• Toxicological Research
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• v.23 no.2
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• pp.135-142
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• 2007

Formononetin is an isoflavonoid phytoestrogen found in certain foodstuffs such as soy and red clover. In this study, we examined the action of formononetin with the carcinogen activation pathway mediated through the aryl hydrocarbon receptor (AhR) in MCF-7 breast carcinoma cells. Treating the cells with formononetin alone caused the accumulation of CYP1A1 mRNA as well as elevation in CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity in a dose dependent manner. However, a concomitant treatment with 7,12-dimethylbenz[a]anthracene (DMBA) and formononetin markedly reduced both the DMBA-inducible EROD activity and CYP1A1 mRNA level. Under the same conditions, formononetin inhibited the DMBA-induced AhR transactivation, as shown by reporter gene analysis using a xenobiotic responsive element (XRE). Additionally, formononetin inhibited both DMBA-inducible nuclear localization of the aryl hydrocarbon receptor (AhR) and metabolic activation of DMBA, as measured by the formation of the DMBA-DNA adducts. Furthermore, formononetin competed with the prototypical AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), for binding to the AhR in an isolated rat cytosol. These results suggest that formononetin might be considered as a natural ligand to bind on AhR and consequently produces a potent protective effect against DMBA-induced genotoxicity. Therefore, that's the potential to act as a chemopreventive agent that is related to its effect on AhR pathway as antagonist/agonist.

• Journal of Microbiology and Biotechnology
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• v.26 no.9
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• pp.1650-1656
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• 2016

The SCO0284 gene of Streptomyces coelicolor A3(2) is predicted to encode an α-galactosidase (680 amino acids) belonging to glycoside hydrolase family 27. In this study, the SCO0284 coding region was cloned and overexpressed in Streptomyces lividans TK24. The mature form of SCO0284 (641 amino acids, 68 kDa) was purified from culture broth by gel filtration chromatography, with 83.3-fold purification and a yield of 11.2%. Purified SCO0284 showed strong activity against p-nitrophenyl-α-D-galactopyranoside, melibiose, raffinose, and stachyose, and no activity toward lactose, agar (galactan), and neoagarooligosaccharides, indicating that it is an α-galactosidase. Optimal enzyme activity was observed at 40℃ and pH 7.0. The addition of metal ions or EDTA did not affect the enzyme activity, indicating that no metal cofactor is required. The kinetic parameters V_max and K_m for p-nitrophenyl-α-D-galactopyranoside were 1.6 mg/ml (0.0053 M) and 71.4 U/mg, respectively. Thin-layer chromatography and mass spectrometry analysis of the hydrolyzed products of melibiose, raffinose, and stachyose showed perfect matches with the masses of the sodium adducts of the hydrolyzed products, galactose (M+Na, 203), melibiose (M+Na, 365), and raffinose (M+Na, 527), respectively, indicating that it specifically cleaves the α-1,6-glycosidic bond of the substrate, releasing the terminal D-galactose.

• Bulletin of the Korean Chemical Society
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• v.32 no.9
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• pp.3372-3376
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• 2011

We have studied the relative stability and atomic structures of five $C_{20}X_2$ regioisomers obtained as diadducts of a $C_{20}$ cage (X = H, F, Cl, Br, and OH). All the regioisomers are geometric isomers, i.e., they differ in their spatial arrangement. Full-geometry optimizations of the regioisomers have been performed using the hybrid density-functional (B3LYP/6-31G(d, p)) method. Our results suggest that the cis-1 regioisomer (the 1,2-diadduct) is the most stable and that the second most stable is the trans-2 (1,13-diadduct) regioisomer, implying that the long-range interaction between the two adducts and the resonance effect are more pronounced than the diadduct-induced strain in the $C_{20}$ cage. The HOMO and LUMO characteristics of each regioisomer with the same symmetry of structural regioisomers except $C_{20}(OH)_2$ are topologically same. This suggests that by using an entirely different set of characteristic chemical reactions for each regioisomer, we can distinguish between the five regioisomers for each $C_{20}$ diadduct derivative.

