Key Words: 7,8-dihydro-8-oxo-2'-deoxyguanosine • glutathione • 17-ethinylestradiol
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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concurrent with a possible increase in male breast cancer (2)
, and sperm quality may be deteriorating (3)
. Mechanisms involving hormone-disrupting chemicals have been suggested (4)
, supported by similar reproductive changes seen in wildlife (5
6
7)
and in sons exposed to diethylstilbestrol in utero (8)
, although the exact mechanisms are obscure. Estrogens are capable of inducing oxidative DNA damage—including the mutagenic (9)
lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG)—in livers and kidneys of experimental animals (10
, 11)
, and this has been related to induction of tumors (12)
. Vitamin C, vitamin E, and ß-carotene have been demonstrated to reduce the formation of 8-oxodG in rat liver after oral exposure to EE, supporting the idea that formation of free radicals may participate in the carcinogenic process (13)
. 8-OxodG is abundant in sperm DNA, where it has been closely correlated to male fertility, sperm function and routine seminal parameters in humans (14)
. Accordingly, oxidative DNA damage may be involved in estrogen-induced effects on cancer risk and male reproduction. In this study, we examined the effect of the synthetic estrogen EE (2.8 and 56 mg EE/kg body weight, or bw) on levels of 8-oxodG in DNA from testes, livers, and kidneys from preadult and adult Wistar rats 1 and 4 h after administration. In vitro, we examined concentration–effect relationships of 8-oxodG after exposure of testicular cells to EE and the effect of the estrogen receptor antagonist ICI 182.780.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
Preadult Wistar rats (30–35 days) or adult Wistar rats (110–120 days) from Charles River, Germany, were used. The animals received standard laboratory diet (Altromin 1314) and tap water ad libitum. The animals were housed in pairs in an environmentally controlled animal facility operating on a 12 h dark/light cycle at 21–23°C and 50–70% humidity.
In vivo design
The preadult or adult animals were divided randomly into six groups of six rats receiving 0, 2.8, or 56 mg EE/kg bw by intraperitoneal injections. After 1 or 4 h, the animals were killed and liver, kidneys, and testes were excised. The tissue was stored at -80°C until analysis.
In vitro design
Preparation of testicular cells
The method described by Bradley and Dysart in 1985 (15)
was used with modifications. Testes from adult Wistar rats (preadult rats were not used due to the small amount of testis tissue) 110–120 days old were excised and decapsulated in cold RPMI 1640 medium containing pyruvic acid (0.5 mM). The tissue was cut up using a pair of scissors and incubated at 32°C in RPMI 1640 with collagenase (100 U/ml) for 20 min. Trypsin (0.25 mg/ml) was added and the suspension was incubated for another 12 min also at 32°C. To stop the trypsination, 8% fetal calf serum was added along with DNase I (0.2 mg/ml). After 15 min the resulting cell suspension was cooled off, filtered, and washed three times in E medium [140 mM sodium chloride (AppliChem, Darmstadt, Germany), 5 mM potassium chloride, 0.35 mM disodium hydrogen phosphate (Riedel-deHaën, Seelze, Germany), 0.35 mM potassium dihydrogen phosphate (Merck, Darmstadt, Germany), 0.8 mM magnesium sulfate (Merck), 20 mM HEPES, and 3 mM calcium chloride (Merck), pH 7.4, with 1% bovine serum albumin]. The tissue was centrifuged at 270 g for 5 min. Cell viability was >95% as measured by trypan blue exclusion. The cells were analyzed by the 8-oxodG analysis.
EE experiment
Pooled testicular cells from one animal were divided into six samples. The cells were incubated at 32°C for 30 min in 0, 10-10, 10-9, 10-8, 10-7, and 10-6 M EE in E medium. After incubation, the cells were washed once in E medium.
