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Inhibition of benzo[a]pyrene- and 1,6-dinitropyrene-DNA adduct formation in human mammary epithelial cells bydibenzoylmethane and sulforaphane
Cancer Letters 155 (2000) 47±54
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Inhibition of benzo[a]pyrene- and 1,6-dinitropyrene-DNA adduct formation in human mammary epithelial cells bydibenzoylmethane and sulforaphane K. Singletary*, C. MacDonald 1 Department of Food Science and Human Nutrition, University of Illinois, 905 South Goodwin Avenue, Urbana, IL 61801, USA Received 20 December 1999; received in revised form 23 February 2000; accepted 24 February 2000
Abstract Numerous phytochemicals have been examined for their capacity to act as cancer chemopreventive agents. Dibenzoylmethane, a minor constituent of licorice and a compound structurally-related to curcumin, recently was identi®ed as an effective inhibitor of chemically-induced rat mammary DNA-adduct formation and tumorigenesis (Carcinogenesis 19(1998)1039-1043). The present studies were conducted to examine the capacity of dibenzoylmethane to inhibit the formation of DNA adducts following exposure to benzo[a]pyrene (BP) and 1,6-dinitropyrene (1,6-DNP), and to stimulate the expression of glutathione-Stransferase (GST) and NAD(P)H-quinone reductase (QR) proteins in the human mammary epithelial cell line MCF-10F. In addition, the ef®cacy of dibenzoylmethane as an enzyme inducer and adduct inhibitor was compared with that of sulforaphane, a potent inducer of phase II detoxi®cation enzymes and inhibitor of chemically-induced rat mammary tumorigenesis. Dibenzoylmethane at concentrations from 0.1 M to 2.0 mM inhibited BP-DNA adduct formation by 63 to 81%. Likewise, sulforaphane inhibited BP-DNA adduct formation by 68 to 80% over the same concentration range. DNA adduct formation following exposure to 1,6-DNP was signi®cantly inhibited by 46 to 61% due to dibenzoylmethane treatment (0.1 to 2.0 mM) and 30 to 56% due to sulforaphane treatment at the same concentrations. The expression of QR and GSTP1-1 proteins were increased by 3 to 4-fold and 3 to 5-fold, respectively, for MCF-10F cells treated with sulforaphane (0.5±2.0 mM). Dibenzoylmethane treatment at the same concentrations did not induce GSTP1-1 expression and signi®cantly stimulated QR expression only at the 2.0 mM concentration. These data indicate that human mammary epithelial MCF-10F cells can convert BP and 1,6-DNP to DNA-binding forms, and that DNA adduct formation can be inhibited by the phytochemicals dibenzoylmethane and sulforaphane. The inhibition of BP-DNA and 1,6-DNP adduct formation by sulforaphane was associated with increases in QR and GST protein expression. The mechanisms underlying the capacity of dibenzoylmethane to inhibit BP-DNA and 1,6-DNP-DNA adduct formation could not be explained by changes in QR or GST expression and remain to be determined. Together these data suggest that dibenzoylmethane and sulforaphane warrant continued evaluation as breast cancer chemopreventive agents. q 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dibenzoylmethane; Sulforaphane; Mammary; DNA adduct; Benzo[a]pyrene; 1,6-Dinitropyrene
1. Introduction * Corresponding author. Fax: 11-217-244-2455. E-mail address: [email protected] (K. Singletary). 1 LNMR, NCI-FDRDC, Building 560, Frederick, MD 21702, USA.
Cancer chemoprevention, the strategy to inhibit or reverse the process of carcinogenesis or to suppress the progression of preneoplastic lesions, has become a
0304-3835/00/$ - see front matter q 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00412-2
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potentially important approach to combating cancer [1]. Both synthetic and naturally occurring chemicals are being examined as possible chemopreventive agents [2]. Recently, we reported that the b-diketone dibenzoylmethane, a minor constituent of licorice and a compound structurally similar to the bioactive phytochemical curcumin, was an effective dietary inhibitor of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary DNA adduct formation and tumorigenesis [3]. These ®ndings were subsequently con®rmed and extended by others [4]. Dibenzoylmethane, appeared to act as a blocking agent, in part, by inducing the activity of the liver phase II detoxi®cation enzymes, NAD(P)H-quinone reductase (QR) and glutathione-S-transferase (GST). The present studies were conducted in order to extend our previous ®ndings with dibenzoylmethane by determining the capacity of dibenzoylmethane to inhibit carcinogen-DNA adduct formation and increase the expression of QR and GST in non-neoplastic human mammary epithelial cells. The adduct inhibitory action of this phytochemical was examined using representatives of two classes of chemical carcinogens, the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BP) and the nitro-polycyclic aromatic hydrocarbon (nitro-PAH) 1,6-dinitropyrene (1,6-DNP). Benzo[a]pyrene is an environmental contaminant and widely-studied chemical carcinogen [5] capable of forming covalent DNA adducts [6]. Although a de®nitive role for BP in breast cancer causation has not been established, there is evidence that BP-like DNA adducts are present in the normal breast tissue of breast cancer patients [7,8], and that BP may be a risk factor for sporadic breast cancer due to its ability to downregulate BRCA-1 in human breast cancer cells [9]. In addition, primary cultures of human mammary epithelial cells, in contrast to rat mammary epithelial cells, have been reported to metabolize BP to DNA-binding metabolites to a greater extent than the synthetic PAH DMBA [10]. NitroPAH also are widely distributed in the environment, apparently present in smaller quantities than PAH, but, nonetheless mutagenic and carcinogenic [11,12]. In Japan, 1,6- and 1,8-dinitropyrene isomers have been identi®ed as major mutagens in organic extract of soil samples [13]. The 1,6-dinitropyrene isomer has been reported to be highly mutagenic in the Ames Salmonella reversion assay and has been shown to be tumorigenic in several animal models, including the
mammary gland [14]. The nitroPAH also can be converted to DNA-reactive forms in several tissues [15,16]. The formation of target tissue carcinogenDNA adducts is considered to be an important prerequisite and a potential early biomarker for the initiation stage of chemically-induced carcinogenesis [17,18]. The capacity of a phytochemical to inhibit carcinogen-DNA binding is determined by the balance between activation and detoxi®cation pathways of metabolism. In particular, the action of a compound in inducing phase II enzymes, such as QR and GST, has been suggested to be an important indicator of its ability to block tumor initiation by environmental chemicals such as PAH [19,20]. Therefore, the effectiveness of dibenzoylmethane in inhibiting BP- and 1,6-DNP-DNA adduct formation and in stimulating the expression of QR and GST in human mammary epithelial cells was investigated. In addition, the ef®cacy of dibenzoylmethane was compared with that of sulforaphane, a constituent of cruciferous vegetables that recently has been identi®ed as a potent enhancer of QR and GST and an inhibitor of chemically-induced breast carcinogenesis in rats [21,22].
