Diallyl sulfide inhibits diethylstilbesterol-induced DNA adducts in the breast of female ACI rats

Food and Chemical Toxicology 43 (2005) 1323–1331 www.elsevier.com/locate/foodchemtox

Diallyl sulfide inhibits diethylstilbesterol-induced DNA adducts in the breast of female ACI rats M. Green a, C. Wilson a, O. Newell b, S. Sadrud-Din b, R. Thomas a

a,*

Environmental Toxicology Program, College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA b Department of Biology, College of Arts and Sciences, Florida A&M University, Tallahassee, FL 32307, USA

Abstract Diethylstilbestrol (DES) is metabolized to reactive intermediates that produce DNA adducts and ultimately cancer. Diallyl sulfide (DAS) has been shown to inhibit the metabolism of several procarcinogens. The ability of DES to produce DNA adducts in microsomal, mitochondrial, and nuclear in vitro metabolic systems and in the breast of female ACI rats, as well as ability of DAS to inhibit DNA adducts were investigated. Microsomes, mitochondria, and nuclei isolated from breast tissue of female ACI rats were used to catalyze oxidation reactions. Female ACI rats were treated i.p. as follows: (1) corn oil, (2) 200 mg/kg DES, (3) 200 mg/kg DES/200 mg/kg of DAS, (4) 200 mg/kg DES/400 mg/kg DAS. DES produced DNA adducts in each metabolic system. The relative adduct levels were 2.1 · 10 4, 6.2 · 10 6, and 2.9 · 10 7 in microsomal, mitochondrial, and nuclear reactions, respectively. DAS inhibited DNA adducts in each metabolic system. The percent inhibition ranged from 86% in microsomes to 93% in nuclei. DES produced DNA adducts in mtDNA and nDNA. DAS completely inhibited the DES-induced mtDNA adducts and caused a dose dependent decrease in nDNA adduct formation. These findings suggest that DAS could inhibit DES-induced breast cancer by inhibiting its metabolism.
2005 Elsevier Ltd. All rights reserved. Keywords: Diethylstilbesterol; Diallyl sulfide; Chemoprevention; DNA adducts

1. Introduction Among women ages 30–60, breast cancer is the second leading cause of death (Greenlee et al., 2001). It has been demonstrated that exposure to estrogens increase the risk for breast cancer (Adami et al., 1989). Diethylstilbesterol (DES) is a hormonal chemical that mimics the activity of estrogen. DES has been shown Abbreviations: DES, diethylstilbesterol; DAS, diallyl sulfide; dAP, deoxyadenosine monophosphate; dGMP, deoxyguanosine monophosphate; RALs, relative adduct levels; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; NP1, nuclease P1; PEI, polyetheleneimine; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; PMSF, phenylmethyl sulfonyl fluoride; PNK, T4 polynucleotide kinase; SPD, spleen phosphodiesterase; TLC, thin layer chromatography. * Corresponding author. Tel./fax: +1 850 599 3347. E-mail address: [email protected] (R. Thomas). 0278-6915/$ - see front matter
2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.02.005

to produce cancer in humans, as well as in animals, particularly breast cancer (Herbst, 1984; Greenberg et al., 1984). The mechanism of estrogen-induced breast cancer is not clearly understood. It was believed that the overstimulation of the estrogen receptor resulted in cell tranformation by altering gene regulation, protein synthesis, and cell growth (Jenson, 1992). However, a lack of correlation between estrogenecity and tumor incidence suggest that factors other than estrogen potency are involved in estrogen-induced neoplasm (Roy and Liehr, 1990). Recent data suggests estrogen metabolism plays a significant role in carcinogenesis. DES undergoes redox-cycling which leads to the formation of reactive intermediates, such as DES-semiquinone and DES quinone (Liehr and Roy, 1990; Roy and Liehr, 1992). These reactive intermediates covalently bind to DNA and nuclear proteins forming adducts (Williams et al.,