EE/ICI experiment
Pooled testicular cells from one animal were divided into five samples; 1 ml ICI 182.780, Tocris Cookson, Bristol, UK (0, 0, 10-10, 10-8, or 10-6 M in E medium, respectively) was added and the cells were incubated for 5 min at 32°C. Subsequently, 2 ml of 1.5 x 10-8 M EE (yielding a final concentration of 10-8 M EE) was added to all the samples except for the control, along with ICI concentrations similar to the first incubation. The samples were incubated an additional 30 min at 32°C. Testicular cells were also incubated with 10-6 M ICI 182.780 alone for 30 min at 32°C.
After incubations, the cells were washed once in E medium. The cells were analyzed by 8-oxodG analysis. The in vitro experiments were repeated five to seven times.
8-oxodG analysis
The analysis used by Helbock et al. (16)
was followed with minor modifications. Kidney-, testis, or liver tissue (100 mg) or cell suspensions were homogenized in 1.5 ml buffer A containing 320 mM sucrose, 5 mM magnesium chloride, 10 mM Trizma®base, 0.1 mM deferoxamine mesylate, and 1% Triton®X-100 (AppliChem) pH 7.5. The homogenizer was washed in 0.5 ml buffer A and the nuclear pellets were collected by centrifuging 1500 g for 10 min. The pellets were washed once in 2 ml buffer A. After centrifugation, 600 µl buffer B containing 5 mM EDTA (AppliChem), 10 mM Trizma®base, and 0.15 mM deferoxamine mesylate, pH 8.0, and 35 µl 10% sodium dodecyl sulfate (Merck) was added and the samples were vigorously agitated. RNase A 30 µl (1 mg/ml) and RNase T1 8 µl (1 U/µl) (both purchased from Boehringer Mannheim, Mannheim, Germany) dissolved in buffer B were added and the samples were incubated at 50°C for 15 min. Proteinase K 30 µl (20 mg/ml) in buffer B was added and the samples were incubated for another 60 min at 37°C. After centrifugation at 5000 g for 15 min, the supernatants were collected. Exactly 1.2 ml of EDTA 20 mM, 7.6 M sodium iodide (AppliChem), 40 mM Trizma®base, and 0.3 mM deferoxamine mesylate, pH 8, and 2 ml of 2-propanol (Romil, Cambridge, UK) were added. The samples were mixed carefully by inversion until signs of DNA precipitation. The DNA was collected by centrifugation at 5000 g for 15 min and washed first in 1 ml of 2-propanol (40%), then in 1 ml ethanol (70%) (Danisco A/S, Aalborg, Denmark). After centrifugation the DNA is dissolved in 100 µl water and digested by 10 µl nuclease P1 (1 U/µl) in 300 mM sodium acetate (Merck), 1 mM zinc chloride (Merck), pH 5.3, for 30 min at 37°C. Twelve microliters of alkaline phosphatase (1 U) (Boehringer Mannheim) dissolved in 500 mM Trizma®base and 1 mM EDTA, pH 8, was added and the solution was incubated for another 60 min. Approximately 50 µl of chloroform (Romil) was added and the samples were centrifuged at 5000 g for 15 min. (In the EE/ICI in vitro experiment, interference with the detection of 8-oxodG by the electrochemical detector was observed. Washing the samples three times in 100 µl chloroform obviated the problem.) An aliquot of the supernatant was injected into the chromatographic column (Progidy, Phenomenex, CA; ODS 25 cm-5 µm particle size). The column was eluted with 4.85% acetonitrile (Romil) in a sodium phosphate buffer, pH 6. The effluent was monitored by a UV detector (Waters, 254 nm) for quantification of dG and by an ESA Coulochem II electrochemical detector (Millipore Corp., Bedford, MA) with a 5011 analytical cell set for quantification of 8-oxodG. The chromatographic data were processed by Merck-Hitachi integration software. Quantification of the oxidized and normal nucleoside was performed by external standardization from injection of known amounts of pure 8-oxodG and dG.
The coefficient of variance for testicular 8-oxodG/106 dG measurements on different samples from the same animal was 18% (n=8), whereas interindividual CVs for the mature and preadult animals were 40% and 21% (n=10), respectively. The CV for in vitro 8-oxodG/106 dG measurements in different preparations from the same testis was 34%. The laboratory participates in the European Standard Committee for Oxidative DNA Damage (ESCODD), where our analytical results of 8-oxodG are in the lowest range of values and variation (17
, 18)
.
