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Diallyl sulfide induces the expression of nucleotide excision repair enzymes in the breast of female ACI rats
Toxicology Letters 168 (2007) 40–44
Diallyl sulfide induces the expression of nucleotide excision repair enzymes in the breast of female ACI rats Mario Green, Oneil Newell, Ayoola Aboyade-Cole, Selina Darling-Reed, Ronald D. Thomas ∗ College of Pharmacy, Florida A&M University, 302 West Pershing Street, Tallahassee, FL 32307, United States Received 7 July 2006; received in revised form 25 October 2006; accepted 25 October 2006 Available online 10 November 2006
Abstract Diethylstilbestrol (DES) causes DNA adducts resulting in breast cancer, whereas diallyl sulfide (DAS) inhibits cancer formation. We hypothesize that DAS induces the expression of nucleotide excision repair genes. To test this hypothesis, female ACI rats were treated for 4 days with corn oil, DES, DAS, and DAS/DES (50 mg/kg). The expression of P53, Gadd45a, PCNA, and DNA polymerase delta was analyzed by real-time PCR. DES decreased the expression of P53, Gadd45a and PCNA. DAS and DAS/DES increased the expression of all four genes. These results suggest that DAS enhances the ability of breast tissue to repair DNA damage thus preventing cancer. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Diallyl sulfide; Diethylstilbestrol; Chemoprevention; DNA repair and breast cancer
1. Introduction Diethylstilbestrol (DES) has been shown to induce breast cancer in female ACI rats (Marselos and Tomatis, 1993; Rothschild et al., 1987). The mechanisms of estrogen-induced carcinogenesis are not clearly understood. However, recent data suggest that estrogen metabolism plays an important role in estrogen-induced cancer. DES is metabolized to reactive intermediates (semiquinone and quinone) by cytochrome P450s (Liehr and Roy, 1990; Roy and Liehr, 1992). These reactive intermediates result in the formation of DNA adducts ∗
Corresponding author. Tel.: +1 850 561 2786/599 3389; fax: +1 850 599 3347. E-mail addresses: [email protected] (M. Green), [email protected] (O. Newell), [email protected] (A. Aboyade-Cole), [email protected] (S. Darling-Reed), [email protected] (R.D. Thomas).
(Gladek and Liehr, 1989). If these DNA adducts undergo one or more cycles of replication without repair, the damage becomes fixed resulting in genomic instability. This genomic instability may lead to the initiation of cancer. DNA repair enzymes maintain DNA integrity by removing damaged bases. The primary mechanism for the removal of stable (bulky) adducts is nucleotide excision repair (NER). Two pathways of NER are transcription couple repair (TCR) and global genomic repair (GGR). TCR is initiated by the stalling of RNA polymerase II by DNA lesions (bulky adducts) during transcription (Hanawalt, 1992; Sweder and Hanawalt, 1992; Selby and Sancar, 1993). GGR involves the scanning of the genome by specific damage recognition proteins such as xeroderma pigmentosum C (XPC), transcription factor IIH (TFIIH), xeroderma pigmentosum A (XPA), and the growth arrest DNA damage-inducible 45 (Gadd45a) in search of DNA damage (Van Steeg, 2001). XPC and Gadd45a have been shown to be regulated by
0378-4274/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2006.10.013
M. Green et al. / Toxicology Letters 168 (2007) 40–44
the tumor suppressor gene, p53 (Wang et al., 1999; Zhan et al., 1994). Once the damage is recognized, the remaining steps involved in NER are similar for both TCR and GGR. DNA is unwound and a repair bubble is created by xeroderma pigmentosum B (XPB) and xeroderma pigmentosum D (XPD) helicases. The repair bubble is followed by excisions on the 5 - and 3 -ends of the damage by xeroderma pigmentosum F (XPF) and xeroderma pigmentosum G (XPG) endonucleases. A new strand of DNA is synthesized by DNA polymerase delta/epsilon. The activity of DNA polymerase delta/epsilon is dependent on the presence of proliferating cell nuclear antigen (PCNA) (De Laat et al., 1999). Estrogens have been shown to inhibit nucleotide excision repair (Evans et al., 2003). This impairment may result in an increased rate of mutations that contribute to the initiation of estrogen-induced cancer. Previously, we have demonstrated that DES is metabolized to reactive intermediates resulting in the formation of DNA adducts in the breast of female ACI rats (Green et al., 2005). Furthermore, we have demonstrated that diallyl sulfide (DAS), an organosulfur compound found in garlic, inhibits DNA adduct formation (Green et al., 2005). The chemopreventive properties of DAS have been attributed to metabolic modulation (Guyonnet et al., 2002; Srivastava et al., 1997). The female ACI rat is a good model for studying estrogen-induced breast cancer because they develop breast cancer upon exposure to estrogens, including 17-beta estradiol and DES, and have a low rate of developing spontaneous tumors (IARC, 1979). We chose DES as our carcinogenic test compound because it has estrogenic activity similar to that of 17-beta estradiol and has been shown to be associated with breast cancer in both animals and humans (Marselos and Tomatis, 1993). In the present study, we have demonstrated for the first time that DAS enhances the expression of p53, Gadd45a, PCNA, and DNA polymerase delta, which are involved in nucleotide excision repair. These results suggest that DAS-induced DNA repair may play a role in the prevention of estrogen-induced breast cancer. 2. Material and methods 2.1. Chemicals DES, RNAlater and DAS were purchased from Sigma Chemical Company (St. Louis, MO). Trizol LS reagent was purchased from Invitrogen (Carlsbad, CA). SYBRgreen RealTime Mastermix (2X) and Stratascript First Strand cDNA Synthesis System were purchased from Stratagene (La Jolla,