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1993; Roy and Pathak, 1993; Bhat et al., 1994). These DNA adducts may lead to the initiation of estrogen-induced carcinogenesis. Redox-cycling of DES also results in the formation of reactive oxygen species that induce DNA damage including DNA base oxidation, DNA strand breaks, chromosomal damage, gene mutations, and lipid DNA adducts (Roy and Liehr, 1999; Cavalieri et al., 2000). In addition to initiation, the mitogenic action of estrogen could stimulate the promotion and progression of estrogen induced cancer (Fishman et al., 1995; Hiraku et al., 2001). Traditionally, metabolic studies have used microsomes. However, it has been demonstrated that mitochondria and nuclei have the metabolic capabilities to metabolize DES to reactive intermediates (Thomas and Roy, 1995; Roy and Thomas, 1994). Recently, we have demonstrated that redox-cycling of DES occurs in microsomes, mitochondria, and nuclei in the breast of female ACI rats, suggesting that DES metabolism occurs not only in the liver, but also in the breast which may subsequently lead to breast cancer (Thomas et al., 2004). Natural dietary products have recently received much attention as chemopreventive agents. Diallyl sulfide (DAS) is an organosulfur compound present in garlic that has been demonstrated to have potent chemopreventive properties. Diallyl sulfide has been demonstrated to inhibit various forms of chemically induced carcinogenesis (Wargovich, 1987; Wargovich et al., 1988; Sparnins et al., 1988; Hays et al., 1987). The chemopreventive properties of DAS are attributed to the modulation of metabolizing enzymes (Yang et al., 2001; Pan et al., 1993; Dragnev et al., 1995; Guyonnet et al., 1999; Guyonnet et al., 2000). We have also demonstrated that DAS inhibits the redox-cycling of DES in the breast of female ACI rats (Thomas et al., 2004). In the present study, the ability of DAS to inhibit the formation of DES induced DNA adducts was investigated in in vitro metabolic systems (mitoplasts, microsomes, and nuclei) as well as in female ACI rats. This study provides direct evidence that DAS inhibits the formation of DES-induced DNA adducts. The ability of DAS to inhibit the formation of DES-induced DNA adducts is significant in that it may help explain the role of estrogen metabolism in DES induced cancer. This data will provide a rationale to investigate DAS as a possible chemopreventive agent in estrogen-induced cancers such as breast, cervical and uterine cancer.

2. Materials and methods 2.1. Chemicals Diethylstilbesterol (DES), diallyl sulfide (DAS), bnaphthoflavone, chloroform:isoamyl alcohol (24:1),

phenol:chloroform:isoamyl alcohol (25:24:1), Proteinase K, Ribonuclease A, sucrose, phenylmethyl sulfonyl fluoride (PMSF), micrococcal nuclease, zinc chloride, sodium acetate, sodium succinate, CHES buffer, urea, lithium formate, sodium phosphate, deoxymonophosphate (DAP), and deoxyguanine monophosphate (dGMP) were purchased from Sigma Chemical Company (St. Louis, MO); carrier-free (32P) phosphate in water was purchased from MP Biomedicals (Irvine, CA); nuclease P1 (NP1) and calf spleen phosphodiesterase (SPD) were purchased from EMD Biosciences, Inc. (La Jolla, CA); T4 polynucleotide kinase (PNK) was purchased from US Biologicals (Swampscott, MA); and polyetheleneimine (PEI)-cellulose TLC plates were purchased from Fisher Scientific (Savannah, GA). 2.2. In vitro study 2.2.1. Animals Female ACI rats weighing 100–115 g (6–7 weeks old) were purchased from Harlan (Indianapolis, IN). The rats were housed in groups of two per cage in a climate-controlled room with a 12-h light/dark cycle. The rats were provided a standard diet (Harlan Teklad, Indianapolis, IN) and water ad libitum. The studies were carried out under established federal guidelines for the care and use of laboratory animals and were approved by the Florida A&M University Animal Care and Use Committee. Ten female ACI rats were treated with b-naphthoflavone (50 mg/kg i.p.) daily for 4 days. The rats were sacrificed by exposure to carbon dioxide. The breast tissue was dissected and homogenized in 5 volumes of 0.25 M sucrose solution containing 1.0 mM phenylmethyl sulfonyl fluoride. The homogenate was kept on ice at all times until used to isolate nuclei, mitoplasts, and microsomes by centrifugation at 4 C. 2.2.2. Nuclei preparation The homogenate was filtered through four layers of cheesecloth and centrifuged for 15 min at 1000 · g. The pellet was resuspended in 0.25 M sucrose and underlayed with 4 volumes of 2.3 M sucrose. The nuclei were pelleted by centrifugation at 100,000 · g for 60 min. The nuclei were then washed with 0.25 M sucrose. The purity of the nuclei was assessed by both morphological and biochemical analyses. Nuclei were stained with hematoxylin and eosin. Phase-contrast microscopy confirmed the presence of intact nuclei. The determination of cytochrome c oxidase (Wharton and Tzagoloff, 1967), an enzymatic marker of mitochondria, showed very low activity (2 lmol/mg protein/min). The activity in purified mitochondria ranged from 100 to 110 lmol/mg protein/min. Microsomal contamination was assessed by measuring the activity of glucose