Glutathione
Testicular cells were prepared as described earlier. The cells from one animal were divided into four samples and incubated with 10-10, 10-8, 10-6, and 0 M EE in E medium for 30 min at 32°C. After incubation, the cells were washed in PBS and resuspended in 1 ml PBS. A suspension of 100 µl was saved for protein content measurement, whereas the remaining 900 µl was used to measure the oxidized (GSSG) and total glutathione (GSSG+GSH) content. The enzymatic method by Floreani et al. (19)
originally proposed by Tietze in 1969 (20)
was later modified by Anderson (21)
.
Protein measurement
A commercially available kit (Coomassie® Protein Assay Reagent Kit, Pierce, Rockford, IL) determined the protein concentrations.
Statistics
In vivo data were tested by multifactorial ANOVA with post hoc comparisons by means of least significant difference. All 8-oxodG values were log transformed for normal distribution and homogeneity of variance. Linear regression analysis on dose effects in the preadult rats was performed by the methods of least squares. To test the effect of age, 8-oxodG levels in the testes from the preadult and the adult animals were compared by pooling the control data after 1 and 4 h and testing these two groups by means of the Student’s t test. The in vitro data was analyzed by two-way ANOVA with repeated measures. Post hoc comparisons were done by means of least significant differences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In fact, the levels were very similar after 1 and 4 h in every pair of groups. In the preadult rats, the testicular 8-oxodG levels after 56 mg EE/kg bw were significantly increased by 1.9-fold (after 1 h of exposure) and 1.6-fold (after 4 h of exposure) compared with the controls. In preadult rats, the 8-oxodG level was significantly higher after 56 mg EE/kg bw than after 2.8 mg EE/kg bw at the 1 h sampling time. In these animals, regression analysis of the testicular 8-oxodG values showed a dose-response relation that was significant after 1 h (P<0.005) and borderline significant after 4 h (P=0.06). In the mature control animals, 8-oxodG levels in testis DNA were 1.5-fold higher than in the corresponding preadult rats (P<0.05). In adult rats, the relationship between 8-oxodG levels and EE showed the same dose-response trend as in preadult rats, but did not reach statistical significance, possibly due to higher background levels and variation in the older rats.
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No significant effects on 8-oxodG levels in liver or kidneys were seen after EE administration in preadult or adult rats (Table 1
). No effects on body weight or absolute organ weights were observed. In preadult rats, the relative total testicular weights decreased in the two experimental groups vs. the control group after 1 h of exposure (Table 1)
, whereas no changes were seen in testes from the 4 h sampling point, testes from the mature rats, or the relative weight of liver and kidneys. There was no significant individual correlation between the total relative testicular weights and log 8-oxodG/106 dG in testicular DNA from the preadult rats (r=-0.19, P=0.27).
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In vitro experiments
After incubation of isolated testicular cells with EE (10-10-10-6 M) in vitro, the level of 8-oxodG increased and then decreased, approximating a bell-shaped concentration-effect curve (Fig. 2
). The maximum level of 8-oxodG was observed at 10-8 M EE and levels similar to control levels were seen at 10-6 M EE.
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Coincubation of isolated testicular cells with increasing concentrations (10-10 to 10-6 M) of ICI 182.780 inhibited the 8-oxodG-inducing effect of 10-8 M EE. Maximum inhibition of EE-induced 8-oxodG formation was achieved at 10-8 M ICI 182.780 (Fig. 3
). Compared with control, no change in the 8-oxodG level from testicular cells incubated with ICI 182.780 alone was observed (results not shown). After EE exposure, no significant effects was recorded with respect to the level of total glutathione per gram protein or the ratio between oxidized and total glutathione in isolated testicular cells (Table 2
).