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CA). The primers for growth arrest and DNA damage 45a (Gadd45a), p53, proliferating cell nuclear antigen (PNCA), and DNA polymerase delta were ordered from SuperArray, Inc. (Detrick, MD). Female ACI rats (5–6 weeks old) were ordered from Harland Sprague, Inc. (Indianapolis, IN). 2.2. Animal treatment Four groups of 10 ACI rats were analyzed for expression of DNA repair genes involved in nucleotide excision repair (Gadd45a, PCNA, p53 and DNA polymerase delta). Rats were treated i.p. daily for 4 days with corn oil (control), DES (50 mg/kg), DAS (50 mg/kg), and DAS/DES (50 mg/kg). The breast tissue was removed, immediately placed in RNAlater and stored in at −80 ◦ C. Total RNA was isolated from the breast tissue and analyzed for gene expression using real-time PCR. 2.3. RNA isolation Fifty milligrams of breast tissue were weighed and homogenized in the presence of 0.75 ml of Trizol LS Reagent. Following homogenization, the samples were incubated at room temperature for 5 min. Chloroform (0.2 ml) was added to the homogenate and vortexed vigorously for 1 min. The homogenate were incubated at room temperature for 15 min followed by centrifugation at 12,000 × g for 15 min at 4 ◦ C. The upper aqueous layer containing RNA was transferred to a new RNase-free tube without disturbing the interphase layer. RNA was precipitated by adding 0.5 ml isopropyl alcohol. The samples were incubated at room temperature for 10 min and centrifuged at 12,000 × g for 10 min at 4 ◦ C. The supernatant was aspirated and RNA pellet collected was washed with 1 ml of 75% ethanol. The pellets were air-dried and reconstituted in 25 l of RNase-free water. The quality of RNA was determined by measuring the OD260/280 ratio with UV–vis spectrophotometry. RNA purified by this method resulted in OD260/280 ratio of >1.7. The quality of RNA was further determined by formaldehyde agarose gel electrophoresis. The presence of 18S and 28S rRNA bands indicated high quality RNA with low degradation. 2.4. cDNA amplification The cDNA was synthesized using the Stratascript FirstStrand cDNA Synthesis System for RT-PCR. The first cDNA strand was synthesized by combining 10 g RNA, 3 l of Oligo (dT) (100 ng/l), 5 l of 10× first-strand buffer, 1 l RNase Block Ribonuclease Inhibitor (40 U/l) and 2 l of 100 mM dNTPs in a microcentrifuge tube. The total volume was adjusted to 38 l with DEPC-treated water. The reaction was mixed gently and incubated at 65 ◦ C for 5 min. The reactions were slowly cooled at room temperature (∼10 min) to allow the primers to anneal to the RNA. One microliter of Stratascript reverse transcriptase (50 U/l) was then added to the reaction mixtures. They were mixed gently and incubated
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M. Green et al. / Toxicology Letters 168 (2007) 40–44
at 42 ◦ C for 1 h. The reactions were terminated by incubation at 90 ◦ C for 5 min. The completed reactions were then chilled on ice and the cDNA was collected by brief centrifugation. The cDNA was used immediately for PCR or stored at −80 ◦ C. 2.5. Real-time RT-PCR analysis Real-time PCR was performed in a 96-well plate. 12.5 l of 2× SYBR Green master mix (SureStart DNA polymerase (5 U/l), 10× PCR reaction buffer, 50 mM magnesium chloride, 20 mM dNTP mix, 100% DMSO, 50% glycerol, SYBR Green I dye), 1 l primer (Gadd45a, p53, PCNA, or DNA polymerase delta (100 ng/l)), 1 l cDNA (250 ng/l), and 10.5 l dd H2 O were combined for a total volume of 25 l per well. The contents of the well were mixed by pipetting gently. The tubes were incubated in a thermocycler at 95 ◦ C for 15 min to activate Taq DNA polymerase. There were 45 cycles of PCR amplification performed consisting of denaturing at 95 ◦ C for 30 s, annealing at 55 ◦ C for 30 s, and extension at 72 ◦ C for 30 s. Relative gene expression was calculated by the following equation: relative gene expression (RGE) = 2
Ctcon −Cttreat
where Ctcon is the cycle in which the fluorescence signal reaches threshold for the control group of animals and Cttreat is the cycle in which the fluorescence signal reaches threshold for the treated group of animals. The relative gene expression was normalized to GAPDH using the following equations: relative GAPDH expression (RGPE) = 2CtconGAPDH −CttreatGAPDH and normalized gene expression = RGE/RGPE. 2.6. Statistics Statistical analysis was performed with GraphPad Prism software using one-way analysis of variance (ANOVA). Tukey’s multiple comparison post test was used to compare the means of treatments to the control with a significance level of 0.05 where p < 0.05 indicated significance.
