Carcinogen-induced Mutagenesis Research Articles

Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis.

The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e., adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer.

Unveiling DNA Polymerase Synthesis, Proofreading, and Mutagenesis Activities - One Molecule at a Time

DNA polymerases must accurately replicate DNA to maintain the integrity of the genome. Carcinogenic adducts, such as 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF), are covalently bound to bases on DNA leading to altered DNA polymerase activity and increased levels of mutagenesis at the adduct site. Although AF and AAF adducts differ only by an acetyl group, the two adducts have different biological effects and the AF adduct is much more easily bypassed by most DNA polymerases. While carcinogen-induced mutagenesis has been extensively studied using ensemble experiments, the exact mechanism for how DNA damage leads to a mutation remains unclear. We have used two complementary single-molecule methods, fluorescence resonance energy transfer (smFRET) and protein-induced fluorescence enhancement (smPIFE), to characterize the interactions between a DNA polymerase and DNA containing an AF or AAF adduct. These studies have allowed us to gain unprecedented insights into the molecular dynamics occurring between a DNA polymerase and a damaged template. We found the degree to which an adduct destabilizes polymerase binding to the DNA depends on the location of the adduct with respect to the primer terminus, the type of adduct, and the nucleotides present in the solution. Not only do the adducts influence the dwell time of the polymerase on the DNA, but also its binding position and orientation. Finally, we have for the first time directly observed the adduct-induced internal transfer of the DNA from the polymerization active site to the 3’-5’ exonuclease site.

Inhibitory Efect of Uprocessed/Processed Aliums under in vitro/in vivo Conditions on Carcinogen Induced Mutagenesis Using Different Assays

Three experiments were carried out to assess the antimutagenic potential of garlic or onion This was tested in, in vitro/in vivo models where mutagenicity was induced either with benzo (a) pyrene (BP)/3-methyl-cholanthrene (3MC), using Ames test or the comet assay. Also the effects of cooking processes like boiling/frying on the antimutagenic potential of these alliums were tested by SOS inhibition assay using 4-nitroquinoline-N-oxide (4 NQO) as the mutagen. In the first experiment, onion feeding at 1 and 5% in the diet inhibited the formation and excretion of urinary mutagens in dose dependent manner as indicated by decreased frequency of revertants in strains TA 98 and TA100 in the presence of urine belonging to rats fed onion and exposed to BP or 3MC. In the second experiment using SOS induction assay, both processed or unprocessed garlic exhibited dose dependent inhibition of SOS induction. However with boiled/unboiled onion this was not statistically significant. Studies with DAS also showed a dose dependent inhibition of SOS induction. No significant differences were observed in inhibition of SOS responses with processing of onion/garlic. These results indicate that the antimutagenic principle in garlic is not destroyed on heat treatment during cooking processes. In the third experiment, BP treatment resulted in comet induction (as judged by the comet ratio¬diameter/length) significantly as compared to control, both in liver and kidney tissues (p<0.001). Allium vegetables fed groups showed values similar to control group. As was observed in the peripheral blood lymphocytes, a trend towards recovery from damage was noted both in liver and kidney tissue due to prior allium feeding. All these results show that alliums whether in the processed or unprocessed form can be used in diets to avert toxic effects of environmental carcinogens/genotoxicants and can be potent chemopreventive agents and cancer prevention is far more desirable alternative than treatment by surgery and drugs. The protection could be achieved even by very low intake level of allium in natural form through diet.

971. Novel Ribozyme-Based Strategy for Cancer Chemoprevention

Top of pageAbstract Recent advances in understanding the molecular mechanisms of carcinogen-induced mutagenesis indicate that most mutations are dependent on the activity of newly-described translesion DNA polymerases, which have reduced base-pairing requirements and are able to replicate past DNA damage with potentially mutagenic consequences. In human cells in culture, reducing the level of one such polymerase, REV1, with a gene-specific ribozyme greatly reduces carcinogen-induced mutagenesis. To examine the hypothesis that reducing the mutagenic load will reduce the incidence of cancer, we are developing strategies to reduce REV1 in vivo in mouse models of carcinogenesis. We constructed vectors that express a ribozyme targeting mouse REV1 in a region homologous to the human mRNA used in our published studies. In order to improve the efficiency of gene-specific knockdown, DNA coding for the ribozyme was cloned into expression cassettes designed to target the transcripts either to the nuclear or to the cytoplasmic compartment. Using quantitative real-time PCR for REV1 mRNA with GAPDH as an internal control, we consistently found that the greatest suppression occurred when both compartments were targeted. When both compartments were targeted, REV1 expression was consistently suppressed by 80-90%. UV-induced mutant frequency at the HPRT locus in these cells was also reduced up to 80%, but UV-induced cytotoxicity was not affected. We plan to deliver these plasmids to the skin of excision repair deficient hairless xpa knockout mice, which develop squamous cell skin cancer with 100% penetrance after low doses of UV. Delivery of these expression cassettes to carcinogen-exposed tissue presents a novel chemopreventive strategy to reduce the frequency of mutations and the incidence of cancer.

