Formation Of carcinogen-DNA Adducts Research Articles

Noni enhances the anticancer activity of cyclophosphamide and suppresses myelotoxicity and hepatotoxicity in tumor-bearing mice

Background and aimMorinda citrifolia fruit juice (noni) is an herbal remedy documented to have antioxidant properties. It has been suggested that prevention of carcinogen-DNA adduct formation and the antioxidant activity of NJ may contribute to the cancer preventive effect. In the present study, the antitumor activity of noni was investigated in the presence of cyclophosphamide (CYL) in vitro and in vivo.MethodsIn vitro breast cancer cells (MDA-MB-468) were used to measure the percentage of inhibition and the IC50. The in vivo antitumor activity of noni was studied by monitoring the mean survival time (MST), percentage increase in life span (%ILS), viable and non-viable cell count, tumor volume, body weight, and hematological and serum biochemical parameters in mice. Treatment with noni and CYL exhibited dose- and time-dependent cytotoxicity toward breast cancer cells.ResultsIndividual treatment of noni and CYL exhibited dose- and time-dependent cytotoxicity on breast cancer cell lines, while in combination therapy of noni and CYL, noni enhances cytotoxic effect of CYL at 48 h than that at 24 h. Similar result was found in in vivo studies, the results of which revealed that alone treatment of CYL and noni suppressed tumor growth. However, combination treatment with CYL and noni presented better tumor inhibition than that of alone treatment of CYL and noni. On the contrary, CYL alone drastically attenuated hematological parameters, i.e., RBC, WBC, and Hb compared to normal and control groups, and this change was reversed and normalized by noni when given as combination therapy with CYL. Moreover, the levels of serum biochemical markers, i.e., AST, ALP, and ALT, were significantly increased in the control and CYL-treated groups than those in the normal group. In the combination treatment of noni and CYL, the above biochemical marker levels significantly decreased compared to CYL alone-treated group.ConclusionsThe present study suggested that CYL treatment can cause serious myelotoxicity and hepatic injury in cancer patients. In conclusion, the combined use of noni with CYL potentially enhances the antitumor activity of CYL and suppresses myelotoxicity and hepatotoxicity induced by CYL in tumor-bearing mice.

Exposure to ambient particulate matter induces oxidative stress in lung and aorta in a size- and time-dependent manner in rats

Exposure to particulate matter (PM) has been implicated in oxidative stress (OxS) and inflammation as underlying mechanisms of lung damage and cardiovascular alterations. PM is a chemical mixture that can be subdivided according to their aerodynamic size into coarse (CP), fine (FP), and ultrafine (UFP) particulates. We investigated, in a rat model, the induction of OxS (protein oxidation and antioxidant response), carcinogen-DNA adduct formation, and inflammatory mediators in lung in response to different airborne particulate fractions, CP, FP, and UFP, after an acute and subchronic exposure. In addition, OxS was evaluated in the aorta to assess the effects beyond the lungs. Exposure to CP, FP, and UFP induced time- and size-dependent lung protein oxidation and DNA adduct formation. After acute and subchronic exposure, nuclear factor erythroid-2 (Nrf2) activation was observed in the lung, by electrophoretic mobility shift assay, and the induction of mRNA antioxidant enzymes in the FP and UFP groups, but not in the CP. Cytokine concentration of interleukin 1β, interleukin 6, and macrophage inflammatory protein-2 was significantly increased in bronchoalveolar lavage fluid after acute exposure to FP and UFP. Activation of Nrf2 and expression of mRNA antioxidant enzymes were observed only after the subchronic exposure to FP and UFP in the aorta. Our results indicate that FP and UFP were mainly accountable for the oxidant toxic effects in the lung; OxS is spread from the lung to the cardiovascular system. We conclude that the biological mechanisms associated with transient OxS and inflammation are particle size and time-dependent exposure resulting in acute lung injury, which later reaches the vascular system.

