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1.
Pretreatment of secondary cultures of Syrian hamster embryo cells with 7,8-benzoflavone (7,8-BF) inhibited both the metabolism of 7,12-dimethylbenz[alpha] anthracene (DMBA) and the formation of DMBA-DNA adducts. The DMBA-deoxyribonucleoside adducts from 7,8-BF-treated cultures had the same elution profiles on Sephadex LH-20 columns as those from cultures exposed to DMBA alone, but 7,8-BF-treated cultures contained smaller amounts of DMBA-DNA adducts per mg DNA. As the concentration of 7,8-BF was increased, the decrease in the amount of DMBA-DNA adducts per mg DNA was logarithmic with respect to the decrease in the amount of DMBA metabolized. The results suggest that more than one metabolic step is required for the binding of DMBA to DNA in hamster embryo cells. 相似文献
2.
El-Bayoumy K Das A Boyiri T Desai D Sinha R Pittman B Amin S 《Chemico-biological interactions》2003,146(2):179-190
1,4-phenylenebis(methylene)selenocyanate (p-XSC) inhibits 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis and DMBA-DNA binding in the rat mammary gland. Tetraselenocyclophane (TSC) was identified in rat feces as a metabolite of p-XSC. This led us to postulate the metabolic pathway: p-XSC-->glutathione conjugate (p-XSeSG)-->aromatic selenol (p-XSeH)-->TSC. Whether p-XSC or one of its metabolites is responsible for cancer prevention is the focus of this study. We utilized the DMBA-DNA binding assay with p-XSC as a positive control to evaluate the chemopreventive potential of p-XSC metabolites at dietary selenium levels of 10 ppm. Rats were fed AIN-76A diet supplemented with various selenium compounds for 1 week prior to the oral administration of a single dose of [3H]DMBA (5 mg per rat, specific activity 51.3 mCi/mmol). The rats were sacrificed 24 h later and DNA was isolated from the mammary fat pads. Relative levels of total binding were: [pmol/mg DNA, mean +/- S.D., n=6]; DMBA [7.2 +/- 1.6]; DMBA+p-XSC [3.5 +/- 2.7]; DMBA+p-XSeSG [2.2 +/- 1.1]; DMBA+TSC [5.6 +/- 2.9]. All selenium compounds, except TSC, significantly inhibited DMBA-DNA adduct formation; however, the difference between p-XSC and p-XSeSG was not statistically significant. The inhibition of total binding was attributed to a reduction in the formation of the three major adducts derived from bay-region diol epoxides of DMBA. On the basis of their chromatographic characteristics, these were identified as anti-diol-epoxide:deoxyguanosine, syn-diol-epoxide:deoxyadenosine, and anti-diol-epoxide:deoxyadenosine. Our results suggest that p-XSeSG, but not TSC, is the likely inhibitor of mammary cancer. Selenium levels measured by atomic absorption spectroscopy in the target organ (mammary fat pads) and in plasma following the dietary administration of selenium compounds were in the order of p-XSeSG congruent with p-XSC>TSC. These results appear to be consistent with their order of inhibitory effects on total DMBA-DNA binding. Further in vitro studies of the effect of selenium compounds on cell proliferation suggest that, depending on the dose and time point selected, p-XSC is comparable to or better than p-XSeSG; but both are more effective than TSC. Collectively, our in vivo and in vitro results indicate that p-XSC and its conjugate are better candidates than TSC for future studies on mammary cancer chemoprevention. 相似文献
3.