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DISCUSSION |
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In a wide range of estrogen receptor-mediated assays, maximal effects of EE and ICI 182.780 were elicited in similar concentration ranges (22)
. Recently it was shown that in estrogen receptor-positive cells, considerable more single strand breaks in the DNA were induced by 4-hydroxyequilenin, a catechol metabolite of the estrogen replacement pharmaceutical equilenin, than in estrogen receptor-negative cells after 3 h of incubation (23)
. The present study suggests that the catechol estrogen bound to the estrogen receptor is transported to estrogen-sensitive genes in the nucleus, where redox cycling may take place. The limited change in oxidized and total glutathione levels is consistent with localized redox cycling of EE in the nucleus, where the pool of glutathione is considered more resistant to depletion than the cytoplasmic pool (24)
.
Estrogen receptors are abundant in the male reproductive tract (25)
; recent findings of the estrogen receptor ß in high numbers in male genitals (26)
emphasize its importance in male reproduction. Other findings support this: the P450 aromatase is present in testicular germ cells and epididymal sperm (27
, 28)
, estrogen receptors exist in human sperm (29)
, and estradiol concentrations in semen and rete testis are extremely high (30
, 31)
.
An estrogen receptor situated in the membrane of sperm (32)
and testicular cells (33)
capable of mediating extremely rapid nongenomic responses to estrogen exposure has recently been found. In human sperm, a rapid and sustained increase in intracellular calcium concentrations and tyrosine phosphorylation of several sperm proteins were observed after estrogen exposure (32
, 34)
. Sperm motility has been connected to second messenger systems—most notably, cyclic adenosine monophosphate (cAMP) (35)
and intracellular calcium (36)
—and incubation of human spermatozoa with estradiol caused an increase in motility, oocyte penetration, and oxidative metabolism (37)
. It is therefore possible that estrogens can mediate acute oxidative damage by increasing the oxidative metabolism in spermatozoa and perhaps in spermatids, spermatogonia, and other cells. Gene activation has been observed at low concentrations after exposure to the natural estrogen 17 ß-estradiol (38)
. Down-regulation of DNA repair enzymes (39)
and antioxidant enzymes (40)
by estrogens is also suspected mechanism. However, this would not be likely to occur as soon as 30 min, as in the present study. In vivo, the 8-oxodG level was increased to the same extent 1 and 4 h after EE dosing, supporting a rapidly acting mechanism.
Catechol estrogens formed by metabolism of estrogens can either be inactivated by catechol-O-methyltransferase or oxidized by cytochrome P450 enzymes. During oxidation, reactive oxygen species (ROS) is produced (41)
. The superoxide radical is reactive, but the main danger presented by the molecule is its conversion to hydrogen peroxide and via the Fenton reaction to the hydroxyl radical (42)
. Previous work has shown that oxidation of catechols of EE and estradiol by Cu2+ produces the hydroxyl radical (43)
. Other metal ions such as Fe3+ may also participate in the oxidation of catechol estrogens, and the presence and transition state of the metal ions along with the concentration of the catechol estrogens seem to determine whether a pro-oxidant or an antioxidant effect will predominate (44
, 45)
. The nonsignificant decrease in the 8-oxodG level observed at low EE concentrations after in vitro incubation (see Fig. 2
) could be the result of antioxidant capacity of EE, as shown by other estrogens with respect to neuroprotective (46)
and atheroprotective effects (47
, 48)
. The decrease in oxidative DNA damage observed at high EE concentrations after in vitro incubation (see Fig. 2
) can also result from an inhibition of EE hydroxylation by 2-hydroxylated EE or EE itself (49)
. Likewise, it cannot be excluded that the reducing effect by ICI 182.780 on the EE-induced 8-oxodG level (see Fig. 3
) was due to inhibition of the catechol metabolism of EE. However, ICI 182.780 has only been shown to affect CYP1A1 2 hydroxylation in MCF-7 cells (50)
. Testicular cells express cytochrome P450s, particularly CYP3A4, in abundance (51)
, which is important in the generation of hydroxylated catechols of estrogens (52)
.