Activation of uPA-uPAR signaling pathway by human CYP1B1 promotes cancer progression and metastasis in human breast cancer cells

Flavonoids are phenolic compounds isolated from plants, and several of them like genistein and quercetin, have been documented to be effective in preventing cancer. Baicalein, a flavonoid extracted from the root of Scutellaria species, is widely used as a health supplement and herbal medicine in Asian countries. In this study, the chemopreventive effect of baicalein on 7,12-dimethylbenz〚a〛anthracene (DMBA)-induced DNA damage was evaluated in an established cell culture model. In a preliminary screening, baicalein was identified to be a strong inhibitor to EROD activities induced by DMBA in MCF-7 cells. Subsequent enzyme kinetic analysis revealed that baicalein was a competitive inhibitor to EROD, and CYP1A1 and CYP1B1 gene expressions were also determined. Baicalein could reduce the CYP1A1/1B1 mRNA expressions induced by DMBA, and the mRNA abundance of CYP1A1 appeared to be more responsive than that of CYP1B1. A XRE–luciferase gene reporter assay indicated that AhR transactivation was suppressed. Since CYP1A1/1B1 were responsible for the biotransformation of polycyclic aromatic hydrocarbons, baicalein also demonstrated its ability to reduce DMBA–DNA adduct formation in MCF-7 cells. This study suggested that the natural occurring baicalein could be an agent preventing carcinogen–DNA adduct formation.

Anticancer potential of emodin.

Traditional Chinese Medicine (TCM) is widely used in clinical research due to its low toxicity, low number of side effects, and low cost. Many components of common fruits and vegetables play well-documented roles as chemopreventive or chemotherapeutic agents that suppress tumorigenesis. Anthraquinones are commonly extracted from the Polygonaceae family of plants, e.g., Rheum palmatum and Rheum officinale. Some of the major chemical components of anthraquinone and its derivatives, such as aloe-emodin, danthron, emodin, chrysophanol, physcion, and rhein, have demonstrated potential anticancer properties. This review evaluates the pharmacological effects of emodin, a major component of Aloe vera. In particular, emodin demonstrates anti-neoplastic, anti-inflammatory, anti-angiogenesis, and toxicological potential for use in pharmacology, both in vitro and in vivo. Emodin demonstrates cytotoxic effects (e.g., cell death) through the arrest of the cell cycle and the induction of apoptosis in cancer cells. The overall molecular mechanisms of emodin include cell cycle arrest, apoptosis, and the promotion of the expression of hypoxia-inducible factor 1α, glutathione S-transferase P, N-acetyltransferase, and glutathione phase I and II detoxification enzymes while inhibiting angiogenesis, invasion, migration, chemical-induced carcinogen-DNA adduct formation, HER2/neu, CKII kinase, and p34cdc2 kinase in human cancer cells. Hopefully, this summary will provide information regarding the actions of emodin in cancer cells and broaden the application potential of chemotherapy to additional cancer patients in the future.

Interactions of chemical carcinogens and genetic variation in hepatocellular carcinoma.

In the etiology of hepatocellular carcinoma (HCC), in addition to hepatitis B virus and hepatitis C virus infections, chemical carcinogens also play important roles. For example, aflatoxin B(1) (AFB(1)) epoxide reacts with guanine in DNA and can lead to genetic changes. In HCC, the tumor suppressor gene p53 codon 249 mutation is associated with AFB(1) exposure and mutations in the K-ras oncogene are related to vinyl chloride exposure. Numerous genetic alterations accumulate during the process of hepatocarcinogenesis. Chemical carcinogen DNA-adduct formation is the basis for these genetic changes and also a molecular marker which reflects exposure level and biological effects. Metabolism of chemical carcinogens, including their activation and detoxification, also plays a key role in chemical hepatocarcinogenesis. Cytochrome p450 enzymes, N-acetyltransferases and glutathione S-transferases are involved in activating and detoxifying chemical carcinogens. These enzymes are polymorphic and genetic variation influences biological response to chemical carcinogens. This genetic variation has been postulated to influence the variability in risk for HCC observed both within and across populations. Ongoing studies seek to fully understand the mechanisms by which genetic variation in response to chemical carcinogens impacts on HCC risk.