Native as well as denatured calf thymus DNA, deoxyguanylic and deoxyadenylic acid, respectively, were reacted with the racemic anti 5,9-dimethylchrysene dihydrodiol epoxide (5,9-DMCDE). The deoxyribonucleoside adducts were separated by HPLC and characterized by CD and NMR. Approximately 17% of the epoxide was trapped by native DNA and 76% of the adducts were derived from the RSSR enantiomer. The ratios of dAdo/dGuo modification in DNA were 14/86 and 19/81 for RSSR and SRRS enantiomers, respectively. By monitoring the product yields of anti 5,9-DMCDE with DNA and deoxyribonucleotides, we hoped to gain further insight into the factors responsible for deoxyguanosine adduct formation by 5-methylchrysene dihydrodiol epoxide (5-MCDE) compared to 5, 6-dimethylchrysene dihydrodiol epoxide (5,6-DMCDE). The adduct yields in deoxyribonucleotide reactions of 5,9-DMCDE were slightly higher than those from 5-MCDE. However, the reaction yields of 5, 9-DMCDE with DNA were lower than those with 5-MCDE in most cases, particularly for the cis and trans deoxyadenosine adducts. It seems that the 9-methyl group of 5,9-DMCDE significantly influences adduct formation with the deoxyadenosine residue in DNA in contrast to the 6-methyl group of 5,6-DMCDE. The 9-methyl group sterically decreases deoxyadenosine adduct yields more in reaction with native DNA than denatured DNA, but it has little effect on deoxyribonucleotide reactions. Adduct formation with deoxyguanosine residues in DNA by all three dihydrodiol epoxides correlate with their respective tumorigenic and mutagenic activities. 相似文献
4.
7,12-Dimethylbenz[a]anthracene (DMBA) is a highly potent experimental carcinogen, that must be transformed to its ultimate carcinogenic form in vivo. The meso-region theory of aromatic hydrocarbon carcinogenesis predicts that 7-hydroxymethyl sulfate (7-HMBA) ester plays a major role in the metabolic activation, benzylic DNA adduct formation and complete carcinogenicity of HMBA and DMBA. This study was undertaken to detect highly lipophilic benzylic DNA adducts resulting from the reaction between 7-hydroxymethy sulfate ester of HMBA (7-SMBA) and DNA as well as determine their DNA base selectivity. Synthetic 7-SMBA was incubated with DNA (800 microg/ml) and individual deoxynucleoside 3'-monophosphates (600 microg/ml) and benzylic adducts were analyzed by 32P-postlabeling/TLC following their enrichment with butanol extraction. Dilute ammonium hydroxide-based solvents were developed to detect the highly lipophilic aralkyl adducts. The reaction with DNA, dGp and dAp gave rise to multiple adducts; dCp and dTp showed no significant adducts. Chromatographic comparison revealed that the major DNA adduct was derived from dG. The methodology developed was also found applicable for highly lipophilic adducts resulting from sulfate esters of structurally-related metabolites of DMBA. 相似文献
5.
Driscoll HC Matson SW Sayer JM Kroth H Jerina DM Brosh RM 《The Journal of biological chemistry》2003,278(42):41126-41135
Helicases are among the first enzymes to encounter DNA damage during DNA processing within the cell and thus are likely to be targets for the adverse effects of DNA lesions induced by environmental chemicals. Here we examined the effect of cis- and trans-opened 3,4-diol 1,2-epoxide (DE) DNA adducts of benzo[c]phenanthrene (BcPh) at N6 of adenine on helicase activity. These adducts are derived from the highly tumorigenic (-)-(1R,2S,3S,4R)-DE as well as its less carcinogenic (+)-(1S,2R,3R,4S)-DE enantiomer in both of which the benzylic 4-hydroxyl group and epoxide oxygen are trans. The hydrocarbon portions of these adducts intercalate into DNA on the 3' or the 5' side of the adducted deoxyadenosine for the 1S- and 1R-adducts, respectively. These adducts inhibited the human Werner (WRN) syndrome helicase activity in a strand-specific and stereospecific manner. In the strand along which WRN translocates, cis-opened adducts were significantly more effective inhibitors than trans-opened isomers, indicating that WRN unwinding is sensitive to adduct stereochemistry. WRN helicase activity was also inhibited but to a lesser extent by cis-opened BcPh DE adducts in the displaced strand independent of their direction of intercalation, whereas inhibition by the trans-opened stereoisomers in the displaced strand depended on their orientation, such that only adducts oriented toward the advancing helicase inhibited WRN activity. A BcPh DE adduct positioned in the helicase-translocating strand did not sequester WRN, nor affect the rate of ATP hydrolysis relative to an unadducted control. Although the Bloom (BLM) syndrome helicase was also inhibited by a cis-opened adduct in a strand-specific manner, this helicase was not as severely affected as WRN. Because BcPh DEs form substantial amounts of deoxyadenosine adducts at dA, their adverse effects on helicases could contribute to genetic damage and cell transformation induced by these DEs. Thus, the unwinding activity of RecQ helicases is sensitive to the strand, orientation, and stereochemistry of intercalated polycyclic aromatic hydrocarbon adducts. 相似文献
6.