Fig. 1. p53 gene expression in breast of female ACI rats treated with DAS and/or DES. Real-time amplification of p53 cDNA was performed as described in Section 2. Results are expressed as mean ± S.E.M. of three independent experiments. The (*) indicates data significantly different from the DES treatment group. The (**) indicates data significantly different from the DAS treatment group (p < 0.05).
The administration of DES decreased mRNA levels of Gadd45a by 92%. The administration of DAS increased Gadd45a expression by 181-fold, whereas coadministration of DAS/DES increased the expression of Gadd45a by 22-fold (Fig. 2). DAS reversed the DESinduced inhibition of the expression of Gadd45a. DES completely inhibited the expression of PCNA whereas DAS resulted in 5.2-fold increase in its expression. The co-administration of DAS/DES synergistically increased the expression of PCNA by 32.2-fold (Fig. 3). DAS reversed the DES-induced inhibition of the expression of PCNA. DES resulted in a 1.8-fold increase in the expression of DNA polymerase delta (Fig. 4). DAS increased the
3. Results The effects of DAS on the expression of DNA repair genes involved in nucleotide excision repair was investigated in the breast of female ACI rats using real-time PCR. The rats were dosed daily with 50 mg/kg of DES and/or DAS via i.p. injections. The administration of DES decreased the relative gene expression of p53 by 75% compared to the control. However, this reduction in gene expression was not statistically significant. The administration of DAS increased relative gene expression of p53 by 12.1-fold, whereas co-administration of DAS/DES increased the relative gene expression of p53 by 4.9-fold (Fig. 1). DAS reversed the DES-induced inhibition of the expression of p53.
Fig. 2. Gadd45a gene expression in breast of female ACI rats treated with DAS and/or DES. Real-time amplification of Gadd4a cDNA was performed as described in Section 2. Results are expressed as mean ± S.E.M. of three independent experiments. The (*) indicates data significantly different from the DES treatment group. The (**) indicates data significantly different from the DAS and the DES treatment groups (p < 0.05).
M. Green et al. / Toxicology Letters 168 (2007) 40–44
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4. Discussion
Fig. 3. PCNA gene expression in breast of female ACI rats treated with DAS and/or DES. Real-time amplification of PCNA cDNA was performed as described in Section 2. Results are expressed as mean ± S.E.M. of three independent experiments. The (*) indicates data significantly different from the DAS and the DES treatment groups (p < 0.05).
expression of DNA polymerase delta by 6.3-fold. Coadministration of DAS/DES synergistically increased the expression of DNA polymerase delta by 61-fold. Based on statistical analysis, DES does not alter the expression of DNA polymerase delta, Gadd45a, PCNA, and p53. The fact that DES causes DNA damage and does not induce the expression of DNA repair enzymes is significant. DES treatments alone can cause significant DNA damage, which may not be repaired. The lack of induction of DNA repair enzymes by DES may play a role in estrogen-induced breast cancer. Conversely, DAS alone and in combination with DES induced the expression of Gadd45a, PCNA, p53, and DNA polymerase delta, which may play a role in DAS-induced cancer prevention.
Fig. 4. Effect of DAS and/or DES on DNA polymerase delta gene expression in breast of female ACI rats. Real-time amplification of DNA polymerase delta cDNA was performed as described in Section 2. Results are expressed as mean ± S.E.M. of three independent experiments. The (*) indicates data significantly different from the DAS and the DES treatment groups (p < 0.05).