2. Materials and methods 2.1. Cell culture These studies were performed using the nonneoplastic human mammary cell line, MCF-10F, which was obtained from the Michigan Cancer Foundation (Detroit, MI). These cells are spontaneouslyimmortalized human breast epithelial cells that behave like normal breast epithelial cells in culture, and that can be transformed to the neoplastic phenotype by short exposure to mammary carcinogens including benzo[a]pyrene [23,24]. For adduct and enzyme experiments, 1 £ 10 5 cells were added to T150 ¯asks containing DMEM/F-12 medium (Gibco, Grand Is-land, NY) supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin, 2.5 mg/ml amphotericin B, 0.5 mg/ml hydrocortisone, 0.02 mg/ml epidermal growth factor, 0.1 mg/ml cholera enterotoxin, 10 mg/ml insulin and 5% equine serum. This number of cells was chosen for plating so that, by the end of the dibenzoylmethane or sulforaphane exposure period, cells would not have reached con¯uence. After
K. Singletary, C. MacDonald / Cancer Letters 155 (2000) 47±54
overnight attachment of cells, the either dibenzoylmethane (98%, Aldrich Chemicals, Milwaukee, WI) or sulforaphane (97%, as d,l-sulforaphane from LKT Labs, St. Paul, MN) were added at concentrations ranging from 0.1 to 2 mM for 7 days, with medium changes every other day. On day 7, cells received fresh medium containing benzo[a]pyrene (BP, Sigma Chemical) at 0.3 mM for 12 h or 1,6-dinitropyrene (DNP, Sigma Chemical) at 10 mM for 22 h. After the carcinogen incubation period, cells were harvested and the DNA isolated or the cytosolic fraction (100 000 £ g supernatant) prepared. 2.2. P-postlabeling as immunoblot analyses Aliquots of DNA were analyzed for either BP-DNA or 1,6-DNP adducts by 32P-postlabeling procedures previously described [25,26]. Nuclease P1 enhancement and butanol enhancement procedures were used for the BP-DNA and 1,6-DNP-DNA adduct analyses, respectively. For quantitation of adducts, adduct spots were removed from TLC plates and radioactivity determined by scintillation counting. Quantities were expressed as nmol adduct/mol DNA assuming 1 mg DNA 3240 pmol deoxyribonucleoside-3 0 -monophosphate. For immunoblot (Western) analysis of GSTP11 and QR proteins, equal quantities of total protein were loaded onto polyacrylamide gels, separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed using either QR antibody (rabbit antiNQO1 E. coli, a generous gift of Dr Shu-Shu Pan, University of Maryland School of Medicine, Baltimore, MD) or GSTP1-1 antibody (from Oxford Biomedical Research, Oxford, MS). Bands were detected by enhanced chemiluminescence and quantitated by densitometry.
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adduct spot was formed from cells treated with BP (Fig. 1). We tentatively identi®ed this adduct spot to be that due to binding of anti-BP-dihydrodiolepoxide with deoxyguanosine. This was determined by reacting deoxyadenosine-3 0 -monophosphate (dAdo) or deoxyguanosine-3 0 -monophosphate (dGuo) with a racemic mixture of (^) anti-BP-dihydrodiolepoxides (Chemsyn Science Laboratories, Lenexa, KS) using the protocol described by Canella et al. [27]. Only the 32P-postlabeled product formed from reaction with dGuo co-migrated during TLC with the MCF10F BP-DNA adduct (data not shown). Also, we determined that BP-DNA adduct formation increased linearly when cells were exposed to BP for 12 h at concentrations from 0.08 to 0.8 mM. BP-DNA adducts formed were 127, 323 and 583 nmol BP/mol DNA for cells incubated for 12 h with BP concentrations of
3. Results Neither dibenzoylmethane nor sulforaphane exhibited cytotoxic effects toward the MCF-10F cells at the concentrations and incubation periods examined. Viability, as determined by Trypan Blue exclusion, always exceeded 95%. The ®rst objective of these studies was to determine the inhibitory effect of dibenzoylmethane and sulforaphane toward DNA adduct formation in the MCF-10F cells resulting from either BP or 1,6 DNP exposure. Predominantly one DNA
Fig. 1. Autoradiographs of 32P-postlabeled DNA from MCF-10F cells exposed to benzo[a]pyrene (A) and 1,6-dinitropyrene (B). Arrows indicate adduct spots quantitated. OR, origin.