Benzo[a]pyrene diol epoxide, a metabolite of benzo[a]pyrene (BaP), and chlorohydrin, the reaction product of chloride and the epoxide, form in vitro the same trans- and cis-stereoisomeric DNA adducts, but in different proportions. In this study, we asked whether the DNA adduct concentration can be kept the same by applying the appropriate dose of (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE)and (+/-)-7r,8t,9t-trihydroxy-10c-chloro-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-BPDCH) to rodent skin and whether the DNA adducts formed differ only in their trans- and cis-stereoisomerism. Skin from C57Bl6 mice, spontaneous hypertension rats (SHR) and Sprague-Dawley (SD) rats was treated ex vivo immediately after the death of the animals with anti-BPDE and its corresponding bay region chlorohydrin trans-BPDCH and the epidermis was analyzed for DNA adducts 1h after the application. We found that adduct formation at the exocyclic amino groups of deoxyguanosine and deoxyadenosine in epidermal DNA followed a linear dose-response within 6--100 nmol/cm(2) with both chemicals. In order to achieve the same adduct concentration in mouse, spontaneous hypertension rat (SHR), and Sprague-Dawley (SD) rat skin, respectively, a 37-, 23- and 10-fold lower dose of anti-BPDE than of trans-BPDCH had to be applied. The order of 2'-deoxyguanosine (dGuo) adduct concentration with anti-BPDE was similar to what has been reported, but the order with trans-BPDCH was (+)-cis-BPDE-N(2)-dGuo adduct>(+)-trans-BPDE-N(2)-dGuo=(-)-trans-BPDE-N(2)-dGuo>(-)-cis-BPDE-N(2)-dGuo in mouse skin. Irrespective of species or strain, a significantly higher proportion of cis-adducts was obtained after treatment with trans-BPDCH than after treatment with anti-BPDE. Therefore, DNA adduct concentration can be kept the same by applying the appropriate dose of anti-BPDE and trans-BPDCH to rodent skin and the DNA adducts formed differ only in their trans- and cis-stereoisomerism. 相似文献
7.
1,1,1-Trichloropropene 2,3-oxide (TCPO), a known inhibitor of the enzyme epoxide hydrase, inhibits binding of the carcinogen, 7,12-dimethylbenz(a)anthracene (DMBA), to the DNA of secondary mouse embryo cell cultures under conditions which do not appreciably decrease the overall metabolism of this carcinogen. This suggests that the formation of a transdihydrodiol is a necessary step in the metabolic pathway leading to DNA binding and that binding probably occurs through the generation of a reactive diol-epoxide. In concert with this, the major DMBA-DNA product isolated by chromatography on Sephadex LH-20 eluted with a methanol-water gradient is resolved into two separate components in a methanol-sodium borate solution gradient suggesting that, as is known for benzo(a)pyrene, two stereoisomeric diol-epoxides are involved in the binding of DMBA to DNA. 相似文献
8.
The effects of various factors, including population doubling number, percent of confluence, serum concentration and storage in liquid nitrogen on the binding of several polycyclic aromatic hydrocarbons to human and hamster embryo cells were studied. The binding of 7,12-dimethylbenz[a]-anthracene (DMBA) to hamster embryo cells DNA, RNA and protein was maximal after 22 h of treatment. In contrast, binding to human embryo cell macromolecules increased for at least 55 h. Treatment of hamster embryo cells at 100% confluence resulted in much less binding than treatment at 70% confluence, whereas with human embryo cells the binding increased, or remained constant, following treatment at the greater confluence. The transforming frequency of hamster embryo cells decreases with increasing population doubling number. Accordingly, we found that the binding of DMBA to hamster embryo DNA, RNA and protein decreased approximately 100-fold between population doubling numbers 8 and 20. In transformable cell cultures, DMBA was bound to hamster embryo cell DNA to a greater extent than to RNA or protein. The binding of DMBA to nucleic acids was much greater than binding by either dibenz[a,h]anthracene (DB[a,h]A) or dibenz-[a,c]anthracene (DB[a,c]A), both of which had low binding values at all population doubling numbers tested. Therefore, the best correlation of binding with carcinogenicity and transforming activity was observed with DMBA. Storage of hamster embryo cells in liquid nitrogen did not alter their binding characteristics. Binding of all three hydrocarbons to human embryo cell nucleic acids was low during all population doubling numbers studied, while binding to cellular protein increased until population doubling number 70 and then decreased sharply. 相似文献
9.