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6-phosphatase, an enzymatic marker of endoplasmic reticulum (Baginski et al., 1974). The activity of glucose 6-phosphatase in nuclei was <1% of that found in microsomes (5.0 pmol/mg protein/min in nuclei versus 563 pmol/mg protein/min in microsomes). This is in agreement with the reports of Niranjan and Avadhani (1980) and Niranjan et al. (1985). These results suggested that nuclei preparations were highly pure.

and three times with chloroform:isoamyl alcohol (24:1). The DNA was precipitated with two volumes of ice-cold ethanol, resuspended in distilled water, and analyzed by 32P-Postlabeling. The purity of DNA was analyzed by measuring absorbance of DNA at 260 and 280 with UV spectrometry. The 260/280 ratio ranged from 1.65 to 1.8 indicating the DNA was relatively pure.

2.2.3. Mitoplasts preparation The supernatant that was remaining after the 1000 · g centrifugation in the isolation of nuclei was used to collect mitoplast. The mitochondria were pelleted by centrifugation at 11,000 · g for 30 min. To exclude the possibility of contamination with endoplasmic reticulum, the outer membrane of mitochondria was removed by treating with 1.6% digitonin. After 30 min of gentle stirring, samples were centrifuged at 11,000 · g for 15 min and mitoplasts were collected. The purity of mitoplasts was assessed by both morphological and biochemical analyses. Analyses suggested that mitoplast preparations were highly pure.

2.3.2. Chemical reactions between DES quinone and dGMP DES quinone was prepared as described by Roy and Liehr (1992). One mg of dGMP (dissolved in 1.4 ml water) was reacted with 1 mg DES quinone (dissolved in 0.6 ml ethanol) for 4 h at 25 C. Reaction mixtures were extracted three times with water-saturated ethyl ether. The concentration of dGMP remaining in the aqueous phase was estimated at 260 nm. The samples were stored at 80 C unless used immediately for adduct analysis.

2.2.4. Microsomal preparation The supernantant remaining from the 11,000 · g centrifugation was centrifuged at 40,000 · g for 60 min to collect microsomes. The purity of microsomes was assessed by both morphological and biochemical analyses. Phase-contrast microscopy did not reveal any cellular contamination. Analyses revealed the presence of microsomes with no mitochondrial or nuclear contamination. The protein concentration of the organelles (nuclei, mitoplasts, and microsomes) was determined using a BioRAD protein assay kit. The organelles were stored at 80 C until needed. 2.3. Oxidation system for the production of DES quinone DNA adducts The oxidation reaction system contained mitoplasts (0.42 mg equivalent mitochondrial protein), microsomes (0.346 mg equivalent microsomal protein), or nuclei (0.450 mg equivalent nuclear protein) cumen hydroperoxide (120 lM), DES (100 lM), and 200 lg of DNA in a final volume of 1 ml 10 mM phosphate buffer, pH 7.5. The DNA was isolated from testes of non-treated male Sprague Dawley rats. Parallel reactions were carried out in the presence of diallyl sulfide (400 lM). The concentrations were determined based on previously published data (Thomas et al., 2004). The oxidation reactions were incubated for 30 min at 37 C. 2.3.1. DNA extraction The DNA from the oxidation reactions was extracted once with phenol:chloroform:isoamyl alcohol (25:24:1)