• IMMUNE NETWORK
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• v.2 no.4
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• pp.208-216
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• 2002

Background: Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can induce mutations in key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity. Key members of the MMR system include MutS${\alpha}$ (comprised of hMSH2 and hMSH6), which can sense and repair single base mismatches and 8-oxoguanine, and MutS${\beta}$ (comprised of hMSH2 and hMSH3), which repairs longer insertion/deletion loops. Methods: To provide further evidence of DNA damage, we analyzed synovial tissues for microsatellite instability (MSI). MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells (PBC) of RA patients using specific primer sequences for 5 key microsatellites. Results: Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis (OA) tissue. Western blot analysis of the same tissues for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP). Western blot analysis demonstrated constitutive expression of hMSH2, 3 and 6 in RA and OA FLS. When FLS were cultured with SNAP, the RA synovial pattern of MMR expression was reproduced (high hMSH3, low hMSH6). Conclusion: Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.

• Nutritional Sciences
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• v.9 no.3
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• pp.212-226
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• 2006

The involvement of arachidonic acid (AA) metabolizing enzyme, lipoxygenase (LOX), in the development of particular tumors in humans has gradually been acknowledged and LOX has emerged as a novel target to prevent or treat human cancers. In the mouse skin carcinogenesis model, which provides an excellent model to study multistage nature of human cancer development, many studies have shown that some of the LOXs are constitutively upregulated in their expression. Moreover, application of LOX inhibitors effectively reduced tumor burdens, which implicates the involvement of LOX in mouse skin tumor development as well. 8S-LOX is a recently cloned LOX, which is specifically expressed in mouse skin after 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment but not in normal skin. Unlike other members of the LOX 'family' expressed in mouse skin, this TPA-induced expression of 8S-LOX is prominent only in the skin of the TPA tumor promotion-sensitive strains of mice (SENCAR, CD-1, and NMRI) but not in the promotion-resistant C57BL/6J mice. This is a very unique phenomenon among strains of mice. Constitutive upregulation of 8S-LOX was also found in early stage papillomas and the expression was gradually reduced as the tumors became malignant. Based on these observations, it has been thought that 8S-LOX is involved in TPA-induced tumor promotion as well as in tumor conversion from papillomas to carcinomas. In accordance with this hypothesis, several studies have suggested possible roles of 8S-hydroxyeicosatetraenoic acid (HETE), an AA metabolite of 8S-LOX, in mouse skin tumor development. A clastogenic activity of 8S-HETE was demonstrated in primary keratinocytes and a close correlation between the levels of etheno-DNA adducts and 8S-HETE during skin carcinogenesis was also reported. On the other hand, it has been reported that 8S-LOX protein expression is restricted to a differentiated keratinocyte compartment Moreover, reported findings on the ability of 8S-HETE to cause keratinocyte differentiation appear to be contrary to the procarcinogenic features of the 8S-LOX expression, presenting a question as to the role of 8S-LOX during mouse skin carcinogenesis. In this review, molecular and biological features of 8S-LOX as well as current views on the functional role of 8S-LOX/8S-HETE during mouse skin carcinogenesis are presented.

• Toxicological Research
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• v.6 no.2
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• pp.167-182
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• 1990