The administration of the placental hormone human chorionic gonadotrophin (HCG) to 50-day-old virgin Sprague--Dawley rats has been shown to reduce the incidence of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary cancer. We now report from studies using rat mammary epithelial cells in culture that the anti-carcinogenic effect of HCG may be related to its effect on DNA binding of DMBA and on DNA repair. The results showed that the level of excision repair in cells derived from young virgin (YV) rats grown in the presence of HCG (10 units/ml) was 2.5-4.0 times higher than the level exhibited by control YV cells and 1.5-2.5 times over that obtained for cells from old virgin and parous rats. The effect of HCG on DMBA-DNA binding was also determined in YV cells cultured in the presence of HCG (10 units/ml). Results from this study indicated that DMBA-DNA binding was inhibited by 30-40% in HCG-treated cells as compared to control cells. DNA binding of DMBA was also determined with mammary epithelial cells from YV rats which were given subcutaneous injections of HCG (5 units/rat) 5 times per week for 4 weeks. Using this in vivo-in vitro protocol, DMBA-DNA binding was 17-51% lower in cells from HCG-treated rats than in cells derived from control saline-treated rats. These results suggest that the protective effect provided by HCG against DMBA-induced mammary tumorigenesis may be attributed to its ability to inhibit binding of the carcinogen to mammary cell DNA and to its ability to increase the level of excision repair in the cells. 相似文献
10.
Regine Kahl Barbara Deckers-Schmelzle Erik Klaus 《Biochemical and biophysical research communications》1978,85(3):938-945
The ability of rat liver microsomes to catalyze the formation of benzo(a)pyrene 7,8-diol-9,10-epoxide — DNA nucleoside adduct was increased threefold by feeding 0.5% ethoxyquin to the animals. Microsomal epoxide hydratase activity was enhanced i parallel by a factor of 3 while aryl hydrocarbon hydroxylase activity was not induced. Liver microsomes from rat pretreated with 3-methylcholanthrene produced an increased proportion of diol epoxide — DNA adduct when ethoxyquin had been fed to the animals. The main chromatographic peak formed by microsomes from 3-methylcholanthrene treated rats which contains DNA adducts of secondary benzo(a)pyrene phenol metabolites is reduced when the animals had received ethoxyquin. 相似文献
11.
Ilknur Ozdemir Zeliha Selamoglu Talas Mehmet Gul Burhan Ates Yetkin Gok Mukaddes Esrefoglu Ismet Yilmaz 《Experimental Animals》2006,55(5):449-455
The balance between prooxidants and antioxidants is crucial to the survival and functioning of aerobic organisms. Partially reduced derivatives of oxygen, which are produced in aerobic organisms as part of normal physiological and metabolic processes, are toxic species, oxidizing numerous biomolecules, which initiate tissue injury and cell death. DMBA (7,12-dimethylbenz[a]anthracene) is a polycyclic aromatic hydrocarbon (PAH) known to cause tumors in rats. DMBA is known to generate DNA-reactive species, which may enhance oxidative stress in cells, during its metabolism. Besides the formation of DNA adducts, oxidative products derived from mutagen metabolism, such as DMBA, might impair vital cellular functions by damaging proteins and lipid membranes. Synthetic organoselenium compounds inhibit the initiation phase of carcinogenesis by inhibiting DMBA-DNA adduct formation in the target organ in vivo. Because of the health problems induced by many environmental pollutants, many efforts have been undertaken to evaluate the relative antioxidant potential of selenium and synthetic organoselenium compounds. We undertook the present study to evaluate the chemopreventive potential of the novel synthetic organoselenium compounds (1-isopropyl-3-methylbenzimidazole-2-selenone (SeI) and 1,3-di-p-methoxybenzylpyrimidine-2-selenone (SeII)) in the well-established DMBA-treated rat model by monitoring the extent of lipid peroxidation and mammary duct damage. In this study, adult female Wistar rats were treated with DMBA and the novel organoselenium compounds (SeI and SeII) in determined doses. In DMBA-treated rats, the effects of the organoselenium compounds on malondialdehyde (MDA) levels and histological changes in the rat mammary lactiferous duct were studied. The ability of the organoselenium compounds to prevent oxidative damage induced by DMBA in rat mammary ducts was demonstrated. Protection against lipid peroxidation measured as MDA in the SeI and SeII treated groups was provided by the novel synthesized organoselenium compounds. SeI and SeII both provided chemoprevention against DMBA-induced oxidative stress in the rat mammary duct. 相似文献
12.