Previously, we have shown that DAS inhibits DESinduced DNA adduct formation in the breast of female ACI rats (Green et al., 2005). The mechanism of this inhibition is not clear, but may be partially attributed to increased detoxification as a result of the induction of phase II metabolism enzymes by DAS (Hu and Singh, 1997; Hu et al., 1996). Additionally, DAS may decrease the level of DES-induced DNA adducts through the upregulation of genes involved in nucleotide excision repair (Gadd45a, PCNA, and DNA polymerase delta). We have demonstrated DAS’s ability to up-regulate nucleotide excision repair genes and thereby provide a potential mechanism for DAS induced increases in DNA repair and DNA adduct removal. Gadd45a is one of many genes regulated by p53 (Smith et al., 2000). DES decreased the expression of both p53 and Gadd45a. Therefore, the DES induced decrease in Gadd45a expression observed in our study may be attributed to the decrease in p53 expression. These results were supported by the fact that estradiol was found to decrease p53 levels in human breast epithelial cells (Somai et al., 2003). Furthermore, DAS has been shown to increase the levels of p53 in Swiss albino mice (Arora et al., 2004). This suggests that the alterations in Gadd45a expression may be associated with alterations in p53 expression. The interaction of Gadd45a with PCNA is essential for nucleotide excision repair. PCNA stimulates DNA polymerase delta to replace damaged nucleotides (Hodgson and Smart, 2001). In our study, PCNA expression in rats treated with DES was completely down-regulated. PCNA expression was up-regulated in rats treated with DAS and DAS/DES. Additionally, we have shown that DES, DAS, and combined treatments of DAS/DES increased the expression of DNA polymerase delta. While all three treatment groups resulted in increased levels of DNA polymerase delta, the DAS/DES combination was significantly higher than the other groups. This up-regulation of both PCNA and DNA polymerase delta will increase DNA repair efficiency particularly in the DAS/DES group. Impairment of DNA repair systems will result in increased levels of DNA damage that may lead to mutations and ultimately result in cancer. We have demonstrated that DAS induces the expression of P53, Gadd45a, PCNA, and DNA polymerase delta. These changes in gene expression will increase the cells ability to remove DES-induced DNA adducts in the breast of female ACI rats providing an additional mechanism by which DAS exerts its anti-carcinogenic activity. Further
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studies are warranted on DAS and its role in DNA repair as there are several other proteins involved in this complex pathway. Nevertheless, enhancement of nucleotide excision repair by DAS provides a promising target for chemoprevention of estrogen-induced breast cancer and other cancers. Acknowledgements This research was generously supported by DOD Grant # DAMD 17-02-1-0381, NIH/RCMI Grant # G12 RR03020, NIH/NCRR Grant # 1C06 RR12512-01 and Merck-UNCF Science Initiative. References Arora, A., Siddiqui, I.A., Shukia, Y., 2004. Modulation of p53 in 7,12dimethylbenz[a]anthracene induced skin tumors by diallyl sulfide in Swiss albino mice. Mol. Cancer Ther. 3, 1459–1466. De Laat, S.W., Boonstra, J., Defize, L.H., Krujer, W., van der Saag, P.T., Tertoolen, I.G., van Zoelen, E.J., den Hertog, J., 1999. Growth factor signalling. Int. J. Dev. Biol. 43 (7), 681–691. Evans, M.D., Butler, J.M., Nicoll, K., Cooke, M.S., Lunec, J., 2003. 17 beta-O-estradiol attenuates nucleotide excision repair. FEBS Lett. 535 (1–3), 153–158. Gladek, A., Liehr, J.G., 1989. Mechanism of genotoxicity of diestilbestrol in vivo. J. Biol. Chem. 264 (28), 16847–16852. Green, M., Wilson, C., Newell, O., Sadrud-Din, S., Thomas, R., 2005. Diallyl sulfide inhibits diethylstilbesterol-induced DNA adducts in the breast of female ACI rats. Food Chem. Toxicol. 43 (9), 1323–1331. Guyonnet, D., Belloir, C., Suschetet, M., Siess, M., Le Bon, A., 2002. Mechanisms of protection against aflatoxin B(1) genotoxicity in rats treated by organosulfur compounds from garlic. Carcinogenesis 23 (8), 335–341. Hanawalt, P.C., 1992. Transcription-dependent and transcriptioncoupled DNA repair responses. In: Bor, V.A., et al. (Eds.), DNA Repair Mechanisms. Munksgaard, Copenhagen, pp. 231–242. Hodgson, E., Smart, R., 2001. Introduction to Biochemical Toxicology, 3rd ed. John Wiley and Sons, New York. Hu, X., Singh, S.V., 1997. Glutathione S-transferases of female A/J mouse lung and their induction by anticarcinogenic organosulfides from garlic. Arch. Biochem. Biophys. 340 (2), 279–286. Hu, X., Benson, P.J., Srivastava, S.K., Mack, L.M., Xia, H., Gupta, V., Zaren, H.A., Singh, S.V., 1996. Glutathione S-transferases of