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K. Singletary, C. MacDonald / Cancer Letters 155 (2000) 47±54
0.08, 0.3 and 0.8 mM, respectively. An intermediate dose of 0.3 mM was thus chosen for these experiments. The formation of BP-DNA adducts was significantly inhibited by 63, 62 and 81% for cells treated with 0.1, 0.5 and 2.0 mM dibenzoylmethane, respectively, compared with controls (Fig. 2). Likewise, sulforaphane inhibited BP-DNA adduct formation by 68, 80 and 70% for cells treated at concentrations of 0.1, 0.5 and 2.0 mM, respectively, compared with controls. When adduct formation following exposure of cells with 1,6-DNP was determined, essentially one major and two minor adduct spots were observed (Fig. 1). We did not determine the identity of these TLC adduct spots. In preliminary studies we deter-
mined that adduct spot formation increased in a linear fashion when cells were incubated for 22 h with 1,6-DNP at concentrations of 5 and 10 but not 50 mM. The 10 mM concentration of 1,6-DNP was thus chosen for these experiments. Total 1,6-DNPDNA adduct spot formation was inhibited by 46, 61 and 50% for samples treated with 0.1, 0.5 and 2.0 mM dibenzoylmethane, respectively, and by 56, 33 and 30% for samples treated with 0.1, 0.5 and 2.0 mM sulforaphane, respectively, compared with controls (Fig. 2). The second part of these studies involved examining the effect of dibenzoylmethane and sulforaphane treatment on the expression of the phase II detoxi®cation enzymes QR and GSTP1-1. The pi class of GST was chosen for examination because it is actively involved in BP-diolepoxide (BPDE) conjugation in humans [19], and because GSTP1-1 in mouse liver has been reported to be more effective in BPDE detoxi®cation than all other GSTs combined [28]. We observed that dibenzoylmethane increased QR protein expression, compared with controls, but only at the 2.0 mM concentration of dibenzoylmethane. Dibenzoylmethane had no effect on GSTP1-1 protein levels (Fig. 3). On the other hand, sulforaphane at concentrations from 0.5 to 2.0 mM signi®cantly increased QR protein and GSTP1-1 protein levels by 3 to 5-fold and 3 to 4-fold, respectively, compared with controls (Fig. 4). 4. Discussion
Fig. 2. Effect of dibenzoylmethane (DBM) and sulforaphane on BPDNA adduct formation (A) and 1,6-DNP adduct formation (B) in MCF-10F cells. Values represent means ^ SEM of three separate samples. Bars indicated by w are signi®cantly different (ANOVA) from controls at P , 0:05.
The present studies provide evidence that cultures of the non-neoplastic human mammary epithelial cell line MCF-10F exposed to BP and 1,6-DNP form carcinogen-DNA adduct spots following isolation and detection by the 32P-postlabeling procedure, with the magnitude of adduct formation following treatment with BP being greater. Our ®ndings that non-neoplastic mammary cells can bioactivate BP are similar to those reported by Moore et al. [6,10,29] in which human mammary cells possessed the capacity to metabolize BP to DNA-binding metabolites. Stampfer et al. [30] also determined that, for primary human mammary epithelial cells exposed to BP, the major adduct formed is derived from binding to dGuo. Our results with 1,6-DNP are similar to those
K. Singletary, C. MacDonald / Cancer Letters 155 (2000) 47±54
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Fig. 3. Effect of dibenzoylmethane (DBM) on the protein expression of GST(A) and QR (B) in MCF-10F cells. Values represent means ^ SEM of three separate samples. Bars indicated by w are signi®cantly different (ANOVA) from controls at P , 0:05.
of Carmichael et al. [26], who observed that suspensions of primary human mammary epithelials treated with 1,6-DNP produced three DNA adduct spots. Although the quantity of DNA binding was not determined, it was observed that adduct formation from 1,6-DNP was less than that resulting from treatment of cells with other environmental agents, such as the hetrocyclic amines and the PAH dibenzo[a,l]pyrene. Elbridge et al. [31] examined the genotoxicity of BP, 1,6-DNP and other environmental agents in normal human mammary cells, and found that treatment of cells with BP and 1,6-DNP resulted in detectable genotoxicity, as indicated by unscheduled DNA synthesis. However, the extent of binding was not quantitated. In F344 rats, administration of 1,6-DNP was reported to produce one major adduct, the N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene in lung, liver, and other tissues [32]. The studies also indicate that the phytochemicals dibenzoylmethane and sulforaphane can inhibit carci-
nogen-DNA binding in cultures of MCF-10F cells treated with either BP or 1,6-DNP. However, the magnitude of the inhibitory effect depended on the carcinogen examined. When cells were treated with BP, both dibenzoylmethane and sulforaphane were effective inhibitors of adduct formation. When cells were treated with 1,6-DNP, both compounds inhibited adduct formation, although to a lesser extent than when BP was used. In other words, at the concentrations of dibenzoylmethane and sulforaphane examined, BP adduct formation was inhibited by greater than 60%. However, 1,6-DNP adduct formation was inhibited at most by 61% following treatment with similar concentrations of the two phytochemicals. When adduct inhibition was compared with the capacity to enhance protein expression of QR and GST, sulforaphane's capacity to stimulate QR and GST protein expression was associated with the inhibition by sulforaphane of DNA adduct formation due to BP treatment. However, although dibenzoylmethane did
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K. Singletary, C. MacDonald / Cancer Letters 155 (2000) 47±54
Fig. 4. Effect of sulforaphane on the protein expression of GST(A) and QR (B) in MCF-10F cells. Values represent means ^ SEM of three separate samples. Bars indicated by w are signi®cantly different (ANOVA) from controls at P , 0:05.