The proportions of individual benzo[a]pyrene (BaP)-DNA adducts present in rodent embryo cell cultures change with the length of time of exposure to BaP; the major alteration is an increase in the proportion of (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP (BaPDE)-deoxyguanosine (dG) adduct (Sebti et al., Cancer Res., 45 (1984) 1594-1600). To determine if this change in the BaP-DNA adducts could result from the induction of enzymes involved in oxidation of BaP, hamster embryo cell cultures were exposed to acetone or BaP for 24 h and then the medium was replaced with fresh medium containing [3H]BaP. After 5 h the BaP-pretreated cells had a 30% higher level of binding of BaP to DNA and formed a greater proportion of (+)-anti-BaPE-dG adduct than the acetone-pretreated control group. Cells pretreated for 24 h with BaP and then exposed to [3H]BaP and Actinomycin D for 5 h had a lower level of binding of BaP to DNA and a lower amount of (+)-anti-BaPDE-deoxyguanosine adduct than cells pretreated with acetone and exposed to [3H]BaP for 5 h. In contrast, pretreatment for 24 h with BaP plus Actinomycin D followed by a 5-h exposure to [3H]BaP resulted in a decrease in overall binding of BaP to DNA but had no effect on the amount of (+)-anti-BaPDE-deoxyguanosine adduct. Actinomycin D treatment had no significant effect on either the total amount of BaP metabolized, the formation of primary and water-soluble BaP metabolites, or cell viability, but reduced [3H]uridine incorporation into RNA by more than 65% at all times. These results suggest that induction of specific isozymes of cytochrome P-450 may be involved in the time-dependent increase in the proportion of (+)-anti-BaPDE-DNA adducts in BaP-treated cells. The state of induction of specific isozymes of cytochrome P-450 and the ability of the BaP dose applied to induce them may be major factors in determining the proportion of BaP metabolized to (+)-anti-BaPDE, the most carcinogenic stereoisomer of BaPDE. 相似文献
13.
Typically, chemopreventive agents either inhibit the cytochrome P450s (CYPs) that are essential for the metabolism of carcinogens or induce phase II detoxifying enzymes. This study examined the chemopreventive effect of eugenol on 7,12-dimethylbenz[a]anthracene (DMBA)-induced DNA damage in MCF-7 cells. Eugenol inhibited the formation of the DMBA-DNA adduct in a dose dependent manner. CYP1A1 and CYP1B1 activity, which catalyze the biotransformation of DMBA, were strongly inhibited by eugenol. Eugenol also suppressed the CYP1A induction by DMBA through decreased aryl hydrocarbon receptor activation and subsequent DNA binding. Furthermore, eugenol increased the expression and activity of NAD(P)H:quinone oxidoreductase (QR), a major detoxifying enzyme for DMBA, through NF-E2 related factor2 binding to antioxidant response element in QR gene. Therefore, eugenol has a potent protective effect against DMBA-induced genotoxicity, presumably through the suppression of the DMBA activation and the induction of its detoxification. These results suggest that eugenol has potential as a chemopreventive. 相似文献
14.