Dietary restriction (caloric restriction) is the only intervention which has been reliably shown to extend the maximum life span of warm-blooded animals and delay the many phenomena associated with aging. It is also one of the most effective modulators of toxicity, especially cancer endpoints. In spite of the known modulator effects of caloric restriction, the biological mechanisms responsible for these effects had not been in vestigated until recently. The National Center for Toxicological Research (NCTR), in a collaborative effort with the National Institute of Aging (NIA), initiated a project whereby nine (9) combinations of rodent species/strains and diets were fed both restricted and ad libitum. The NIA's initiative was to identify biomarkers of aging whereas NCTR's initiative was to identify the biological effects associated with the profound effects caloric restriction has in protecting against both spontaneous (age-related) and chemically-induced toxic endpoints. Independent of sex or species, caloric restriction has similar effects on body temperature, oxygen consumption and $CO_2$production. Caloric restriction also decreased lipid glycolysis and metabolism in rats and mice, which suggest decreased production of metabolites which could lead to fatty acid epoxide formation. The age-associated loss of ciradian regulation of intermediate enzymes is also significantly reduced. Moreover, caloric restriction reduced the age-associated feminization of sexually dimorphic liver isozymes, increased several glucocorticoid responsive isozymes, elevated glucagon/insulin ratios, produced less microsomal superoxide and enhanced the capacity for utilzing detoxicating metabolic pathways. Calorically restricted rats have less than half the number of aflatoxin ($AFB_1$)-DNA adducts than ad libitum animals and urinary excretion of $AFB_1$ was increased significantly. Finally, DNA repair mechanisms are enhanced and oncogene expression is decreased in calorically restricted animals.

• Journal of Life Science
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• v.8 no.4
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• pp.400-409
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• 1998

Formation of adduct was studied in benzo(a)pyrene(BP)- and doxorubicin(Dx)-treated human NC-37 cells and isolated nuclei. Major adducts formed were determined by fluorescence absorption spectrophotometery and DNA-lin-ked protein assay. When isolated nuclei were exposed to carcinogens BP and DMBA, and anticancer drugs m-AMSA, ellipticine and Dx, varying degrees of adduct formation occured between DNA-protein complex and these drugs. When the mixture was centrifuged 1.7 M sucrose solution, binding BP and DMBA appeared to be similar between the sediment and the supernatant. When the sediment was centrifuged again with 0.35% polymin-P, the amount of BP bound was 2-fold greater in the protein(1077$\pm$55cpm) than in DNA fraction (470$\pm$20cpm), whereas that of DMBA was 1.6-fold greater in the DNA than in protein fraction. In the case of m-AMSA, ellipticine and Dx, the amount of binding was slightly greater in supernatant than in sediment in centrifugation with 1.7 M sucrose, and more than 3 times greater in the DNA- than in protein- fraction in centrifugation with 0.35% polymin P. DNA fractions which associated with a subset of nonhistone chromosomal protein were isolated from NC-37 cells exposed to $^{3}$H-BP and $^{14}$C-Dx. They were separated into two distince components DNA-S and DNA-P by centrifugation with 2M Nacl chromatin extraction. The results indicated that the amount of $^{3}$H-BP bound was 6.0-fold greater in DNA-P as compared with DNA-S, while that of $^{14}$C-Dx binding appreaed to be 6.2-fold greater in DNA-S than in DNA-P fraction. When $^{3}$H-BP binding wasdetermined in the presence of cold Dx, the amount of binding was reduced only in the DNA-P fraction, indicating that the interaction between DNA and protein is decreased. Gene expression by these drugs, BP treated cells were increased to compare with nomal cells but reduced by treatment with BP-Dx. These results suggest that the protein moiety which tightly bound to DNA-P fraction may play an important role in the regulation of gene expression.

• Journal of Ginseng Research
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• v.13 no.1
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• pp.37-41
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• 1989