exhibit adduct inhibitory effects similar to sulforaphane toward BP- and 1,6-DNP-induced DNA adduct formation, dibenzoylmethane treatment did not increase GST protein expression in MCF-10F cells and only stimulated QR protein expression at the highest concentration of dibenzoylmethane examined, 2.0 mM. This is in contrast to dietary dibenzoylmethane's capacity to stimulate QR and GST activity in the livers of female rats [3]. The reason for the inability of dibenzoylmethane to induce GST in these human mammary cells is not known, but may be due to tissue-related differences in the regulation of QR and GST gene expression. This suggests that mechanisms other than those involving GST- and QR-induction are contributing to the inhibition of BP-DNA adduct formation in MCF-10F cells treated with dibenzoylmethane, especially at dibenzoylmethane concentrations ,2.0 mM. The effect of dibenzoyl-
methane treatment on cytochrome P450 enzymes of MCF-10F cells may be important to determine, since cytochrome P450 1A is known to participate in the metabolism of polycyclic aromatic hydrocarbons like BP, and cytochrome P450 1B1 is a new P450 family 1 member present in rodent and human tissues that can convert numerous chemicals including PAHs and nitroPAH to genotoxic intermediates in liver and extrahepatic tissue [33±35]. In addition, dibenzoylmethane may be acting, in part, via antioxidant and/or antiin¯ammatory processes. It has been suggested that BP can be metabolically activated to DNA-binding species by a process involving free radicals [36,37]. Although dibenzoylmethane has been reported to be inactive as an antioxidant and free-radical scavenger in vitro [4], the b-diketone moiety has been shown to be important for the antioxidant action of other biological compounds [38,39]. With regard to 1,6-DNP adduct formation, both dibenzoylmethane and sulforaphane exhibited more modest degrees of inhibition compared with their inhibition of BP-DNA adduct formation. The adduct inhibitory action of sulforaphane toward 1,6-DNP could be due, in part, to its GST- and QR-inducing effect. However, although the QR-inducing effect of dibenzoylmethane may be contributing partly to the inhibition of 1,6-DNP adduct spot formation (particularly at the 2.0 mM dose), other pathways for metabolism of 1,6-DNP may be affected by dibenzoylmethane. It has been reported that nitroreductase and O-acetylation activities may be involved in the activation of dinitropyrenes [40]. There is evidence that MCF-10 cells may possess the capacity to O-acetylate, since Swaminathan et al. [41] have reported that the human mammary epithelial cell line MCF-10A can activate the mammary gland carcinogen N-hydroxy-4-acetylaminobiphenyl by acetyl transferase(s) enzyme systems. Localization of acyltransferases [42] and their role in activation of carcinogens in the rat mammary gland have been observed [43]. The extent of nitroreductase activity in mammary tissue and whether dibenzoylmethane (and sulforaphane) can inhibit this activity as well as alter acyl transferase activity warrants further study. The present data for sulforaphane are consistent with that of Zhang et al. [21,22] who reported that sulforaphane is a potent inducer of GST and QR in cytosols from several organs of CD-1 mice, and that sulforaphane can inhibit both tumor multiplicity and
K. Singletary, C. MacDonald / Cancer Letters 155 (2000) 47±54
incidence in female Sprague±Dawley rats initiated with the PAH DMBA. Our results are consistent with these reports and provide additional evidence that sulforaphane can be effective as an inducer of QR and GST, and be a blocker of carcinogen-DNA adducts in human mammary epithelial cells. In summary, we report that the non-neoplastic human mammary epithelial cell line MCF-10F can convert BP and 1,6-DNP, two widespread environmental contaminants, to DNA-binding forms. At the doses and times examined BP binding to mammary DNA was greater than that due to 1,6-DNP. In addition, the formation of DNA adducts due to the two carcinogens was inhibited signi®cantly by treatment of cells with the cancer chemopreventive phytochemicals dibenzoylmethane and sulforaphane. Treatment of cells with sulforaphane resulted in a signi®cant stimulation of both GST and QR protein expression, whereas, dibenzoylmethane was ineffective in inducing GST and only effective in inducing QR protein expression at the highest concentration examined. The inhibitory action of sulforaphane toward BP-DNA and 1,6-DNP-DNA adduct formation could be explained by the phytochemical's capacity to stimulate QR and GST protein expression. This was not the case for dibenzoylmethane. Thus, other pathways for metabolizing BP and 1,6-DNP in MCF-10F cells are likely to be affected by dibenzoylmethane. These results provide additional evidence that sulforaphane and dibenzoylmethane warrant further evaluation as potential human breast cancer chemopreventive agents.
