Crude Edible Fig (Ficus carica) Leaf Extract Prevents Diethylstilbestrol (DES)-Induced DNA Strand Breaks in Single-Cell Gel Electrophoresis (SCGE)/Comet Assay: Literature Review and Pilot Study.

Fig (Ficus carica) trees are among the oldest plants on earth. The chemopreventive properties of constituent polyphenols and fiber that implicate figs in having a functional role in averting cancer have not been fully elucidated. We therefore hypothesized that fig leaf extract would inhibit (or attenuate) DES-induced DNA single-strand breakage in MCF10A human breast epithelial cells. To test this hypothesis, MCF10A cells were treated with DES (1, 10, 100 µM), crude fig leaf extract (5, 10, 15 µL), or concomitant doses of DES (100 µM)/fig leaf extract (5, 10, 15 µL). The cells were analyzed for DNA strand breakage using the SCGE/COMET assay with mean olive tail moment as a marker of DNA damage. DES induced DNA strand breaks at all treatment levels compared to DMSO and non-treatment controls. DES at concentrations of 1, 10, and 100 µM produced mean olive tail moments of 1.2082 (177.6%), 1.2702 (186.7%), and 1.1275 (165.7%), respectively, which were statistically significantly (p<0.05) higher than the DMSO control value (0.6803). Exposure to fig leaf extract produced no DNA damage. Rather, a desirable dose-dependent reduction in DES-induced DNA strand breaks was observed. Composite treatment of MCF10A cells with DES and fig leaf extract attenuated DES-induced DNA strand breaks. Taken together, these results suggest a potential mechanism for cancer chemoprevention. Additional studies are necessary to identify relevant active ingredients, confirm the mechanism of action, and further elucidate the therapeutic potential of fig leaf extract for early-stage breast cancer chemoprevention.

Diallyl sulfide inhibits diethylstilbestrol induced DNA damage in human breast epithelial cells (MCF-10A).

Breast cancer is the second leading cause of cancer deaths in women in the United States. Diethylstilbestrol (DES) is a synthetic estrogen that has been shown to cause cancer in animals and humans, altering cell viability as well as inducing DNA damage. Diallyl sulfide (DAS) is a garlic organosulfide that has been shown to inhibit both the initiation and promotion phases of cancer in vivo and in vitro, as well as reduce the risk of cancer in epidemiological studies. MCF-10A cells, regarded as a normal breast epithelial cell line, were treated with varying concentrations of DES, DAS or various dose combinations of DES and DAS concomitantly, and assessed for cell viability, DNA strand breaks, and lipid peroxidation. DES (10µM) in combination with 1, 10, or 100µM DAS resulted in a 31%, 34%, or 36% respective increase in cell viability compared to the DES treatment alone, after 24h. At the same time point, 1, 10, and 100µM DAS were all effective in significantly reducing DES (100µM)-induced strand breaks to near that of the vehicle control. Additionally, 1µM DAS was effective in significantly reducing DES (100µM)-induced lipid peroxidation after 3h. The results of this research suggest that DAS is effective in recovering cell viability, attenuating DNA strand breaks, and decreasing lipid peroxidation in MCF-10A cells.

Induction of cell death, DNA strand breaks, and cell cycle arrest in DU145 human prostate carcinoma cell line by benzo[a]pyrene and benzo[a]pyrene-7,8-diol-9,10-epoxide.

Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon, is a major environmental pollutant. In this study, the effects of this carcinogen/mutagen and one of its metabolites, benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), on human prostate carcinoma cell line DU145, were examined. Cell viability, DNA damage, and cell cycle progression were evaluated as toxic end-points. We have shown that B[a]P and BPDE inhibited cell viability following 48 hr of exposure. Furthermore, comet assay analyses revealed that both B[a]P and BPDE induced DNA strand breaks in a concentration-dependent fashion. Flow cytometric analyses showed that about 70% of DU145 cells were arrested by B[a]P at the G1 phase, while about 76% were arrested at G1 phase by BPDE. These data reveal that B[a]P and BPDE are cytotoxic and genotoxic to DU145 prostate cancer cells.

Diallyl sulfide induces the expression of estrogen metabolizing genes in the presence and/or absence of diethylstilbestrol in the breast of female ACI rats.

Diethylstilbestrol (DES) induces mammary tumors in female ACI rats and is associated with an increased risk of developing breast cancer in humans. Diallyl sulfide (DAS) has been shown to prevent cancer in animals. Previously, we have shown that DAS inhibits the production of DES induced DNA adducts when given prior to DES. We hypothesize that DAS alters the expression of genes responsible for DES metabolism. To test this hypothesis, four groups of 10 female ACI rats were treated daily for four days as follows: (1) corn oil, (2) 50mg/kg DES, (3) 50mg/kg DAS, and (4) 50mg/kg DAS+50mg/kg DES. RNA was isolated from breast tissue and mRNA levels of CYP1A1, CYP1B1, glutathione-S-transferase (GST) and superoxide dismutase (SOD) were analyzed by real-time PCR. DES, DAS, and DES/DAS treatments increased the expression of CYP1A1 by 2.1-, 4.7-, and 12.7-fold, respectively. Similar results were seen for CYP1B1. DES decreased the expression of GST by 23%, whereas DAS and DAS/DES treatments increased the expression of GST by 12- and 16.7-fold, respectively. Similar results were seen with SOD. These results suggests that DAS may prevent the formation of DES induced DNA damage by altering the expression of DES metabolizing genes.