It has been widely discussed whether estrogens and estrogen-like chemicals are carcinogens; although EE is a carcinogen in both male and female rodents (53)
, many of these chemicals are negative in short-term assays for the induction of gene mutations in both prokaryotic (54
, 55)
and eukaryotic cells (56
, 57)
. Estrogens can damage DNA as measured by a wide range of different assays (12)
. Lipid–DNA adducts, DNA strand breaks, and genome alterations (especially 8-hydroxylation of guanine) have been emphasized in addition to estrogen–DNA adducts. 8-OxodG is premutagenic if base pairing with adenine instead of cytosine. A dG:dC to dT:dA transversion can occur on replication if 8-oxodG is formed within the DNA, or a dA:dT to dC:dG transversion may be induced if 8-oxodG is incorporated from the nucleotide pool into DNA (58)
. DNA from invasive ductal carcinoma tissue had a lower ratio of ring-opened products (e.g., 4,6-diamino-5-formamidopyrimidine) to hydroxy adducts of adenine and guanine than DNA from mammaplasty tissue (59)
. Male Syrian hamsters, which have a high incidence of renal tumors after chronic estradiol administration, had an increased 8-oxodG level in liver and kidney tissue vs. control after acute estradiol treatment (10)
. This strongly indicates a connection between estrogen exposure, formation of oxidated guanine residues in the DNA, and tumor development. Previous work suggests that oncogenes (60)
and tumor suppressor genes, such as p53 (61)
, may be involved in testicular carcinogenesis and that 8-oxodG might cause some of the changes in these genes. According to the IARC database on changes in p53 in testicular tissue (www.IARC.fr), dG:dC to dT:dA and dA:dT to dC:dG transversions exist even though they do not seem to be the most abundant.
Women using the contraceptive pill consume up to 50 µg EE per day. Thus, even the lower dose of 2.8 mg EE/kg used in the in vivo study is 
3000-fold higher than the daily human dose. However, during pregnancy, the combined concentration of plasma estrogens exceeds 100 nM (62)
, whereas peak plasma concentrations of EE were 34 nM after administration of 3 mg EE to a woman (63)
. Accordingly, the EE doses used in the in vivo study are not irrelevant, for example, to fetal estrogen exposure.
In addition to increased oxidative DNA damage, EE exposure caused a decrease in the relative paired testis weight from the preadult animals in the two experimental groups after 1 h of exposure. This was caused mainly by a decrease in the absolute paired testis weights, which was significant in the two experimental groups only if the 1 h sampling point was selected for analysis. These data support that estrogens have acute effects in the testes although the mechanism for the weight change is unknown.
In this study, no change in either kidney or liver oxidative DNA damage with age was observed, in agreement with previous results (64)
although others suggest an increase in nuclear 8-oxodG in these tissues (65
66
67)
. Conversely, an age-associated increase in the amount of nuclear testicular oxidative DNA damage in terms of 8-oxodG was found in this study. No association between age and increase in nuclear 8-oxodG had previously been found in rat testes (65)
. However, background levels of 8-oxodG were 10-fold higher, which may have obscured the age-related differences. In that study, Fischer 344 rats between 1 and 24 months of age were used whereas Wistar rats of 30–35 or 110–120 days were used in the present study. The use of Wistar rats and the age span of 30 to 120 days in the present study may have contributed to the different results.
In conclusion, EE induced oxidative DNA damage in rat testicular cells. The concentration-effect relationship and inhibition by an antiestrogen suggest that the effect is receptor mediated, although inhibition of metabolism of EE by ICI 182.780 cannot be excluded. The mechanism may involve second messenger systems activated by receptors situated in the cell membrane.
It is possible that estrogens have different effects on the individual cell populations in the testis. Future works should address this area.
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ACKNOWLEDGMENTS |
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Received for publication May 22, 2001.
Revision received October 17, 2001.
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significantly increased compared with the preadult control animals after 4 h of exposure (P<0.05), #significantly increased compared with the preadult animals exposed to 2.8 mg EE/kg bw for 1 h (P<0.05).