2.4. In vivo study 2.4.1. Animal treatment Four groups of 10 ACI rats were dosed to determine the ability of DAS to inhibit DES-induced DNA adducts. The rats were dosed i.p. with corn oil as vehicle, DES, and/or DAS as follows: Group 1 (control) received corn oil; Group 2 received DES (200 mg/kg); Group 3 received DES (200 mg/kg) and DAS (200 mg/ kg); Group 4 received DES (200 mg/kg) and DAS (400 mg/kg). The doses were determined based on previously published data (Gued et al., 2003; Green et al., 2003). Both mtDNA and nDNA from breast tissue was isolated from each group and analyzed by 32Ppostlabeling. 2.4.2. DNA isolation Breast tissue from female ACI rats was homogenized in 5 volumes of 0.25 M sucrose solution containing 1.0 mM phenylmethyl sulfonyl fluoride and then centrifuged at 1000 · g for 30 min to collect the nuclei. The remaining supernatant was centrifuged at 11,000 · g for 30 min to collect the mitochondria. To isolate the DNA, the nuclei and mitochondria were then suspended in 50 mM Tris, 10 mM EDTA, pH 8.0 containing 1% SDS and homogenized. The homogenate was treated with proteinase K (500 lg/ml) for 16 h at 37 C and then RNAase-A (150 lg/ml) and RNAase TI (20 U/ml) for 30 min at 37 C. The DNA was extracted once with phenol:chloroform:isoamyl alcohol (25:24:1) and three times with chloroform isoamyl alcohol (24:1). The purity of DNA was checked by ultraviolet spectroscopy and agarose gel electrophoresis.

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2.4.3. 32P-Postlabeling assay Analysis of covalent modification in DNA was carried out by 32P-postlabeling as follows. DNA (10 lg) was digested with micrococcal nuclease/spleen phosphodiesterase (0.133 unit Mn + 1.33 lg SPD/ll) in 10 mM CaCl2, 30 mM Na Succinate (pH 6.0) in a total volume of 10 ll for 3.5 h at 37 C. The samples were further digested by adding 5.0 ll of a NP1 cocktail (nuclease P1 (4 lg/ll), 1.60 ll of 1 mM ZnCl2, 0.9 ll of 1 mM Na acetate (pH 5.0)) and incubated for 40 min at 37 C. Following incubation, 3.0 ll of 500 mM of Tris (pH 8.0) was added to each sample. The nucleotides resulting from the digest as well as dGMP standards were labeled with 5 ll of P32-post label reaction mixture consisting of cP32 ATP (100 lCi/ll), T4 polynucleotide kinase (PNK) (30 mM/ll) buffered with kinase buffer (pH 9.8) and incubated for 40 min at 37 C. To produce a dAP standard for the quantitation of DNA adducts, 5.0 ll dAP (2 pmol/ll), 2.5 ll of CHES (50 mM pH 9), and 2.5 ll of P32 labeling reaction mixture was incubated for 40 min at 37 C. The ATP was synthesized as described by Gupta et al. (1982). The samples from DNA digest mixture were spotted on PEI-cellulose TLC plates. The unmodified nucleotides were separated from the adducted nucleotides on PEI-cellulose TLC plates using D1 solvent (2.3 M sodium phosphate, pH 5.7). The resolution of 32Ppostlabeled adducts was carried out on PEI-cellulose TLC plates with D3 (4.2 M lithium formate and 6.8 M urea, pH 3.3) and D4 (0.8 M sodium phosphate, 0.5 M Tris and 8.5 M urea) solvents. The 32P-postlabeled adducts were detected by autoradiography using X-Omat film. The spots containing 32P-postlabeled adducts were excised from the TLC plates, and levels of radioactivity were determined by Cerenkov counting. The relative adduct levels (RALs) were calculated as follows: CPMs in adducted nucleotides  CPMs/mole dAP = mole adducts, and mole of adducts/mole of digested nucleotides = RALs (Reddy and Randerath, 1986).