Reactive metabolites generated by benzo(a)pyrene(BP) monooxygenase(AHH) interact with nucleophiles in DNA and cause mutation and carcinogenesis. We studied the effect of Panax ginseng C.A. Meyer, which induce epoxide hydratase(EH) activity without concomitant induction of AHH activity, on the binding of BP metabolites to DNA in uitro in Sprague Dawley rats. DNA-BP metabolite adducts can be resolved into at least five distinct peaks by elution of a Sephadex LH-20 column with a water methanol gradieNt. These peaks are arbitrarily designated A(most polar) through I(least polar). Of the 5 peaks tentatively assigned to 7,8 biol-9,10-oxide(A),7,8·oxide(B),4,5-oxide(C), and further metabolites of 9-OH-BP(D & E), peaks A, C, D, and I were reduced to 70, 85, 80, and 30% of controls, respectively, and there was no significant change in peak B. In connection with this DNA binding study, BP metabolizing enzymes including AHH, EH, demethylase(DM) activity and cyt. P-450 contents were also investigated in order to compare the BP treated control with ginseng and BP treated test groups. The results showed that the EH activity was increased by 139% over the BP control, the Cyt. P-450 content was increased by 180% over the control value, and DM and AHH activities were also increased to some degree for the BP test group, but there was no significant effect of the ginseng treatment.

• Archives of Pharmacal Research
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• v.22 no.5
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• pp.474-478
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• 1999

Modulation of unscheduled DNA synthesis by dehydroepiandrosterone (DHEA) after exposure to various chemical carcinogens was investigated in the primary rat hepatocytes. Unscheduled DNA synthesis was induced by treatment of such direct acting carcinogens as methly methanesulfonate (MMS) and ethyl methanesulfonate (EMS) or procarcinogens including benzo(a)pyrene (BaP) and 7, 12-dimethylbenz(a)anthracene (DMBA). Unscheduled DNA synthesis was determined by measuring [methyl-3H]thymidine radioactivity incorporated into nuclear DNA of hepatocytes treated with carcinogens in the presence or absence of DHEA. Hydroxyurea $(5{\times}10^{-3} M)$was added to growth medium to selectively suppress normal replication. DHEA at concentrations ranging from $(1{\times}10^{-6} M)$ to$(5{\times}10^{-4} M)$ did not significantly inhibit unscheduled DNA synthesis induced by either MMS $(1{\times}10^{-4} M)$ or EMS $(1{\times}10^{-2} M)$. In contrast, DHEA-significantly inhibited unscheduled DNA synthesis induced by BaP $(6.5{\times}10^{-5} M)$ and DMBA.$(2{\times}10^{-5} M)$. DHEA-induced hepatotoxicity in rats was examined using lactate dehydrogenase (LDH) release as an indicator of cytotoxicity. DHEA exhibit no significant increase in LDH release compared with the control at 18 h. These data suggest that nontoxic concentration of DHEA does not affect the DNA excision repair process, but it probably influence the enzymatic system responsible for the metabolic activation of procarcinogens and thereby decreases the amount of the effective DNA adducts formed by the ultimate reactive carcinogenic species.

• Journal of the Korean Chemical Society
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• v.38 no.9
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• pp.676-681
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• 1994

Diels-Alder adducts, carbacephems were obtained when 4-acetoxyazetidine-2-one or (3R,4R)-4-acetoxy-3-[(1R)-1-(tert-butyldimethylsilyloxy)ethyl]azetidin-2-one was reacted with 2-(tert-butyldimethylsilyloxy)-1,3-butadiene or with 3-(tert-butyldimethylsilyloxy)-1,3-pentadiene. When tert-butyldiemethylsilyl acrylate was used as the diene, products in which was acetoxy group of 4-acetoxyazetidin-2-one derivatives was substituted by an acryloyloxy group were isolated. When the same reaction was carried out with 4-phenylsulfonylazetidin-2-one as a dienophile with 2-(tert-butyldimethylsilyloxy)-1,3-butadiene, 4-phenylsulfoyl-2-butanone was obtained instead of the expected carbacephem. When dimeric form of thiochalcone was reacted with 4-acetoxyazetidin-2-one in the presence of zinc chloride, the $\beta-lactam$ ring of the azetidin-2-one was destructed and no product was isolated. Also, the reaction of 2-trimethylsilyloxy-1-aza-1,3-butadiene, which was obtained from N-methylacrylamide by treatment of trimethylsilyl trifluoromethanesulfonate in the presence of triethylamine, with 4-acetoxyazetidin-2-one did not give any products.