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These studies were funded in part by a grant (50312) from the University of Illinois Agricultural Experiment Station.
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www.elsevier.com/locate/canlet
Inhibition of benzo[a]pyrene- and 1,6-dinitropyrene-DNA adduct formation in human mammary epithelial cells bydibenzoylmethane and sulforaphane K. Singletary*, C. MacDonald 1 Department of Food Science and Human Nutrition, University of Illinois, 905 South Goodwin Avenue, Urbana, IL 61801, USA Received 20 December 1999; received in revised form 23 February 2000; accepted 24 February 2000
Abstract Numerous phytochemicals have been examined for their capacity to act as cancer chemopreventive agents. Dibenzoylmethane, a minor constituent of licorice and a compound structurally-related to curcumin, recently was identi®ed as an effective inhibitor of chemically-induced rat mammary DNA-adduct formation and tumorigenesis (Carcinogenesis 19(1998)1039-1043). The present studies were conducted to examine the capacity of dibenzoylmethane to inhibit the formation of DNA adducts following exposure to benzo[a]pyrene (BP) and 1,6-dinitropyrene (1,6-DNP), and to stimulate the expression of glutathione-Stransferase (GST) and NAD(P)H-quinone reductase (QR) proteins in the human mammary epithelial cell line MCF-10F. In addition, the ef®cacy of dibenzoylmethane as an enzyme inducer and adduct inhibitor was compared with that of sulforaphane, a potent inducer of phase II detoxi®cation enzymes and inhibitor of chemically-induced rat mammary tumorigenesis. Dibenzoylmethane at concentrations from 0.1 M to 2.0 mM inhibited BP-DNA adduct formation by 63 to 81%. Likewise, sulforaphane inhibited BP-DNA adduct formation by 68 to 80% over the same concentration range. DNA adduct formation following exposure to 1,6-DNP was signi®cantly inhibited by 46 to 61% due to dibenzoylmethane treatment (0.1 to 2.0 mM) and 30 to 56% due to sulforaphane treatment at the same concentrations. The expression of QR and GSTP1-1 proteins were increased by 3 to 4-fold and 3 to 5-fold, respectively, for MCF-10F cells treated with sulforaphane (0.5±2.0 mM). Dibenzoylmethane treatment at the same concentrations did not induce GSTP1-1 expression and signi®cantly stimulated QR expression only at the 2.0 mM concentration. These data indicate that human mammary epithelial MCF-10F cells can convert BP and 1,6-DNP to DNA-binding forms, and that DNA adduct formation can be inhibited by the phytochemicals dibenzoylmethane and sulforaphane. The inhibition of BP-DNA and 1,6-DNP adduct formation by sulforaphane was associated with increases in QR and GST protein expression. The mechanisms underlying the capacity of dibenzoylmethane to inhibit BP-DNA and 1,6-DNP-DNA adduct formation could not be explained by changes in QR or GST expression and remain to be determined. Together these data suggest that dibenzoylmethane and sulforaphane warrant continued evaluation as breast cancer chemopreventive agents. q 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dibenzoylmethane; Sulforaphane; Mammary; DNA adduct; Benzo[a]pyrene; 1,6-Dinitropyrene
1. Introduction * Corresponding author. Fax: 11-217-244-2455. E-mail address: [email protected] (K. Singletary). 1 LNMR, NCI-FDRDC, Building 560, Frederick, MD 21702, USA.
Cancer chemoprevention, the strategy to inhibit or reverse the process of carcinogenesis or to suppress the progression of preneoplastic lesions, has become a
0304-3835/00/$ - see front matter q 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00412-2
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potentially important approach to combating cancer [1]. Both synthetic and naturally occurring chemicals are being examined as possible chemopreventive agents [2]. Recently, we reported that the b-diketone dibenzoylmethane, a minor constituent of licorice and a compound structurally similar to the bioactive phytochemical curcumin, was an effective dietary inhibitor of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary DNA adduct formation and tumorigenesis [3]. These ®ndings were subsequently con®rmed and extended by others [4]. Dibenzoylmethane, appeared to act as a blocking agent, in part, by inducing the activity of the liver phase II detoxi®cation enzymes, NAD(P)H-quinone reductase (QR) and glutathione-S-transferase (GST). The present studies were conducted in order to extend our previous ®ndings with dibenzoylmethane by determining the capacity of dibenzoylmethane to inhibit carcinogen-DNA adduct formation and increase the expression of QR and GST in non-neoplastic human mammary epithelial cells. The adduct inhibitory action of this phytochemical was examined using representatives of two classes of chemical carcinogens, the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BP) and the nitro-polycyclic aromatic hydrocarbon (nitro-PAH) 1,6-dinitropyrene (1,6-DNP). Benzo[a]pyrene is an environmental contaminant and widely-studied chemical carcinogen [5] capable of forming covalent DNA adducts [6]. Although a de®nitive role for BP in breast cancer causation has not been established, there is evidence that BP-like DNA adducts are present in the normal breast tissue of breast cancer patients [7,8], and that BP may be a risk factor for sporadic breast cancer due to its ability to downregulate BRCA-1 in human breast cancer cells [9]. In addition, primary cultures of human mammary epithelial cells, in contrast to rat mammary epithelial cells, have been reported to metabolize BP to DNA-binding metabolites to a greater extent than the synthetic PAH DMBA [10]. NitroPAH also are widely distributed in the environment, apparently present in smaller quantities than PAH, but, nonetheless mutagenic and carcinogenic [11,12]. In Japan, 1,6- and 1,8-dinitropyrene isomers have been identi®ed as major mutagens in organic extract of soil samples [13]. The 1,6-dinitropyrene isomer has been reported to be highly mutagenic in the Ames Salmonella reversion assay and has been shown to be tumorigenic in several animal models, including the
mammary gland [14]. The nitroPAH also can be converted to DNA-reactive forms in several tissues [15,16]. The formation of target tissue carcinogenDNA adducts is considered to be an important prerequisite and a potential early biomarker for the initiation stage of chemically-induced carcinogenesis [17,18]. The capacity of a phytochemical to inhibit carcinogen-DNA binding is determined by the balance between activation and detoxi®cation pathways of metabolism. In particular, the action of a compound in inducing phase II enzymes, such as QR and GST, has been suggested to be an important indicator of its ability to block tumor initiation by environmental chemicals such as PAH [19,20]. Therefore, the effectiveness of dibenzoylmethane in inhibiting BP- and 1,6-DNP-DNA adduct formation and in stimulating the expression of QR and GST in human mammary epithelial cells was investigated. In addition, the ef®cacy of dibenzoylmethane was compared with that of sulforaphane, a constituent of cruciferous vegetables that recently has been identi®ed as a potent enhancer of QR and GST and an inhibitor of chemically-induced breast carcinogenesis in rats [21,22].