3. Results 3.1. In vitro study 3.1.1. Microsomal reactions DES was incubated in the presence of DNA, microsomes, and cumene hydroperoxide. In this oxidation system, seven DNA adducts were produced as identified by 32P-postlabeling (Fig. 1). The intensities of the spots on the autoradiograph in Fig. 1 corresponds to the relative adduct levels in Table 1. The total relative adduct level was 2.1 · 10 4. No adducts were produced in the control reaction which contained no cumen hydroperoxide. The addition of DAS to the reaction decreased the total relative adduct level to 2.9 · 10 5 representing an 86% inhibition of DNA adduct formation. Only adduct number 4 was completely inhibited by DAS. 3.1.2. Nuclear reactions DES was incubated in the presence of DNA, nuclei, and cumene hydroperoxide. In this oxidation system, 5 DNA adducts were produced as identified by 32P-postla-

Table 1 Relative adduct levels (RALs) of DES-DNA adducts produced by microsomes Adduct no.

Control DES + microsomes DES + microsomes + DAS

1 2 3 4 5 6 7

ND ND ND ND ND ND ND

6.7 · 10 4.4 · 10 8.8 · 10 6.4 · 10 3.2 · 10 1.9 · 10 4.5 · 10

5

Total adducts % Inhibition

ND

2.1 · 10

4

5 5 6 6 7 6

1.5 · 10 9.4 · 10 1.3 · 10 ND 1.2 · 10 2.1 · 10 9.0 · 10

5

2.9 · 10 86%

5

6 6

6 7 7

This table shows the RALs for each adduct in Fig. 1. ND = not detected (sensitivity: we are able to detect adducts at the level of 1 adduct per 1012 nucleotides).

Fig. 1. P32 autoradiograph of DES-DNA adducts produced by microsomes. The complete oxidation reaction system contained microsomes (0.346 mg equivalent microsomal protein), cumene hydroperoxide (120 lM), DES (100 lM), and DNA (200 lg) in a final volume of 1 ml (10 mM phosphate buffer, pH 7.5). Adducts shown in panel B are the results of the complete oxidation reaction. Panel A represents control reactions which did not contain any cumene hydroperoxide or diallyl sulfide. Adducts shown in Panel C are the results of the complete oxidation reaction with the addition of diallyl sulfide (400 lM).

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Fig. 2. P32 autoradiograph of DES-DNA adducts produced by nuclei. The complete oxidation reaction system contained nuclei (0.450 mg equivalent nuclear protein), cumene hydroperoxide (120 lM), DES (100 lM), and DNA (200 lg) in a final volume of 1 ml (10 mM phosphate buffer, pH 7.5). Adducts shown in panel B are the results of the complete oxidation reaction. Panel A represents control reactions which did not contain any cumene hydroperoxide or diallyl sulfide. Adducts shown in panel C are the results of the complete oxidation reaction with the addition of diallyl sulfide (400 lM).

beling (Fig. 2). The intensities of the spots on the autoradiograph in Fig. 2 corresponds to the relative adduct levels in Table 2. The total relative adduct level was 2.9 · 10 7. No adducts were produced in the control reaction which contained no cumene hydroperoxide. The addition of DAS to the reaction decreased the total relative adduct level to 1.9 · 10 8 representing a 93% inhibition of DNA adduct formation. Adducts 3, 5, and 6 were completely inhibited by DAS.