Endogenous cytosine methylation and the formation of carcinogen carcinogen-DNA adducts

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine ((Me)C, X = Me in Scheme 1). The same sites (e.g. p53 codons 157, 158, 245, 248, and 273) are mutational hotspots in smoking induced lung cancer, suggesting that methylated CG dinucleotides may be preferentially targeted by the reactive metabolites of tobacco carcinogens. We employed a stable isotope labeling HPLC-ESI-MS/MS approach to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diolepoxide metabolites of bay region polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene diol epoxide (BPDE). In contrast, cytosine methylation was protective against O(6)-guanine alkylation by tobacco tobacco-specific nitrosamines, e.g. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), To investigate the mechanisms behind these effects, a series of structural analogs of (Me)C were prepared, and their effects on reactivity of base the paired dG towards BPDE was examined. We found that the presence of the C-5 substituent on cytosine influences the reactivity of its partner guanine towards BPDE and modifies the stereoisomeric composition of the resulting N(2)-BPDE-dG adducts.

Sequence Distribution of Acetaldehyde-Derived N2-Ethyl-dG Adducts along Duplex DNA

Acetaldehyde (AA) is the major metabolite of ethanol and may be responsible for an increased gastrointestinal cancer risk associated with alcohol beverage consumption. Furthermore, AA is one of the most abundant carcinogens in tobacco smoke and induces tumors of the respiratory tract in laboratory animals. AA binding to DNA induces Schiff base adducts at the exocyclic amino group of dG, N2-ethylidene-dG, which are reversible on the nucleoside level but can be stabilized by reduction to N2-ethyl-dG. Mutagenesis studies in the HPRT reporter gene and in the p53 tumor suppressor gene have revealed the ability of AA to induce G-->A transitions and A-->T transversions, as well as frameshift and splice mutations. AA-induced point mutations are most prominent at 5'-AGG-3' trinucleotides, possibly a result of sequence specific adduct formation, mispairing, and/or repair. However, DNA sequence preferences for the formation of acetaldehyde adducts have not been previously examined. In the present work, we employed a stable isotope labeling-HPLC-ESI+-MS/MS approach developed in our laboratory to analyze the distribution of acetaldehyde-derived N2-ethyl-dG adducts along double-stranded oligodeoxynucleotides representing two prominent lung cancer mutational "hotspots" and their surrounding DNA sequences. 1,7,NH 2-(15)N-2-(13)C-dG was placed at defined positions within DNA duplexes derived from the K-ras protooncogene and the p53 tumor suppressor gene, followed by AA treatment and NaBH 3CN reduction to convert N2-ethylidene-dG to N2-ethyl-dG. Capillary HPLC-ESI+-MS/MS was used to quantify N2-ethyl-dG adducts originating from the isotopically labeled and unlabeled guanine nucleobases and to map adduct formation along DNA duplexes. We found that the formation of N2-ethyl-dG adducts was only weakly affected by the local sequence context and was slightly increased in the presence of 5-methylcytosine within CG dinucleotides. These results are in contrast with sequence-selective formation of other tobacco carcinogen-DNA adducts along K-ras- and p53-derived duplexes and the preferential modification of endogenously methylated CG dinucleotides by benzo[a]pyrene diol epoxide and acrolein.

Leukemogenic AML1-ETO fusion protein increases carcinogen-DNA adduct formation with upregulated expression of cytochrome P450-1A1 gene