female A/J mouse liver and forestomach and their differential induction by anti-carcinogenic organosulfides from garlic. Arch. Biochem. Biophy. 336 (2), 199–214. International Agency for Research on Cancer (IARC), 1979. Diethylstilbestrol and diethylstilbestrol dipropionate. IARC Monogr. Eval. Carcinog. Risk Chem. Hum. 21, 173–231. Liehr, J., Roy, D., 1990. Free radical generation by redox-cycling of estrogens. Free Radic. Biol. Med. 8, 415–423. Marselos, M., Tomatis, L., 1993. Diethylstilbesterol II: pharmacology, toxicology, and carcinogenecity in experimental animals. Eur. J. Cancer 29A, 149–155. Rothschild, T., Boyland, E., Calhoon, R., Vonderhaar, B., 1987. Transplacental effects of diethystilbestrol on mammary development and tumorigenesis in female ACI rats. Cancer Res. 47, 4508–4516. Roy, D., Liehr, J.G., 1992. Inhibition of estrogen-induced kidney carcinogenesis in Syrian hamsters by modulators of estrogen metabolism. Carcinogenesis 11 (4), 567–570. Selby, C.P., Sancar, A., 1993. Molecular mechanism of transcription– repair coupling. Science 260, 53–58. Smith, M.L., Ford, J.M., Hollander, M.C., Bortnick, R.A., Amundson, S.A., Seo, Y.R., Deng, C.X., Hanawalt, P.C., Fornace Jr., A.J., 2000. p53-mediated DNA repair responses to UV radiation: studies of mouse cells lacking p53, p21, and/or gadd45 genes. Mol. Cell. Biol. 20 (10), 3705–3714. Somai, S., Chaouat, M., Jacob, D., Perrot, J.Y., Rostene, W., Forgez, P., Gompel, A., 2003. Antiestrogens are pro-apototic in normal human breast epithelial cells. Int. J. Cancer 105 (5), 607–612. Srivastava, S.K., Hu, X., Hong, X., Howard, A.Z., Chatterjee, M.L., Agarwal, R., Singh, S.V., 1997. Mechanism of differential efficacy of garlic organosulfides in preventing benzo(a)pyrene induced cancer in mice. Cancer Lett. 118, 61–67. Sweder, K.S., Hanawalt, P.C., 1992. Preferential repair cyclobutane pyrimidine dimers in the transcribed strand of a gene in yeast chromosomes and plasmids is dependent on transcription. Proc. Natl. Acad. Sci. 89, 10696–10700. Van Steeg, H., 2001. The role of nucleotide excision repair and loss of p53 in mutagenesis and carcinogenesis. Toxicol. Lett. 120 (1–3), 209–219 (review). Wang, X.Y., Zhan, Q., Coursen, J.D., Khan, M.A., Kontny, H.U., Yu, L., Hollander, M.C., O’Connor, P.M., Fornace Jr., A.J., Harris, C.C., 1999. GADD45 induction of a G2 /M cell cycle checkpoint. Proc. Natl. Acad. Sci. 96, 3706–3711. Zhan, Q., Fan, S., Bae, I., Guillouf, C., Liebermann, D.A., O’Connor, P.M., Fornace Jr., A.J., 1994. Induction of BAX by genotoxic stress in human cells correlates with normal p53 status and apoptosis. Oncogene 9, 3743–3751.
Diallyl sulfide induces the expression of nucleotide excision repair enzymes in the breast of female ACI rats Mario Green, Oneil Newell, Ayoola Aboyade-Cole, Selina Darling-Reed, Ronald D. Thomas ∗ College of Pharmacy, Florida A&M University, 302 West Pershing Street, Tallahassee, FL 32307, United States Received 7 July 2006; received in revised form 25 October 2006; accepted 25 October 2006 Available online 10 November 2006
Abstract Diethylstilbestrol (DES) causes DNA adducts resulting in breast cancer, whereas diallyl sulfide (DAS) inhibits cancer formation. We hypothesize that DAS induces the expression of nucleotide excision repair genes. To test this hypothesis, female ACI rats were treated for 4 days with corn oil, DES, DAS, and DAS/DES (50 mg/kg). The expression of P53, Gadd45a, PCNA, and DNA polymerase delta was analyzed by real-time PCR. DES decreased the expression of P53, Gadd45a and PCNA. DAS and DAS/DES increased the expression of all four genes. These results suggest that DAS enhances the ability of breast tissue to repair DNA damage thus preventing cancer. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Diallyl sulfide; Diethylstilbestrol; Chemoprevention; DNA repair and breast cancer
1. Introduction Diethylstilbestrol (DES) has been shown to induce breast cancer in female ACI rats (Marselos and Tomatis, 1993; Rothschild et al., 1987). The mechanisms of estrogen-induced carcinogenesis are not clearly understood. However, recent data suggest that estrogen metabolism plays an important role in estrogen-induced cancer. DES is metabolized to reactive intermediates (semiquinone and quinone) by cytochrome P450s (Liehr and Roy, 1990; Roy and Liehr, 1992). These reactive intermediates result in the formation of DNA adducts ∗
Corresponding author. Tel.: +1 850 561 2786/599 3389; fax: +1 850 599 3347. E-mail addresses: [email protected] (M. Green), [email protected] (O. Newell), [email protected] (A. Aboyade-Cole), [email protected] (S. Darling-Reed), [email protected] (R.D. Thomas).