Table 2 Relative adduct levels (RALs) of DES-DNA adducts produced by nuclei Adduct no.

Control

DES + nuclei

1 2 3 5 6

ND ND ND ND ND

1.4 · 10 3.5 · 10 1.3 · 10 7.8 · 10 2.4 · 10

7

Total adducts % Inhibition

ND

2.9 · 10

7

8 8 8 8

DES + nuclei + DAS 1.1 · 10 7.8 · 10 ND ND ND

8

1.9 · 10 93%

8

9

This table shows the RALs for each adduct in Fig. 2. ND = not detected (sensitivity: we are able to detect adducts at the level of 1 adduct per 1012 nucleotides).

3.1.3. Mitochondrial reactions DES was incubated in the presence of DNA, mitoplasts, and cumene hydroperoxide. In this oxidation system, 3 DNA adducts were produced as identified by 32 P-postlabeling (Fig. 3). The intensities of the spots on the autoradiograph in Fig. 3 corresponds to the relative adduct levels in Table 3. The total relative adduct level was 6.2 · 10 6. No adducts were produced in the control reaction which contained no cumene hydroperoxide (data not shown). The addition of DAS to the reaction decreased the total relative adduct level to 7.1 · 10 7 representing an 88% inhibition of DNA adduct formation. Only adduct number 1 was completely inhibited by DAS. DES produced DNA adducts in all three in vitro metabolic systems (microsomal, mitochondrial, and nuclear). The microsomal metabolic system produced the highest level of DNA adducts when compared to the mitochondrial and nuclear systems. 3.1.4. In vivo study Administration of DES (200 mg/kg) produced both nDNA and mtDNA adducts in the breast of female ACI rats. There were 5 distinguishable nDNA adducts (Fig. 4). The pattern of the nDNA adducts in vivo

Fig. 3. P32 autoradiograph of DES-DNA adducts produced by mitoplast. The complete oxidation reaction system contained mitoplasts (0.42 mg equivalent mitochondrial protein), cumene hydroperoxide (120 lM), DES (100 lM), and DNA (200 lg) in a final volume of 1 ml (10 mM phosphate buffer, pH 7.5). Adducts shown in panel B are the results of the complete oxidation reaction. Adducts shown in panel A represents reactions in which dGMP was reacted with DES-quinone. Adducts shown in panel C are the results of the complete oxidation reaction with the addition of diallyl sulfide (400 lM).

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Table 3 Relative adduct levels (RALs) of DES-DNA adducts produced by mitoplast Adduct no.

DGMP + DESQ

1 2 3 4

9.6 · 10 7.4 · 10 4.1 · 10 1.3 · 10

Total adducts % Inhibition

5 4 6 5

DES + mitoplast 3.4 · 10 5.1 · 10 ND 7.1 · 10

7

6.2 · 10

6

6

7

DES + mitoplast + DAS ND 6.3 · 10 ND 8.9 · 10 7.1 · 10 88%

7

8 7

Table 4 Relative adduct levels (RALs) of nDNA adducts Adduct no.

DES only

1 2 3 5 6

1.2 · 10 9.0 · 10 2.4 · 10 4.0 · 10 1.8 · 10

7

Total adducts % Inhibition

2.9 · 10

7

8 8 8 8

200 mg/kg DAS/DES 2.6 · 10 6.0 · 10 ND ND ND

8

8.6 · 10 71

8

8

400 mg/kg DAS/DES 3.0 · 10 1.3 · 10 ND ND ND

7

4.3 · 10 85.3

8

7

This table shows the RALs for each adduct in Fig. 3. ND = not detected (sensitivity: we are able to detect adducts at the level of 1 adduct per 1012 nucleotides).