2. Materials and methods 2.1. Cell culture These studies were performed using the nonneoplastic human mammary cell line, MCF-10F, which was obtained from the Michigan Cancer Foundation (Detroit, MI). These cells are spontaneouslyimmortalized human breast epithelial cells that behave like normal breast epithelial cells in culture, and that can be transformed to the neoplastic phenotype by short exposure to mammary carcinogens including benzo[a]pyrene [23,24]. For adduct and enzyme experiments, 1 £ 10 5 cells were added to T150 ¯asks containing DMEM/F-12 medium (Gibco, Grand Is-land, NY) supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin, 2.5 mg/ml amphotericin B, 0.5 mg/ml hydrocortisone, 0.02 mg/ml epidermal growth factor, 0.1 mg/ml cholera enterotoxin, 10 mg/ml insulin and 5% equine serum. This number of cells was chosen for plating so that, by the end of the dibenzoylmethane or sulforaphane exposure period, cells would not have reached con¯uence. After
K. Singletary, C. MacDonald / Cancer Letters 155 (2000) 47±54
overnight attachment of cells, the either dibenzoylmethane (98%, Aldrich Chemicals, Milwaukee, WI) or sulforaphane (97%, as d,l-sulforaphane from LKT Labs, St. Paul, MN) were added at concentrations ranging from 0.1 to 2 mM for 7 days, with medium changes every other day. On day 7, cells received fresh medium containing benzo[a]pyrene (BP, Sigma Chemical) at 0.3 mM for 12 h or 1,6-dinitropyrene (DNP, Sigma Chemical) at 10 mM for 22 h. After the carcinogen incubation period, cells were harvested and the DNA isolated or the cytosolic fraction (100 000 £ g supernatant) prepared. 2.2. P-postlabeling as immunoblot analyses Aliquots of DNA were analyzed for either BP-DNA or 1,6-DNP adducts by 32P-postlabeling procedures previously described [25,26]. Nuclease P1 enhancement and butanol enhancement procedures were used for the BP-DNA and 1,6-DNP-DNA adduct analyses, respectively. For quantitation of adducts, adduct spots were removed from TLC plates and radioactivity determined by scintillation counting. Quantities were expressed as nmol adduct/mol DNA assuming 1 mg DNA 3240 pmol deoxyribonucleoside-3 0 -monophosphate. For immunoblot (Western) analysis of GSTP11 and QR proteins, equal quantities of total protein were loaded onto polyacrylamide gels, separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed using either QR antibody (rabbit antiNQO1 E. coli, a generous gift of Dr Shu-Shu Pan, University of Maryland School of Medicine, Baltimore, MD) or GSTP1-1 antibody (from Oxford Biomedical Research, Oxford, MS). Bands were detected by enhanced chemiluminescence and quantitated by densitometry.
49
adduct spot was formed from cells treated with BP (Fig. 1). We tentatively identi®ed this adduct spot to be that due to binding of anti-BP-dihydrodiolepoxide with deoxyguanosine. This was determined by reacting deoxyadenosine-3 0 -monophosphate (dAdo) or deoxyguanosine-3 0 -monophosphate (dGuo) with a racemic mixture of (^) anti-BP-dihydrodiolepoxides (Chemsyn Science Laboratories, Lenexa, KS) using the protocol described by Canella et al. [27]. Only the 32P-postlabeled product formed from reaction with dGuo co-migrated during TLC with the MCF10F BP-DNA adduct (data not shown). Also, we determined that BP-DNA adduct formation increased linearly when cells were exposed to BP for 12 h at concentrations from 0.08 to 0.8 mM. BP-DNA adducts formed were 127, 323 and 583 nmol BP/mol DNA for cells incubated for 12 h with BP concentrations of
3. Results Neither dibenzoylmethane nor sulforaphane exhibited cytotoxic effects toward the MCF-10F cells at the concentrations and incubation periods examined. Viability, as determined by Trypan Blue exclusion, always exceeded 95%. The ®rst objective of these studies was to determine the inhibitory effect of dibenzoylmethane and sulforaphane toward DNA adduct formation in the MCF-10F cells resulting from either BP or 1,6 DNP exposure. Predominantly one DNA
Fig. 1. Autoradiographs of 32P-postlabeled DNA from MCF-10F cells exposed to benzo[a]pyrene (A) and 1,6-dinitropyrene (B). Arrows indicate adduct spots quantitated. OR, origin.