Diallyl sulfide induces the expression of nucleotide excision repair enzymes in the breast of female ACI rats.

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 (50mg/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.

Cytochrome P450 expression and metabolic activation of cooked food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in MCF10A breast epithelial cells.

The cytochrome P450 expression profile was determined in the MCF10A human breast epithelial cell line, as was the ability of this cell line to catalyze the bioactivation of the cooked food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Using non-quantitative reverse transcription-polymerase chain reaction (RT-PCR), transcripts for CYP1B1, CYP2J2, CYP2R1, CYP2U1, CYP2W1, CYP4B1, CYP4F, CYP4V2, CYP4X1 and CYP4Z1 were detected in both sub-confluent and confluent MCF10A cells. By contrast, CYP1A2 mRNA was detected only in confluent MCF10A cells, while CYP1A1, CYP2S1 and CYP2F1 were detected predominantly or exclusively in sub-confluent cultures. 2,3,7,8-Tetrachlorodibenzo-p-dioxin treatment of confluent MCF10A cells markedly induced microsomal ethoxyresorufin O-deethylase activity and CYP1A1, CYP1A2 and CYP1B1 mRNA levels, as determined by real-time RT-PCR, while treatment with 10(-4)M PhIP had little effect on these P450 transcript levels. Treatment of confluent MCF10A cells with PhIP (10(-4)M) for 24, 48 or 72 h produced time-dependent increases in the amounts of DNA adducts, as measured by (32)P-post-labeling. These results indicate that multiple P450s, including those known to catalyze PhIP N-oxidation, are expressed in MCF10A cells, and that this non-neoplastic human breast epithelial cell line contains sufficient enzymatic machinery to support PhIP bioactivation and generate DNA damage.

Diallyl sulfide inhibits the oxidation and reduction reactions of stilbene estrogens catalyzed by microsomes, mitochondria and nuclei isolated from breast tissue of female ACI rats.

Previously, it has been demonstrated that microsomal, mitochondrial and nuclear enzymes isolated from the liver of male Sprague-Dawley rats catalyzed the oxidation of diethylstilbestrol (DES) to DES quinone. In the present study we have shown that diallyl sulfide (DAS) inhibits the oxidation of DES to DES quinone in all three subcellular fractions (microsomes, mitochondria and nuclei) isolated from breast tissue of female ACI rats. UV analysis of mitochondrial and microsomal fractions revealed that DAS decreased the rate of DES oxidation to DES quinone and DAS also decreased the rate in which DES quinone was reduced to DES. Lineweaver-Burk plots of the rate of DES quinone formation at various DES and DAS concentrations demonstrated that DAS inhibited the oxidation of DES and the reduction of DES quinone in a non-competitive fashion. In both microsomal and mitochondrial oxidation reactions the K(m) remained constant whereas the V(max) decreased with increasing DAS (0, 186 and 373 microM) concentrations (microsomes K(m) = 80 microM; V(max) = 5.56, 4.16 and 3.33 nmol/mg protein/min; mitochondria K(m) = 35.7 microM; V(max) = 3.45, 2.44 and 1.82 nmol/mg protein/min). Results were similar for reduction reactions. HPLC analysis revealed that a concentration of 186 microM DAS inhibited the mitochondrial, microsomal and nuclear oxidation by 27, 35 and 40%, respectively. A concentration of 373 microM DAS inhibited the mitochondrial, microsomal and nuclear oxidation by 50, 52 and 60% respectively. The data provide direct evidence that the breast tissue contain the metabolic machinery required to oxidize DES to reactive intermediates that may lead to genetic instability and cancer. This inhibition may play a role in the chemoprevention of stilbene estrogen-induced breast cancer.

Diallyl sulfide inhibits diethylstilbestrol-induced lipid peroxidation in breast tissue of female ACI rats: implications in breast cancer prevention.

Diallyl sulfide (DAS) is a component of garlic and prevents cancer in several animal models in various organs. The chemopreventive effects of DAS are attributed to modulation of enzymes to alter the bioactivation of xenobiotics. Diethylstilbestrol (DES) is a synthetic estrogen that causes breast cancer in female ACI rats subsequent to metabolism with concurrent free radical production. This study assessed the effect of DAS on DES-induced reactive oxygen species (ROS) using lipid peroxidation as an empirical endpoint. We have demonstrated that acute exposure to DES results in a significant increase in lipid hydroperoxides (LPH) in breast tissue and DAS attenuated DES-induced LPH concentrations. Two-week exposure to DES caused significant increases in LPH concentrations in breast and liver tissues. DES-induced LPH concentrations were decreased by coadministration of DAS at this time point. There were no statistical differences in the concentrations of LPH in breast and liver tissues of rats treated for 4/6 weeks with DAS/DES. These results demonstrate that DAS inhibits the production of ROS which suggests that DAS effectively inhibits DES bioactivation in female ACI rats which may have implications for chemopreventive intervention strategies. Our results suggest that garlic consumption might be useful for the prevention of human breast cancers.