This table shows the RALs for each adduct in Fig. 4. ND = not detected (sensitivity: we are able to detect adducts at the level of 1 adduct per 1012 nucleotides).

was comparable to that of the in vitro nuclear metabolic system. The total relative adduct level was 2.9 · 10 7. DAS inhibited the formation of nDNA adducts in a dose dependents fashion. A dose of 200 mg/kg reduced relative adduct levels to 8.6 · 10 8 and a dose of 400 mg/kg reduced the relative adduct level to 4.3 · 10 8 representing a 71% and 85% inhibition in DNA adduct formation, respectively (Table 4). Although complete inhibition was not observed, adducts 3, 5, and 6 were completely inhibited by 200 mg/kg of DAS. Two mtDNA adducts were produced with a total relative adduct level of 8.9 · 10 8 (Table 5). The formation of mtDNA adducts was completely inhibited by 200 mg/kg of DAS (Fig. 5). No adducts were detected in the control group which received only corn oil. In the mitochondrial oxidation system, the pattern of the adducts were very similar to DES-quinone dGMP adducts. Also, the pattern of mtDNA adduct number 4 produced in vivo was very similar to a DES-quinone dGMP adduct. Based on this data and previous experiments, we propose that these adducts are dGMP adducts (Roy and Thomas, 2001). These dGMP adducts are important as they are the most difficult to repair. The chromatographic mobility of the adducts produced in the microsomal and nuclear oxidation systems, as well as nDNA adducts produced in vivo were not compara-

Table 5 Relative adduct levels (RALs) of mtDNA adducts Adduct no.

Control

DES only

4 5

ND ND

5.4 · 10 3.5 · 10

8

Total adducts % Inhibition

ND

8.9 · 10

8

8

200 mg/kg DAS/DES ND ND ND 100

This table show the RALs for each adduct in Fig. 5. ND = not detected (sensitivity: we are able to detect adducts at the level of 1 adduct per 1012 nucleotides).

ble to the dGMP adducts. Based on the chromatographic mobility, we have no reason to believe that these adducts are dGMP adducts. Therefore, no further analysis was performed.

4. Discussion For the first time, we have demonstrated that microsomes, mitochondria, and nuclei isolated from the breast of female ACI rats generate DES-induced DNA adducts. We have also demonstrated that DES administered to female ACI rats produce nDNA and mtDNA adducts in the breast. More significantly, DAS was demonstrated to inhibit DES-induced DNA adduct

Fig. 4. P32 autoradiograph of nDNA adducts produced by DES. Adducts shown in panel A represents animals administered 200 mg/kg DES only. Adducts shown in panel B represents animals administered 200 mg/kg DAS 30 min prior to 200 mg/kg DES. Adducts shown in panel C represents animals administered 400 mg/kg DAS 30 min prior to 200 mg/kg DES.

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Fig. 5. P32 autoradiograph of mtDNA adducts produced by DES. Panel A represents control animals that were administered corn oil. Adducts shown in panel B represents animals administered 200 mg/kg DES only. Panel C represents animals administered 200 mg/kg DAS 30 min prior to 200 mg/kg DES.

formation in all three in vitro metabolic systems as well as in the breast of female ACI rats. Our findings show the microsomal metabolic system produced more adducts when compared to the mitochondrial and nuclear metabolic systems. These differences are expected because microsomes are the major organelle of biotransformation and contain a higher level of cytochrome P450s. However, the mitochondria and nuclei do have some metabolic capabilities as demonstrated in this study by the production of DNA adducts. The in vitro results are supported by previous studies investigating the metabolism of DES. Thomas and Roy (1995) demonstrated that liver mitoplast metabolize DES to reactive intermediates which bind to DNA forming DNA adducts. DES was also converted to reactive metabolites by nuclei, which covalently bind to nuclear proteins and DNA (Roy and Thomas, 1994). In the present study, DAS inhibited DES-induced DNA adducts in all three systems. The percent inhibition ranged from 86% in the microsomes to 93% in the nuclei. This finding is of interest because the highest level of inhibition is found in the nucleus, which is the organelle that contains the genomic DNA, a critical target for carcinogens. DES administered to female ACI rats was shown to produce nDNA adducts in vivo, which was decreased by DAS in a dose dependent fashion. These results were similar to a previous study in which DAS was shown to inhibit DES-induced DNA adduct formation in the liver of male Sprague Dawley rats (Green et al., 2003). In addition, DES was demonstrated to produce mtDNA adducts, which was completely inhibited by DAS. This is an important finding as alterations to the mitochondria and mitochondrial genome are associated with carcinogenesis. Thomas and Roy (2001a,b) reported that DES produce mtDNA adducts in the liver and kidney of male Sprague Dawley rats. Fischer-344 rats administered a single dose or multiple doses 100 mg/kg 2amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), produced mtDNA adducts in the liver, with a significant higher adduct level observed with multiple doses (Davis et al., 1994). Benzo(a)pyrene, 2-acetylaminofluorene, and cigarette smoke produce higher level of mtDNA ad-