Four adducts that would result from trans opening at C-1 of benzo[c]phenanthrene 3,4-diol 1,2-epoxide (B[c]PhDE) isomers (i.e., DE-1 enantiomers, where the epoxide oxygen and benzylic hydroxyl group are cis, and DE-2 enantiomers, where they are trans) by the N(6)-amino group of dAdo, together with the two cis opened N(6)-dAdo adducts of B[c]PhDE-1, were incorporated into two oligonucleotides at the underlined site in 5'-TTTAGAGTCTGCTCCC [context I(A)] and 5'-CAGATTTAGAGTCTGC [context II(A)]. After ligation of these, and the corresponding unsubstituted oligonucleotides, into single-stranded M13mp7L2 bacteriophage and transfection into SOS-induced Escherichia coli SMH77, base substitution mutations induced by the different B[c]PhDE-dAdo adducts were determined. These findings were compared with data [Pontén et al. (1999) Biochemistry 38, 1144-1152] for cis opened B[c]PhDE-2-dAdo adducts in the same sequence contexts. In most cases, adducts with S absolute configuration at the site of attachment of the nucleoside to the hydrocarbon had higher mutation frequencies (1.9-56.5%) than the corresponding adducts with R configuration (0.05-5.6%). For adducts derived from B[c]PhDE-1, the predominant mutations were A-->T transversions in context I(A) and A-->G transitions for most of these adducts in context II(A). For adducts derived from B[c]PhDE-2, A-->T base substitutions predominated for most of the trans adducts, but A-->G mutations were favored by the cis adduct with S configuration in either context. Thus, the structural feature that most dramatically affected mutagenic activity was the configuration of the carbon at the attachment point, with S configuration mostly being associated with greater mutagenicity than the R configuration. However, other structural variations and sequence context also affected mutagenicity, indicating that a combination of structure and context effects define mutagenicity. 相似文献
15.
P Simon B Epe P Mützel D Schiffmann D Wild H Ottenw?lder N Fedtke H M Bolt D Henschler 《Journal of biochemical toxicology》1986,1(2):43-55
Acetoxyoxirane, the epoxide of vinyl acetate and a potential reactive intermediate, was synthesized and characterized by 13C-nuclear magnetic resonance (13C-NMR) and mass spectroscopy. The compound induced lesions (endonuclease-sensitive and alkali-labile sites) in supercoiled PM2 DNA in vitro and was directly mutagenic toward Salmonella typhimurium TA100. The mutagenicity of the epoxide in phosphate buffer (pH 7.4, 37 degrees C) decreased, with an initial half-life of 2.8 minutes, and mutagenicity was completely abolished by addition of S-9 mix. Acetoxyoxirane did not induce unscheduled DNA synthesis on incubation with Syrian hamster embryo fibroblasts (SHE cells). These findings may possibly be explained by an effective inactivation of acetoxyoxirane by esterases when these are present in the biological system. This view is consistent with the lack of acetoxyoxirane detected in rat liver microsomal incubations of vinyl acetate. 相似文献
16.
The polycyclic aromatic hydrocarbon (PAH) benzo[ghi]perylene (BghiP) lacks a "classic" bay-region and is therefore unable to form vicinal dihydrodiol epoxides thought to be responsible for the genotoxicity of carcinogenic PAHs like benzo[a]pyrene. The bacterial mutagenicity of BghiP increases considerably after inhibition of the microsomal epoxide hydrolase (mEH) indicating arene oxides as genotoxic metabolites. Two K-region epoxides of BghiP, 3,4-epoxy-3,4-dihydro-BghiP (3,4-oxide) and 3,4,11,12-bisepoxy-3,4,11,12-tetrahydro-BghiP (3,4,11,12-bisoxide) identified in microsomal incubations of BghiP are weak bacterial mutagens in strain TA98 of Salmonella typhimurium with 5.5 and 1.5 his+-revertant colonies/nmol, respectively. After microsomal activation of BghiP in the presence of calf thymus DNA three DNA adducts were detected using 32P-postlabeling. The total DNA binding of 2.1 fmol/microg DNA, representing 7 adducts in 10(7) nucleotides, was raised 3.6-fold when mEH was inhibited indicating arene oxides as DNA binding metabolites. Co-chromatography revealed the identity between the main adduct of metabolically activated BghiP and the main adduct of the 3,4-oxide. DNA adducts of BghiP originating from the 3,4,11,12-bisoxide were not found. Therefore, a K-region epoxide is proposed to be responsible for the genotoxicity of BghiP and possibly of other PAHs without a "classic" bay-region. 相似文献
17.