AML1-ETO fusion protein is a product of chromosome translocation t(8;21) frequently occurred in acute myeloid leukemia (AML), but its sole expression appears to fail to cause overt leukemia in vivo. In this study, we investigated whether AML1-ETO expression impinged on action of chemical carcinogens-DNA adduct formation. AML1-ETO fusion protein was conditionally induced in engineered U937-A/E 9/14/18 cells. The formation of polycyclic aromatic hydrocarbon (PAH)-DNA adducts and the expression of PAH-metabolizing enzymes cytochrome P450 (CYP) 1A1 and arylhydrocarbon receptor (AhR) were detected by Western blot and/or quantitative RT-PCR. Luciferase reporter system was used to detect the regulation of AML1-ETO on CYP1A1 transcription. Our results showed that AML1-ETO induction significantly increased the formation of carcinogen benzopyrene-DNA adducts in leukemic cells. In line with the effect, we also found that AML1-ETO induction upregulated CYP1A1 expression, which was dependent on AML1-binding motif in the promotor of CYP1A1 gene. Additionally, AML1-ETO protein also increased AhR expression, a ligand-activated transcription factor that mediates PAHs-induced CYP1A1 gene expression. These data, combined with its inhibitory effect on DNA repair as reported previously, propose that the presence of AML1-ETO increases the susceptibility of cells to chemical carcinogens, which favors the development of additional genetic alterations.

Anthocyanin-Rich Grape Extract Blocks Breast Cell DNA Damage

Anthocyanins, belonging to the flavonoid family of phytochemicals, have received attention as agents that may have potential in preventing chronic diseases such as cardiovascular diseases and certain cancers. In the present study, an anthocyanin-rich extract from Concord grapes [referred to as Concord grape extract (CGE)] and the anthocyanin delphinidin were evaluated for their capacity to inhibit DNA adduct formation due to the environmental carcinogen benzo[a]pyrene (BP) in MCF-10F cells, a noncancerous, immortalized human breast epithelial cell line. CGE at 10 and 20 microg/mL and delphinidin at 0.6 microM concentrations significantly inhibited BP-DNA adduct formation. This was associated with a significant increase in activities of the phase II detoxification enzymes glutathione S-transferase and NAD(P)H:quinone reductase 1. In addition, these grape components also suppressed reactive oxygen species (ROS) formation, but did not induce antioxidant response element-dependent transcription. Taken together, these data suggest that CGE and a component grape anthocyanin have breast cancer chemopreventive potential due in part to their capacity to block carcinogen-DNA adduct formation, modulate activities of carcinogen-metabolizing enzymes, and suppress ROS in these noncancerous human breast cells.

Oltipraz chemoprevention trial in Qidong, People's Republic of China: Unaltered oxidative biomarkers

Aflatoxin, which leads to formation of carcinogen-DNA adducts as well as oxidized DNA, is a well-known risk factor for development of hepatocellular carcinoma. The aim of the present study was to investigate if the chemopreventive agent oltipraz had an effect on DNA oxidation measured as oxidized guanine derivatives in urine among healthy individuals living in a region of China at high risk of exposure to aflatoxin and development of hepatocellular carcinoma. Two hundred thirty-three healthy residents of Qidong, PRC, were randomized to 8 weeks treatment with placebo, oltipraz 125 mg daily, or oltipraz 500 mg weekly, with a subsequent 8-week follow-up period. Urine samples were collected as overnight voids. Samples collected 4 weeks into the treatment period and 6 weeks into the follow-up period were analyzed for oxidized guanine derivatives with a HPLC-MS/MS method. A repeated-measures analysis of variance showed no significant differences between the randomization groups regarding changes in oxidized guanine derivatives. In the present double-blind, randomized, placebo-controlled trial performed among healthy individuals, oltipraz had no major effect on oxidative DNA damage. Mechanisms other than prevention of oxidative DNA damage may be of higher importance when oltipraz is used as a chemopreventive agent in humans.