ducts when compared to nDNA adducts (Balasky et al., 1996). These procarcinogens require metabolic activation to the ultimate carcinogen that bind mtDNA. Although there is no direct link between mtDNA damage and carcinogenesis, it is a recurring theme in the induction of cancer. In previous studies, we have demonstrated that DAS administered prior to DES inhibits DES-induced lipid peroxidation in the breast of female ACI rats (Gued et al., 2003). This suggests that DAS inhibits the metabolism of DES to DES-quinone which leads to the formation of DNA adducts and other types of DNA damage in vivo. We have also shown that DAS inhibits the redox cycling of DES in microsomes, mitochondria, and nuclei isolated from the breast of female ACI rats (Thomas et al., 2004). These studies correlate with the present study in which DES produced DNA adducts and DAS inhibited the formation of these adducts. DES quinone produces DNA adducts and superoxide radicals produce lipid peroxides. By inhibiting the oxidation of DES with DAS, this will prevent the formation of DNA adducts and lipid peroxides. The inhibition of DNA adducts is of utmost importance, as DNA adducts are known as a precursor lesion for mutations. Mutations may occur at or near the site of DNA adducts as a result of replication errors during DNA synthesis. DNA adducts may result in frameshifts, deletions, or base substitutions (Garner, 1998). These mutations may activate oncogenes or inactivate tumor suppressor genes resulting in aberrant protein expression and/or alterations in cell cycle control (Garner, 1998; Kozack et al., 2000). In addition, mutations in genes that code for DNA-repair enzymes may result in impaired function of these enzymes which will result in new mutations, and subsequently, genomic instability and dysfunction (Garner, 1998). The results of this study provide evidence that DAS alters the metabolism of DES by directly inhibiting the activity of cytochrome P450 enzymes in such a manner as to inhibit the formation of DNA adducts. Yang et al. (2001) reported DAS is a competitive inhibitor of CYP 2E1. Since cytochrome P450 2E1 is not involved in the metabolism of DES to DES-quinone, it is likely

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that DAS inhibits other cytochrome P450 enzymes. In addition, there may be other protective mechanisms that occur in vivo such as the induction of phase II enzymes, which would detoxify the DES reactive intermediates. DAS has been reported to induce glutathione S-transferase, microsomal epoxide hydrolase, and UDP glucuronosyltransferase in the liver (Guyonnet et al., 1999). Sparnins et al. (1988) reported that DAS induced glutathione S-transferase (GST) activity in the forestomach, but varied in their capacity to induce GST activity in the lung, liver, and small bowel. In conclusion, DAS was demonstrated to inhibit DES-induced DNA adducts in the breast, in vivo and in vitro. This inhibition may be due to alterations in metabolism of DES. By inhibiting DNA adduct formation, DAS may prevent the initiation of estrogeninduced breast cancer. Further studies are on-going to assess the anti-carcinogenic activity of DAS.

Acknowledgments This research was generously supported by DOD Grant # DAMD 17-02-1-0381, NIH/RCMI Grant # G12 RR03020, and Merck-UNCF Science Initiative.

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