Choudhary S Doherty KM Handy CJ Sayer JM Yagi H Jerina DM Brosh RM 《The Journal of biological chemistry》2006,281(9):6000-6009
RecQ helicases are believed to function in repairing replication forks stalled by DNA damage and may also play a role in the intra-S-phase checkpoint, which delays the replication of damaged DNA, thus permitting repair to occur. Since little is known regarding the effects of DNA damage on RecQ helicases, and because the replication and recombination defects in Werner syndrome cells may reflect abnormal processing of damaged DNA associated with the replication fork, we examined the effects of specific bulky, covalent adducts at N(6) of deoxyadenosine (dA) or N(2) of deoxyguanosine (dG) on Werner (WRN) syndrome helicase activity. The adducts are derived from the optically active 7,8-diol 9,10-epoxide (DE) metabolites of the carcinogen benzo[a]pyrene (BaP). The results demonstrate that WRN helicase activity is inhibited in a strand-specific manner by BaP DE-dG adducts only when on the translocating strand. These adducts either occupy the minor groove without significant perturbation of DNA structure (trans adducts) or cause base displacement at the adduct site (cis adducts). In contrast, helicase activity is only mildly affected by intercalating BaP DE-dA adducts that locally perturb DNA double helical structure. This differs from our previous observation that intercalating dA adducts derived from benzo[c]phenanthrene (BcPh) DEs inhibit WRN activity in a strand- and stereospecific manner. Partial unwinding of the DNA helix at BaP DE-dA adduct sites may make such adducted DNAs more susceptible to the action of helicase than DNA containing the corresponding BcPh DE-dA adducts, which cause little or no destabilization of duplex DNA. The single-stranded DNA binding protein RPA, an auxiliary factor for WRN helicase, enabled the DNA unwinding enzyme to overcome inhibition by either the trans-R or cis-R BaP DE-dG adduct, suggesting that WRN and RPA may function together to unwind duplex DNA harboring specific covalent adducts that otherwise block WRN helicase acting alone. 相似文献
18.
M Shoyab 《Chemico-biological interactions》1979,25(1):71-85
A rapid procedure to study the interaction of carcinogens with DNA in cultured cells has been developed. The cells, which are labeled with 7,12-[3H]dimethylbenz[a] anthracene ([3H]DMBA), are lysed with 0.24 M phosphate buffer (pH 6.8), 1% sodium dodecyl sulfate (SDS), 8 M urea and 0.01 M ethylenediamine-tetraacetate (EDTA) and sonicated. The cell lysates are fractionated on columns of hydroxylapatite. Proteins and RNA are removed with 8 M urea in 0.24 M phosphate buffer (pH 6.8). DMBA-bound DNA is eluted with 0.4 M phosphate buffer (pH 6.8). DMBA-DNA isolated by this procedure is virtually free from proteins and RNA. Thermal stability, ultraviolet spectra and the density of DNA is not altered by DMBA binding. The uptake of DMBA by mouse epidermal cells is rapid and the binding of DMBA to DNA is linear for the first 8 h of exposure. DMBA binds to DNA in all phases of the cell cycle. However, the highest binding occurs immediately following maximum DNA synthesis. 相似文献
19.
Neoplastic transformation of cultured Syrian hamster embryo cells by DNA of herpes simplex virus type 2.
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Syrian hamster embryo cells were transformed to a neoplastic phenotype after exposure to herpes simplex virus type 2 (S-1) DNA at concentrations (less than or equal to 0.01 microgram per 60-mm dish) at which infectivity was no longer demonstrable. Transformed cells manifested in vitro phenotypic properties characteristic of the neoplastic state, expressed herpes simplex virus-specific antigens, and induced invasive tumors in vivo. Transfection and transformation of Syrian hamster embryo cells with herpes simplex virus type 2 DNA or its fragments is a suitable system for investigating the structure and function of herpes simplex virus-transforming gene(s). 相似文献
20.
Ribosomal protein differences between animal cells 总被引:1,自引:0,他引:1
Ribosomal proteins of human (HeLa), Syrian hamster, Chinese hamster, chick (embryo) and rat (Novikoff hepatoma) cells have been examined by two-dimensional polyacrylamide gel electrophoresis. The results show that although there are many similarities between the electrophoretic patterns, species-specific marker proteins can be identified for Syrian hamster, chick, rat and possibly HeLa cells, which could be used in genetic analysis. No specific protein marker has been identified for Chinese hamster. The similarity in electrophoretic mobility of the hamster, chick and rat marker proteins suggests an overall structural relationship between them. 相似文献