Induction of CYP1A1 and CYP1B1 and formation of carcinogen–DNA adducts in normal human mammary epithelial cells treated with benzo[a]pyrene

Inter-individual variation in formation of carcinogen–DNA adducts and induction of cytochrome P450 genes was measured in 23 cultured normal human mammary epithelial cell (NHMEC) strains established from reduction mammoplasty tissue. Semi-confluent cells were exposed to 4 μM benzo[a]pyrene (BP) for 12 h and BP–DNA adduct levels were measured by chemiluminescence immunoassay using antiserum elicited against DNA modified with r7, t8-dihydroxy-t-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). BP–DNA adduct levels for 22 of 23 different cell strains ranged from non-detectable (three samples) to about 15 adducts/10 8 nucleotides. Increases in levels of CYP1A1 and CYP1B1 were detected using both oligonucleotide arrays and reverse transcription/quantitative real-time polymerase chain reactions (RT-PCRs). For CYP1A1 and CYP1B1, the oligonucleotide array data and RT-PCR data were highly correlated ( r=0.73 and 0.70, respectively), suggesting that oligonucleotide arrays are a suitable gene discovery tool, and demonstrating that the complementary and efficient RT-PCR may be used to confirm microarray data for a specific gene in a large number of samples. As measured by RT-PCR, inter-individual variation in CYP1A1 induction was 100-fold, while the variation in CYP1B1 induction was almost 40-fold. On a per-person basis, CYP1A1 and CYP1B1 induction were well-correlated ( r=0.88, P<0.001), which is to be expected as they are under the control of a common transcriptional regulation mechanism in response to BP exposure. Inter-individual variation in carcinogen–DNA adduct formation could not be explained only by variation in levels of CYP1A1 or CYP1B1 induction, as neither was well-correlated with BPDE–DNA adduct level ( r=0.40 and 0.50 for CYP1A1 and CYP1B1, respectively). Evaluation of glutathione- S-transferase M1 genotype ( GSTM1 positive or null) revealed an apparent correlation between positive GSTM1 genotype and BPDE–DNA adduct levels ( r=0.84 and 0.77 for CYP1A1 and CYP1B1, respectively); however, after removal of the single outlier this relationship was not significant. Overall the data suggest that BPDE–DNA adduct levels in normal human breast tissue may be modulated by multiple factors that include, but are not exclusive to, CYP1A1 and CYP1B1 inducibility and the presence or absence of GSTM1.

P5 The analysis of genetic polymorphisms in CYP1B1 and COMT genes in breast and endometrial cancer patients

Inter-individual variation in formation of carcinogen–DNA adducts and induction of cytochrome P450 genes was measured in 23 cultured normal human mammary epithelial cell (NHMEC) strains established from reduction mammoplasty tissue. Semi-confluent cells were exposed to 4 μM benzo[a]pyrene (BP) for 12 h and BP–DNA adduct levels were measured by chemiluminescence immunoassay using antiserum elicited against DNA modified with r7, t8-dihydroxy-t-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). BP–DNA adduct levels for 22 of 23 different cell strains ranged from non-detectable (three samples) to about 15 adducts/108 nucleotides. Increases in levels of CYP1A1 and CYP1B1 were detected using both oligonucleotide arrays and reverse transcription/quantitative real-time polymerase chain reactions (RT-PCRs). For CYP1A1 and CYP1B1, the oligonucleotide array data and RT-PCR data were highly correlated (r=0.73 and 0.70, respectively), suggesting that oligonucleotide arrays are a suitable gene discovery tool, and demonstrating that the complementary and efficient RT-PCR may be used to confirm microarray data for a specific gene in a large number of samples. As measured by RT-PCR, inter-individual variation in CYP1A1 induction was 100-fold, while the variation in CYP1B1 induction was almost 40-fold. On a per-person basis, CYP1A1 and CYP1B1 induction were well-correlated (r=0.88, P<0.001), which is to be expected as they are under the control of a common transcriptional regulation mechanism in response to BP exposure. Inter-individual variation in carcinogen–DNA adduct formation could not be explained only by variation in levels of CYP1A1 or CYP1B1 induction, as neither was well-correlated with BPDE–DNA adduct level (r=0.40 and 0.50 for CYP1A1 and CYP1B1, respectively). Evaluation of glutathione-S-transferase M1 genotype (GSTM1 positive or null) revealed an apparent correlation between positive GSTM1 genotype and BPDE–DNA adduct levels (r=0.84 and 0.77 for CYP1A1 and CYP1B1, respectively); however, after removal of the single outlier this relationship was not significant. Overall the data suggest that BPDE–DNA adduct levels in normal human breast tissue may be modulated by multiple factors that include, but are not exclusive to, CYP1A1 and CYP1B1 inducibility and the presence or absence of GSTM1.