(Gladek and Liehr, 1989). If these DNA adducts undergo one or more cycles of replication without repair, the damage becomes fixed resulting in genomic instability. This genomic instability may lead to the initiation of cancer. DNA repair enzymes maintain DNA integrity by removing damaged bases. The primary mechanism for the removal of stable (bulky) adducts is nucleotide excision repair (NER). Two pathways of NER are transcription couple repair (TCR) and global genomic repair (GGR). TCR is initiated by the stalling of RNA polymerase II by DNA lesions (bulky adducts) during transcription (Hanawalt, 1992; Sweder and Hanawalt, 1992; Selby and Sancar, 1993). GGR involves the scanning of the genome by specific damage recognition proteins such as xeroderma pigmentosum C (XPC), transcription factor IIH (TFIIH), xeroderma pigmentosum A (XPA), and the growth arrest DNA damage-inducible 45 (Gadd45a) in search of DNA damage (Van Steeg, 2001). XPC and Gadd45a have been shown to be regulated by
0378-4274/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2006.10.013
M. Green et al. / Toxicology Letters 168 (2007) 40–44
the tumor suppressor gene, p53 (Wang et al., 1999; Zhan et al., 1994). Once the damage is recognized, the remaining steps involved in NER are similar for both TCR and GGR. DNA is unwound and a repair bubble is created by xeroderma pigmentosum B (XPB) and xeroderma pigmentosum D (XPD) helicases. The repair bubble is followed by excisions on the 5 - and 3 -ends of the damage by xeroderma pigmentosum F (XPF) and xeroderma pigmentosum G (XPG) endonucleases. A new strand of DNA is synthesized by DNA polymerase delta/epsilon. The activity of DNA polymerase delta/epsilon is dependent on the presence of proliferating cell nuclear antigen (PCNA) (De Laat et al., 1999). Estrogens have been shown to inhibit nucleotide excision repair (Evans et al., 2003). This impairment may result in an increased rate of mutations that contribute to the initiation of estrogen-induced cancer. Previously, we have demonstrated that DES is metabolized to reactive intermediates resulting in the formation of DNA adducts in the breast of female ACI rats (Green et al., 2005). Furthermore, we have demonstrated that diallyl sulfide (DAS), an organosulfur compound found in garlic, inhibits DNA adduct formation (Green et al., 2005). The chemopreventive properties of DAS have been attributed to metabolic modulation (Guyonnet et al., 2002; Srivastava et al., 1997). The female ACI rat is a good model for studying estrogen-induced breast cancer because they develop breast cancer upon exposure to estrogens, including 17-beta estradiol and DES, and have a low rate of developing spontaneous tumors (IARC, 1979). We chose DES as our carcinogenic test compound because it has estrogenic activity similar to that of 17-beta estradiol and has been shown to be associated with breast cancer in both animals and humans (Marselos and Tomatis, 1993). In the present study, we have demonstrated for the first time that DAS enhances the expression of p53, Gadd45a, PCNA, and DNA polymerase delta, which are involved in nucleotide excision repair. These results suggest that DAS-induced DNA repair may play a role in the prevention of estrogen-induced breast cancer. 2. Material and methods 2.1. Chemicals DES, RNAlater and DAS were purchased from Sigma Chemical Company (St. Louis, MO). Trizol LS reagent was purchased from Invitrogen (Carlsbad, CA). SYBRgreen RealTime Mastermix (2X) and Stratascript First Strand cDNA Synthesis System were purchased from Stratagene (La Jolla,
41
CA). The primers for growth arrest and DNA damage 45a (Gadd45a), p53, proliferating cell nuclear antigen (PNCA), and DNA polymerase delta were ordered from SuperArray, Inc. (Detrick, MD). Female ACI rats (5–6 weeks old) were ordered from Harland Sprague, Inc. (Indianapolis, IN). 