Translesion DNA replication proteins as molecular targets for cancer prevention

Mutations in DNA are generally considered to have an etiologic role in the development of cancer. If so, it follows that reducing the frequency of such mutations will reduce the incidence of cancer induced by mutagens. Recent advances in elucidating the molecular mechanisms of carcinogen-induced mutagenesis indicate that replication of DNA templates that contain replication-blocking adducts is accomplished with error-prone DNA polymerases. These polymerases have relaxed base-pairing requirements, and can insert bases across from adducted templates, but with potentially mutagenic consequences. In principle, these proteins present new and attractive molecular targets to reduce mutagenesis. If this can be done in vivo without increasing cytotoxic responses to carcinogens, then novel chemopreventive strategies can be designed to reduce the risk of cancer in exposed populations prior to the appearance of disease symptoms.

Effects of Daidzein, Genistein, and 17β-Estradiolon 7,12-Dimethylbenz[a]anthracene-Induced Mutagenicityand Uterine Dysplasia in Ovariectomized Rats

: Phytoestrogens, primarily isoflavones daidzein (DZ) and genistein (GE), are increasingly used by postmenopausal women as an alternative to hormone replacement therapy due to reports that estrogen therapy increases the risk of breast and endometrial cancers. These compounds, as estrogen receptor agonists, may influence chemical carcinogenesis in estrogen-responsive tissues such as the uterus. We utilized ovariectomized (OVX) rats to model menopause and assessed the effects of dietary DZ, GE, or 17β-estradiol (E2) on carcinogen-induced mutagenesis and carcinogenesis in the rat uterus. Big Blue® transgenic rats (derived from Fischer 344 strain) were exposed to 7,12-dimethylbenz[a]anthracene (DMBA) in the presence or absence of the supplements. At 16- or 20-wk sacrifice, the uteri were removed and processed to determine mutant frequencies (MFs) and immunohistochemical or histopathological parameters, respectively. In rats treated with DMBA alone, a significant increase in lacI MFs (P < 0.01) in both OVX and intact (INT) rats was observed. The DMBA-induced MFs were not significantly altered by dietary DZ, GE, or E2 in both OVX and INT rats. Although dysplasia was not induced in the uterus of OVX and INT rats treated with DMBA alone, it was detected in 55% of OVX rats fed E2 alone and in 100% of OVX rats fed E2 along with DMBA exposure. Cell proliferation also was significantly higher in OVX rats fed E2 and treated with DMBA. In rats fed the isoflavones and treated with DMBA, the incidence of dysplasia was either reduced or virtually absent in both OVX and INT groups. These results indicate that a high incidence of dysplasia was associated with E2 feeding with or without DMBA treatment in the OVX rats, whereas the incidence was low in rats fed DZ or GE and treated with DMBA, suggesting a weak estrogen receptor agonist of DZ or GE in the rat uterus. The absence of dysplasia in OVX rats exposed to DMBA alone also suggests, in part, a promotional mechanism via estrogen- or isoflavone-driven cell proliferation.

Polyamines modulate carcinogen-induced mutagenesis in vivo

Elevated polyamine levels as a consequence of targeted overexpression of ornithine decarboxylase (ODC) to murine skin enhance susceptibility to tumorigenesis in this tissue. A possible mechanism for the enhanced susceptibility phenotype is an increased sensitivity of tissues with elevated polyamine levels to the mutagenic action of carcinogens. To test this hypothesis, a transgenic mouse model containing the Big Blue transgene and also expressing a K6/ODC transgene was developed. Incorporation of the K6/ODC transgene into the Big Blue model did not affect the spontaneous lacI mutant frequency in either skin or epidermis of the double-transgenic mice. After skin treatment with single doses of either 7,12-dimethylbenz[a]anthracene or N-methyl-N'-nitro-N-nitrosoguanidine, however, the mutant frequency was significantly increased in the skin of double-transgenic Big Blue;K6/ODC mice compared to Big Blue controls. The increases in mutant frequency were clearly due to ODC transgene activity, since treatment of mice with the ODC inhibitor, alpha-difluoromethylornithine, completely abolished the difference in mutant frequencies between double-transgenic and Big Blue mice. These results demonstrate that intracellular polyamine levels modulate mutation induction following carcinogen exposure.