Preferential carcinogen-DNA adduct formation at codons 12 and 14 in the human K-ras gene and their possible mechanisms.

In the ras gene superfamily, codon 12 (-TGGTG-) of the K-ras gene is the most frequently mutated codon in human cancers. Recently, we have found that bulky chemical carcinogens preferentially form DNA adducts at codons 12 and 14 (-CGTAG-) in the K-ras gene in normal human bronchial epithelial (NHBE) cells. Furthermore, DNA adducts formed at codon 12 of the K-ras gene are poorly repaired compared with those at other codons including codon 14. These results suggest that targeted carcinogen-DNA adduct formation is a major reason for the observed high mutation frequency at codon 12 of the K-ras gene in human cancers. This preferential carcinogen-DNA adduct formation at codons 12 and 14 could result from effects of (1) primary sequences of these codons and their surrounding codons in the K-ras gene, (2) the chromatin structure, and/or (3) epigenetic factors such as C5 cytosine methylation or other DNA modifications at these codons and their surrounding codons. To distinguish these possibilities, we have introduced modifications with benzo[a]pyrene diol epoxide, N-hydroxy-2-aminofluorene, and aflatoxin B1 8,9-epoxide in (1) naked intact genomic DNA isolated from NHBE cells, (2) fragmented genomic DNA digested by restriction enzymes, and (3) in vitro synthesized DNA fragments containing the K-ras gene exon 1 sequence with or without methylation of the cytosines at CpG sites and the cytosines pairing with the guanines of codons 12 and 14. The distribution of carcinogen-DNA adducts in the K-ras gene was mapped at the nucleotide sequence level using the UvrABC nuclease incision method with or without the ligation-mediated polymerase chain reaction technique. We have found that carcinogens preferentially form adducts at codons 12 and 14 in the K-ras gene exon 1 in intact as well as in fragmented genomic DNA. In contrast, this preferential DNA adduct formation at codons 12 and 14 was not observed in PCR-amplified DNA fragments containing the K-ras gene exon 1 sequence. Methylation of the cytosine at the CpG site of codon 14, or the cytosine pairing with guanine of codon 14, greatly enhanced carcinogen-DNA adduct formation at codon 14 but did not affect carcinogen-DNA adduct formation at codon 12. Methylation of the cytosine pairing with the guanine of codon 12 also did not enhance carcinogen-DNA adduct formation at codon 12. Furthermore, we found that the cytosine at the CpG site of codon 14 is highly methylated in NHBE cells. These results suggest that cytosine methylation at the CpG site is the major reason for the preferential DNA damage at codon 14 and that epigenetic modification(s) other than cytosine methylation may contribute to the preferential DNA damage at codon 12 of the K-ras gene.

A cross-sectional study of polycyclic aromatic hydrocarbon-DNA adducts and polymorphism of glutathione S-transferases among heavy smokers by race/ethnicity

Differences in lung cancer risk by race/ethnicity have been observed among smokers. To determine whether these observations might reflect differences in the formation of carcinogen-DNA adducts, we analysed blood specimens (n =151) collected from smokers who were recruited for possible participation in an antioxidant vitamin intervention study. Mononuclear cells were analysed for polycyclic aromatic hydrocarbon (PAH)-DNA adducts by competitive enzyme-linked immunosorbent assay. Genotypes of glutathione S-transferase M1 and P1 (GSTM1 and GSTP1), enzymes involved in the detoxification of PAH metabolites, were determined by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism, respectively. GSTM1 was present in 65 out of 88 (73.4%), 16 out of 32 (50.0%) and 16 out of 29 (54.8%) of African-Americans, Caucasians and Latinos, respectively (p =0.022). Homozygosity for the GSTP1 codon 105 variant was found in 25.6%, 6.3% and 10.0% of African-Americans, Caucasians and Latinos, respectively (p =0.023). Regression analysis of the log-transformed adduct levels confirmed that Caucasian and Latino subjects had lower PAH-DNA adduct levels than African-American subjects, after adjustment for gender, education,α -tocopherol and β-carotene levels, and GSTM1 status. Further adjustment for age and current smoking habits had no impact on these findings. Although crude analysis suggested that the GSTM1-positive genotype may be associated with lower PAH-DNA levels in Caucasians (but not in African-Americans or Latinos), a formal test for interaction between GSTM1 and ethnicity was not significant. We found no association between adduct levels and GSTP1 genotype. Although the mechanism is unclear, ethnic differences in DNA damage levels may in part explain why African-Americans have higher lung cancer incidence rates than other ethnic groups.