2.2. Animal treatment Four groups of 10 ACI rats were analyzed for expression of DNA repair genes involved in nucleotide excision repair (Gadd45a, PCNA, p53 and DNA polymerase delta). Rats were treated i.p. daily for 4 days with corn oil (control), DES (50 mg/kg), DAS (50 mg/kg), and DAS/DES (50 mg/kg). The breast tissue was removed, immediately placed in RNAlater and stored in at −80 ◦ C. Total RNA was isolated from the breast tissue and analyzed for gene expression using real-time PCR. 2.3. RNA isolation Fifty milligrams of breast tissue were weighed and homogenized in the presence of 0.75 ml of Trizol LS Reagent. Following homogenization, the samples were incubated at room temperature for 5 min. Chloroform (0.2 ml) was added to the homogenate and vortexed vigorously for 1 min. The homogenate were incubated at room temperature for 15 min followed by centrifugation at 12,000 × g for 15 min at 4 ◦ C. The upper aqueous layer containing RNA was transferred to a new RNase-free tube without disturbing the interphase layer. RNA was precipitated by adding 0.5 ml isopropyl alcohol. The samples were incubated at room temperature for 10 min and centrifuged at 12,000 × g for 10 min at 4 ◦ C. The supernatant was aspirated and RNA pellet collected was washed with 1 ml of 75% ethanol. The pellets were air-dried and reconstituted in 25 l of RNase-free water. The quality of RNA was determined by measuring the OD260/280 ratio with UV–vis spectrophotometry. RNA purified by this method resulted in OD260/280 ratio of >1.7. The quality of RNA was further determined by formaldehyde agarose gel electrophoresis. The presence of 18S and 28S rRNA bands indicated high quality RNA with low degradation. 2.4. cDNA amplification The cDNA was synthesized using the Stratascript FirstStrand cDNA Synthesis System for RT-PCR. The first cDNA strand was synthesized by combining 10 g RNA, 3 l of Oligo (dT) (100 ng/l), 5 l of 10× first-strand buffer, 1 l RNase Block Ribonuclease Inhibitor (40 U/l) and 2 l of 100 mM dNTPs in a microcentrifuge tube. The total volume was adjusted to 38 l with DEPC-treated water. The reaction was mixed gently and incubated at 65 ◦ C for 5 min. The reactions were slowly cooled at room temperature (∼10 min) to allow the primers to anneal to the RNA. One microliter of Stratascript reverse transcriptase (50 U/l) was then added to the reaction mixtures. They were mixed gently and incubated
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at 42 ◦ C for 1 h. The reactions were terminated by incubation at 90 ◦ C for 5 min. The completed reactions were then chilled on ice and the cDNA was collected by brief centrifugation. The cDNA was used immediately for PCR or stored at −80 ◦ C. 2.5. Real-time RT-PCR analysis Real-time PCR was performed in a 96-well plate. 12.5 l of 2× SYBR Green master mix (SureStart DNA polymerase (5 U/l), 10× PCR reaction buffer, 50 mM magnesium chloride, 20 mM dNTP mix, 100% DMSO, 50% glycerol, SYBR Green I dye), 1 l primer (Gadd45a, p53, PCNA, or DNA polymerase delta (100 ng/l)), 1 l cDNA (250 ng/l), and 10.5 l dd H2 O were combined for a total volume of 25 l per well. The contents of the well were mixed by pipetting gently. The tubes were incubated in a thermocycler at 95 ◦ C for 15 min to activate Taq DNA polymerase. There were 45 cycles of PCR amplification performed consisting of denaturing at 95 ◦ C for 30 s, annealing at 55 ◦ C for 30 s, and extension at 72 ◦ C for 30 s. Relative gene expression was calculated by the following equation: relative gene expression (RGE) = 2
Ctcon −Cttreat
where Ctcon is the cycle in which the fluorescence signal reaches threshold for the control group of animals and Cttreat is the cycle in which the fluorescence signal reaches threshold for the treated group of animals. The relative gene expression was normalized to GAPDH using the following equations: relative GAPDH expression (RGPE) = 2CtconGAPDH −CttreatGAPDH and normalized gene expression = RGE/RGPE. 2.6. Statistics Statistical analysis was performed with GraphPad Prism software using one-way analysis of variance (ANOVA). Tukey’s multiple comparison post test was used to compare the means of treatments to the control with a significance level of 0.05 where p < 0.05 indicated significance.
Fig. 1. p53 gene expression in breast of female ACI rats treated with DAS and/or DES. Real-time amplification of p53 cDNA was performed as described in Section 2. Results are expressed as mean ± S.E.M. of three independent experiments. The (*) indicates data significantly different from the DES treatment group. The (**) indicates data significantly different from the DAS treatment group (p < 0.05).