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K. Singletary, C. MacDonald / Cancer Letters 155 (2000) 47±54
0.08, 0.3 and 0.8 mM, respectively. An intermediate dose of 0.3 mM was thus chosen for these experiments. The formation of BP-DNA adducts was significantly inhibited by 63, 62 and 81% for cells treated with 0.1, 0.5 and 2.0 mM dibenzoylmethane, respectively, compared with controls (Fig. 2). Likewise, sulforaphane inhibited BP-DNA adduct formation by 68, 80 and 70% for cells treated at concentrations of 0.1, 0.5 and 2.0 mM, respectively, compared with controls. When adduct formation following exposure of cells with 1,6-DNP was determined, essentially one major and two minor adduct spots were observed (Fig. 1). We did not determine the identity of these TLC adduct spots. In preliminary studies we deter-
mined that adduct spot formation increased in a linear fashion when cells were incubated for 22 h with 1,6-DNP at concentrations of 5 and 10 but not 50 mM. The 10 mM concentration of 1,6-DNP was thus chosen for these experiments. Total 1,6-DNPDNA adduct spot formation was inhibited by 46, 61 and 50% for samples treated with 0.1, 0.5 and 2.0 mM dibenzoylmethane, respectively, and by 56, 33 and 30% for samples treated with 0.1, 0.5 and 2.0 mM sulforaphane, respectively, compared with controls (Fig. 2). The second part of these studies involved examining the effect of dibenzoylmethane and sulforaphane treatment on the expression of the phase II detoxi®cation enzymes QR and GSTP1-1. The pi class of GST was chosen for examination because it is actively involved in BP-diolepoxide (BPDE) conjugation in humans [19], and because GSTP1-1 in mouse liver has been reported to be more effective in BPDE detoxi®cation than all other GSTs combined [28]. We observed that dibenzoylmethane increased QR protein expression, compared with controls, but only at the 2.0 mM concentration of dibenzoylmethane. Dibenzoylmethane had no effect on GSTP1-1 protein levels (Fig. 3). On the other hand, sulforaphane at concentrations from 0.5 to 2.0 mM signi®cantly increased QR protein and GSTP1-1 protein levels by 3 to 5-fold and 3 to 4-fold, respectively, compared with controls (Fig. 4). 4. Discussion
Fig. 2. Effect of dibenzoylmethane (DBM) and sulforaphane on BPDNA adduct formation (A) and 1,6-DNP adduct formation (B) in MCF-10F cells. Values represent means ^ SEM of three separate samples. Bars indicated by w are signi®cantly different (ANOVA) from controls at P , 0:05.
The present studies provide evidence that cultures of the non-neoplastic human mammary epithelial cell line MCF-10F exposed to BP and 1,6-DNP form carcinogen-DNA adduct spots following isolation and detection by the 32P-postlabeling procedure, with the magnitude of adduct formation following treatment with BP being greater. Our ®ndings that non-neoplastic mammary cells can bioactivate BP are similar to those reported by Moore et al. [6,10,29] in which human mammary cells possessed the capacity to metabolize BP to DNA-binding metabolites. Stampfer et al. [30] also determined that, for primary human mammary epithelial cells exposed to BP, the major adduct formed is derived from binding to dGuo. Our results with 1,6-DNP are similar to those
K. Singletary, C. MacDonald / Cancer Letters 155 (2000) 47±54
51
Fig. 3. Effect of dibenzoylmethane (DBM) on the protein expression of GST(A) and QR (B) in MCF-10F cells. Values represent means ^ SEM of three separate samples. Bars indicated by w are signi®cantly different (ANOVA) from controls at P , 0:05.
of Carmichael et al. [26], who observed that suspensions of primary human mammary epithelials treated with 1,6-DNP produced three DNA adduct spots. Although the quantity of DNA binding was not determined, it was observed that adduct formation from 1,6-DNP was less than that resulting from treatment of cells with other environmental agents, such as the hetrocyclic amines and the PAH dibenzo[a,l]pyrene. Elbridge et al. [31] examined the genotoxicity of BP, 1,6-DNP and other environmental agents in normal human mammary cells, and found that treatment of cells with BP and 1,6-DNP resulted in detectable genotoxicity, as indicated by unscheduled DNA synthesis. However, the extent of binding was not quantitated. In F344 rats, administration of 1,6-DNP was reported to produce one major adduct, the N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene in lung, liver, and other tissues [32]. The studies also indicate that the phytochemicals dibenzoylmethane and sulforaphane can inhibit carci-
nogen-DNA binding in cultures of MCF-10F cells treated with either BP or 1,6-DNP. However, the magnitude of the inhibitory effect depended on the carcinogen examined. When cells were treated with BP, both dibenzoylmethane and sulforaphane were effective inhibitors of adduct formation. When cells were treated with 1,6-DNP, both compounds inhibited adduct formation, although to a lesser extent than when BP was used. In other words, at the concentrations of dibenzoylmethane and sulforaphane examined, BP adduct formation was inhibited by greater than 60%. However, 1,6-DNP adduct formation was inhibited at most by 61% following treatment with similar concentrations of the two phytochemicals. When adduct inhibition was compared with the capacity to enhance protein expression of QR and GST, sulforaphane's capacity to stimulate QR and GST protein expression was associated with the inhibition by sulforaphane of DNA adduct formation due to BP treatment. However, although dibenzoylmethane did
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K. Singletary, C. MacDonald / Cancer Letters 155 (2000) 47±54
Fig. 4. Effect of sulforaphane on the protein expression of GST(A) and QR (B) in MCF-10F cells. Values represent means ^ SEM of three separate samples. Bars indicated by w are signi®cantly different (ANOVA) from controls at P , 0:05.