Preferential DNA damage and poor repair determine ras gene mutational hotspot in human cancer.

Mutations in ras genes are commonly found in human cancers and in animal models. Although mutations at codons 12, 13, and 61 of H-, N- and K-ras genes can activate their oncogenic function, mutations at codon 12 of K-ras are the most common mutations found among the three ras genes in human cancers. To investigate whether codon 12 of human K-ras is especially susceptible to carcinogens and/or whether carcinogen-DNA adducts at this codon are repaired less efficiently, we examined tobacco smoke carcinogen-induced DNA damage in normal human bronchial epithelial and fibroblast cells. We used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction to map the distribution of DNA adducts induced by benzo[a]pyrene diol epoxide (BPDE) and other bulky carcinogens within exons 1 and 2 in H-ras, N-ras, and K-ras. We also analyzed BPDE-DNA adduct repair efficiency in these three genes using the same method. Codons 12 and 14 of the K-ras gene were hotspots for carcinogen-DNA adduct formation, with little and no adduct formation at codons 13 and 61, respectively. The BPDE-DNA adducts formed at codon 14 were repaired almost twice as quickly as those formed at codon 12. There was some BPDE-DNA adduct formation at codons 12 of H-ras and N-ras, but this codon was not a hotspot. Furthermore, no substantial difference in repair rates between codon 12 and the other codons analyzed (codons 3 and 18) was observed in either the H-ras or N-ras genes. These findings link the human cancer mutational hotspot at codon 12 of K-ras to preferential DNA damage and poor repair.

Baicalein inhibits DMBA-DNA adduct formation by modulating CYP1A1 and CYP1B1 activities.

Flavonoids are phenolic compounds isolated from plants, and several of them like genistein and quercetin, have been documented to be effective in preventing cancer. Baicalein, a flavonoid extracted from the root of Scutellaria species, is widely used as a health supplement and herbal medicine in Asian countries. In this study, the chemopreventive effect of baicalein on 7,12-dimethylbenz[a]anthracene (DMBA)-induced DNA damage was evaluated in an established cell culture model. In a preliminary screening, baicalein was identified to be a strong inhibitor to EROD activities induced by DMBA in MCF-7 cells. Subsequent enzyme kinetic analysis revealed that baicalein was a competitive inhibitor to EROD, and CYP1A1 and CYP1B1 gene expressions were also determined. Baicalein could reduce the CYP1A1/1B1 mRNA expressions induced by DMBA, and the mRNA abundance of CYP1A1 appeared to be more responsive than that of CYP1B1. A XRE-luciferase gene reporter assay indicated that AhR transactivation was suppressed. Since CYP1A1/1B1 were responsible for the biotransformation of polycyclic aromatic hydrocarbons, baicalein also demonstrated its ability to reduce DMBA-DNA adduct formation in MCF-7 cells. This study suggested that the natural occurring baicalein could be an agent preventing carcinogen-DNA adduct formation.