The administration of DES decreased mRNA levels of Gadd45a by 92%. The administration of DAS increased Gadd45a expression by 181-fold, whereas coadministration of DAS/DES increased the expression of Gadd45a by 22-fold (Fig. 2). DAS reversed the DESinduced inhibition of the expression of Gadd45a. DES completely inhibited the expression of PCNA whereas DAS resulted in 5.2-fold increase in its expression. The co-administration of DAS/DES synergistically increased the expression of PCNA by 32.2-fold (Fig. 3). DAS reversed the DES-induced inhibition of the expression of PCNA. DES resulted in a 1.8-fold increase in the expression of DNA polymerase delta (Fig. 4). DAS increased the
3. Results The effects of DAS on the expression of DNA repair genes involved in nucleotide excision repair was investigated in the breast of female ACI rats using real-time PCR. The rats were dosed daily with 50 mg/kg of DES and/or DAS via i.p. injections. The administration of DES decreased the relative gene expression of p53 by 75% compared to the control. However, this reduction in gene expression was not statistically significant. The administration of DAS increased relative gene expression of p53 by 12.1-fold, whereas co-administration of DAS/DES increased the relative gene expression of p53 by 4.9-fold (Fig. 1). DAS reversed the DES-induced inhibition of the expression of p53.
Fig. 2. Gadd45a gene expression in breast of female ACI rats treated with DAS and/or DES. Real-time amplification of Gadd4a cDNA was performed as described in Section 2. Results are expressed as mean ± S.E.M. of three independent experiments. The (*) indicates data significantly different from the DES treatment group. The (**) indicates data significantly different from the DAS and the DES treatment groups (p < 0.05).
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4. Discussion
Fig. 3. PCNA gene expression in breast of female ACI rats treated with DAS and/or DES. Real-time amplification of PCNA cDNA was performed as described in Section 2. Results are expressed as mean ± S.E.M. of three independent experiments. The (*) indicates data significantly different from the DAS and the DES treatment groups (p < 0.05).
expression of DNA polymerase delta by 6.3-fold. Coadministration of DAS/DES synergistically increased the expression of DNA polymerase delta by 61-fold. Based on statistical analysis, DES does not alter the expression of DNA polymerase delta, Gadd45a, PCNA, and p53. The fact that DES causes DNA damage and does not induce the expression of DNA repair enzymes is significant. DES treatments alone can cause significant DNA damage, which may not be repaired. The lack of induction of DNA repair enzymes by DES may play a role in estrogen-induced breast cancer. Conversely, DAS alone and in combination with DES induced the expression of Gadd45a, PCNA, p53, and DNA polymerase delta, which may play a role in DAS-induced cancer prevention.
Fig. 4. Effect of DAS and/or DES on DNA polymerase delta gene expression in breast of female ACI rats. Real-time amplification of DNA polymerase delta cDNA was performed as described in Section 2. Results are expressed as mean ± S.E.M. of three independent experiments. The (*) indicates data significantly different from the DAS and the DES treatment groups (p < 0.05).
Previously, we have shown that DAS inhibits DESinduced DNA adduct formation in the breast of female ACI rats (Green et al., 2005). The mechanism of this inhibition is not clear, but may be partially attributed to increased detoxification as a result of the induction of phase II metabolism enzymes by DAS (Hu and Singh, 1997; Hu et al., 1996). Additionally, DAS may decrease the level of DES-induced DNA adducts through the upregulation of genes involved in nucleotide excision repair (Gadd45a, PCNA, and DNA polymerase delta). We have demonstrated DAS’s ability to up-regulate nucleotide excision repair genes and thereby provide a potential mechanism for DAS induced increases in DNA repair and DNA adduct removal. Gadd45a is one of many genes regulated by p53 (Smith et al., 2000). DES decreased the expression of both p53 and Gadd45a. Therefore, the DES induced decrease in Gadd45a expression observed in our study may be attributed to the decrease in p53 expression. These results were supported by the fact that estradiol was found to decrease p53 levels in human breast epithelial cells (Somai et al., 2003). Furthermore, DAS has been shown to increase the levels of p53 in Swiss albino mice (Arora et al., 2004). This suggests that the alterations in Gadd45a expression may be associated with alterations in p53 expression. The interaction of Gadd45a with PCNA is essential for nucleotide excision repair. PCNA stimulates DNA polymerase delta to replace damaged nucleotides (Hodgson and Smart, 2001). In our study, PCNA expression in rats treated with DES was completely down-regulated. PCNA expression was up-regulated in rats treated with DAS and DAS/DES. Additionally, we have shown that DES, DAS, and combined treatments of DAS/DES increased the expression of DNA polymerase delta. While all three treatment groups resulted in increased levels of DNA polymerase delta, the DAS/DES combination was significantly higher than the other groups. This up-regulation of both PCNA and DNA polymerase delta will increase DNA repair efficiency particularly in the DAS/DES group. Impairment of DNA repair systems will result in increased levels of DNA damage that may lead to mutations and ultimately result in cancer. We have demonstrated that DAS induces the expression of P53, Gadd45a, PCNA, and DNA polymerase delta. These changes in gene expression will increase the cells ability to remove DES-induced DNA adducts in the breast of female ACI rats providing an additional mechanism by which DAS exerts its anti-carcinogenic activity. Further
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