exhibit adduct inhibitory effects similar to sulforaphane toward BP- and 1,6-DNP-induced DNA adduct formation, dibenzoylmethane treatment did not increase GST protein expression in MCF-10F cells and only stimulated QR protein expression at the highest concentration of dibenzoylmethane examined, 2.0 mM. This is in contrast to dietary dibenzoylmethane's capacity to stimulate QR and GST activity in the livers of female rats [3]. The reason for the inability of dibenzoylmethane to induce GST in these human mammary cells is not known, but may be due to tissue-related differences in the regulation of QR and GST gene expression. This suggests that mechanisms other than those involving GST- and QR-induction are contributing to the inhibition of BP-DNA adduct formation in MCF-10F cells treated with dibenzoylmethane, especially at dibenzoylmethane concentrations ,2.0 mM. The effect of dibenzoyl-
methane treatment on cytochrome P450 enzymes of MCF-10F cells may be important to determine, since cytochrome P450 1A is known to participate in the metabolism of polycyclic aromatic hydrocarbons like BP, and cytochrome P450 1B1 is a new P450 family 1 member present in rodent and human tissues that can convert numerous chemicals including PAHs and nitroPAH to genotoxic intermediates in liver and extrahepatic tissue [33±35]. In addition, dibenzoylmethane may be acting, in part, via antioxidant and/or antiin¯ammatory processes. It has been suggested that BP can be metabolically activated to DNA-binding species by a process involving free radicals [36,37]. Although dibenzoylmethane has been reported to be inactive as an antioxidant and free-radical scavenger in vitro [4], the b-diketone moiety has been shown to be important for the antioxidant action of other biological compounds [38,39]. With regard to 1,6-DNP adduct formation, both dibenzoylmethane and sulforaphane exhibited more modest degrees of inhibition compared with their inhibition of BP-DNA adduct formation. The adduct inhibitory action of sulforaphane toward 1,6-DNP could be due, in part, to its GST- and QR-inducing effect. However, although the QR-inducing effect of dibenzoylmethane may be contributing partly to the inhibition of 1,6-DNP adduct spot formation (particularly at the 2.0 mM dose), other pathways for metabolism of 1,6-DNP may be affected by dibenzoylmethane. It has been reported that nitroreductase and O-acetylation activities may be involved in the activation of dinitropyrenes [40]. There is evidence that MCF-10 cells may possess the capacity to O-acetylate, since Swaminathan et al. [41] have reported that the human mammary epithelial cell line MCF-10A can activate the mammary gland carcinogen N-hydroxy-4-acetylaminobiphenyl by acetyl transferase(s) enzyme systems. Localization of acyltransferases [42] and their role in activation of carcinogens in the rat mammary gland have been observed [43]. The extent of nitroreductase activity in mammary tissue and whether dibenzoylmethane (and sulforaphane) can inhibit this activity as well as alter acyl transferase activity warrants further study. The present data for sulforaphane are consistent with that of Zhang et al. [21,22] who reported that sulforaphane is a potent inducer of GST and QR in cytosols from several organs of CD-1 mice, and that sulforaphane can inhibit both tumor multiplicity and
K. Singletary, C. MacDonald / Cancer Letters 155 (2000) 47±54
incidence in female Sprague±Dawley rats initiated with the PAH DMBA. Our results are consistent with these reports and provide additional evidence that sulforaphane can be effective as an inducer of QR and GST, and be a blocker of carcinogen-DNA adducts in human mammary epithelial cells. In summary, we report that the non-neoplastic human mammary epithelial cell line MCF-10F can convert BP and 1,6-DNP, two widespread environmental contaminants, to DNA-binding forms. At the doses and times examined BP binding to mammary DNA was greater than that due to 1,6-DNP. In addition, the formation of DNA adducts due to the two carcinogens was inhibited signi®cantly by treatment of cells with the cancer chemopreventive phytochemicals dibenzoylmethane and sulforaphane. Treatment of cells with sulforaphane resulted in a signi®cant stimulation of both GST and QR protein expression, whereas, dibenzoylmethane was ineffective in inducing GST and only effective in inducing QR protein expression at the highest concentration examined. The inhibitory action of sulforaphane toward BP-DNA and 1,6-DNP-DNA adduct formation could be explained by the phytochemical's capacity to stimulate QR and GST protein expression. This was not the case for dibenzoylmethane. Thus, other pathways for metabolizing BP and 1,6-DNP in MCF-10F cells are likely to be affected by dibenzoylmethane. These results provide additional evidence that sulforaphane and dibenzoylmethane warrant further evaluation as potential human breast cancer chemopreventive agents.
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Acknowledgements
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These studies were funded in part by a grant (50312) from the University of Illinois Agricultural Experiment Station.
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