The effect of paclitaxel on 2-aminofluorene–DNA adducts formation and arylamine N-acetyltransferase activity and gene expression in human lung tumor cells (A549)

In this study, paclitaxel was used to determine inhibition of arylamine N-acetyltransferase (NAT) activity, gene expresssion and 2-aminofluorene–DNA adduct formation in a human lung tumor cell line (A549). The activity of NAT was measured by HPLC assaying for the amounts of N-acetyl-2-aminofluorene (2-AAF) and remaining 2-aminofluorene (2-AF). Human lung tumor cell cytosols and intact cells were used for examining NAT activity and carcinogen–DNA adduct formation. The results demonstrated that NAT activity, gene expression (NAT1 mRNA) and 2-AF–DNA adduct formation in human lung tumor cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The effects of paclitaxel on the values of the apparent K m and V max of NAT from human lung tumor cells were also determined in both examined systems. The result also indicated that paclitaxel decreased the apparent values of K m and V max from human lung tumor cells in both cytosol and intact cells. Thus, paclitaxel is an uncompetitive inhibitor to NAT enzyme.

Effects of butylated hydroxyanisole and butylated hydroxytoluene on DNA adduct formation and arylamine N-acetyltransferase activity in human bladder tumour cells.

In this study, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were used to determine the inhibition of arylamine N-acetyltransferase (NAT) activity and DNA adduct formation in human bladder tumour cell line T-24. The activity of NAT was measured by high-performance liquid chromatography, assaying for the amounts of N-acetyl-2-aminofluorene and N-acetyl-p-aminobenzoic acid and remaining 2-aminofluorene and p-aminobenzoic acid. Human bladder tumour cell line T-24 cytosols and intact cells were used for examining NAT activity and carcinogen-DNA adduct formation. The results demonstrated that NAT activity and 2-aminofluorene-DNA adduct formation in human bladder tumour cells were inhibited and decreased by BHA and BHT in a dose-dependent manner. The effects of BHA and BHT on the values of the apparent K(m) and V(max) also were determined in both systems examined. The results indicated that BHA and BHT decreased the apparent values of K(m) and V(max) from human bladder tumour cells in both cytosol and intact cells.

Inhibition by vitamin C of DNA adduct formation and arylamine N-acetyltransferase activity in human bladder tumor cells.

Previous studies have already demonstrated the protective role of vitamin C (ascorbic acid) in certain types of cancer. This study reports on the effects of vitamin C on arylamine N-acetyltransferase (NAT) activity and DNA adduct formation in a human bladder tumor cell (T24) line. The activity of NAT was measured using high-performance liquid chromatography (HPLC), by assaying for the amounts of acetylated 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) and the remaining amounts of AF and PABA. T24 cells were used for examining NAT activity and carcinogen DNA adduct formation. The results demonstrated that NAT activity and 2-aminofluorene DNA adduct formation in T24 cells were inhibited and decreased by vitamin C in a dose-dependent manner. The apparent kinetic parameters (apparent values of Km and Vmax) from T24 cells were also determined with and without vitamin C cotreatment. The data also indicated that vitamin C decreased the apparent values of Km and Vmax from T24 cells.

Epidemiology of tobacco carcinogenesis.

Tobacco smoking is a worldwide epidemic. Tobacco smoke is an established human carcinogen that contains more than 50 carcinogens, among the most potent of which are polycyclic aromatic hydrocarbons (PAHs) and tobacco- specific nitrosamines (TSNs). Over the last 40 years, the level of tar and nicotine in cigarettes has decreased, along with the level of PAHs, but the level of TSNs has increased. Also, decreases in nicotine content can lead to an attendant increase in smoking in order for an individual to maintain his or her blood nicotine levels. Several factors determine the biologically effective dose of carcinogens, including the number of cigarettes smoked per day, type of cigarette, smoking topography, carcinogen metabolism, and DNA repair. Many studies have shown a relationship between tobacco smoke exposure, carcinogen-DNA adduct formation, tumor specific mutations (eg, p53 mutational spectra), and cancer risk. Genetically determined host capacity can influence these outcomes and the risk for tobacco addiction. Current areas of interest include determining whether women are indeed at greater risk of lung cancer compared with men, and if blacks are at higher risk than women. Also, newer methods can probably clarify the role of environmental tobacco smoke in carcinogenesis.