Comparative effects of eight varieties of blackberry on mutagenesis

Diets containing large amounts of fruits and vegetables are known to decrease the probability of developing cancer. The chemical composition of fruits can vary with their genetic characteristics and the environmental conditions under which they are cultivated. Because of this variability, different varieties of the same fruit could be expected to have different effects on processes leading to carcinogenesis. Blackberries have been shown to have anti-carcinogenic potential. Since somatic mutations play a major role in the initiation and progression of cancer, we have compared eight varieties of blackberry grown under the same conditions for their abilities to inhibit carcinogen-induced mutagenesis. Using the Ames assay, we have measured the effects of each of the eight varieties on: 1) mutation induction by 2-amino anthracene (2AA), 2) mutation induction by methyl methanesulfonate (MMS) and 3) cell survival. All varieties were found to strongly suppress 2AA mutagenesis, but have minimal effect on MMS mutagenesis. Experiments were performed with berry juice and with homogenized berries. In addition, berries extracts were acidified to simulate changes which might be caused by the digestive process.

Fisetin induces transcription of NADPH:quinone oxidoreductase gene through an antioxidant responsive element-involved activation.

Induction of phase II enzymes such as NADPH:quinone oxidoreductase (QR) can reduce carcinogen-induced mutagenesis and tumor formation. In our search for novel dietary anticarcinogens, fisetin, a flavonol widely distributed in fruits and vegetables was found to induce QR activity in murine hepatoma 1c1c7 cells. The cells were treated with various concentrations of fisetin, and then were assessed for cell growth, QR activity, QR mRNA expression and transcription activation of the QR gene. The results showed that fisetin induced QR activity in time- and dose-dependent manner in the concentration range of 0.1 to 10 microM, and the activity induction was associated with QR mRNA expression as detected by reverse transcription-PCR. Furthermore, transfection studies using a human QR antioxidant/electrophile-response element (ARE/EpRE) reporter construct demonstrated that fisetin activated the ARE/EpRE. These results show that fisetin increases QR activity by transcriptional activation of the ARE/EpRE, suggesting a novel mechanism by which dietary fisetin may be implicated in cancer chemoprevention.

Retroviral expression of the hepatitis B virus x gene promotes liver cell susceptibility to carcinogen-induced site specific mutagenesis

Mutational inactivation of the tumor suppressor gene p53 is common in hepatocellular carcinomas (HCC). AGG to AGT transversion in codon 249 of exon 7 of the p53 gene occurs in over 50% of HCC from endemic regions, where both chronic infection with the hepatitis B virus (HBV) and exposure to carcinogens such as aflatoxin B1 (AFB1) prevail. In this study, we report the effect of the HBV x protein (HBx) on carcinogen-induced cytotoxicity and AGG to AGT mutation in codon 249 of the p53 gene in the human liver cell line CCL13. Expression of HBx, as revealed by its transactivation function, results in enhanced cell susceptibility to cytotoxicity induced by the AFB1 active metabolite, AFB1-8,9-epoxide, and benzo(a)pyrene diol-epoxide. Under similar conditions, expression of HBx promotes apoptosis in a subset of cell population. Exposure to AFB1-8,9-epoxide alone induces a low frequency of AGG to AGT mutation in codon 249 of the p53 gene, as determined by an allele-specific polymerase chain reaction (AS-PCR) assay. However, expression of HBx enhances the frequency of AFB1-epoxide-induced AGG to AGT mutation compared to control cells. In summary, this study demonstrates that expression of HBx enhances liver cell susceptibility to carcinogen-induced mutagenesis, possibly through alteration of the balance between DNA repair and apoptosis, two cellular defense mechanisms against genotoxic stress.

The early detection of frameshift mutations induced by a food-borne carcinogen in rats: a new tool for molecular epidemiology.

The accumulation of genetic changes is considered as the main factor that determines the development of cancer. Recent progresses in genetics and molecular biology led to the discovery of many new molecular markers and to the development of techniques able to monitor these markers. As a consequence, molecular epidemiology has emerged as a powerful approach to study the ternary relationship between the environment, the behaviour and the genetic predisposition of each individual. Susceptibility to cancer is determined at different levels such as the genetic polymorphism of enzymes involved in the activation and detoxification of carcinogens, the polymorphism of genes that maintains the genome stability, like those involved in DNA repair or recombination processes, and finally the polymorphism in oncogenes or tumour suppressor genes. Consequently, the full assessment of each individual's genetic predisposition is a long and difficult task. As the accumulation of mutations in somatic cells integrates all these parameters, its measurement would facilitate the evaluation of the individual predisposition status, provided that a marker common to a large spectrum of carcinogens could be found. Our current studies on the molecular mechanisms of carcinogen-induced mutagenesis has revealed that G-rich repetitive sequences are mutational hot spots for several major classes of environmental genotoxins such as aromatic and heterocyclic amines, polycyclic hydrocarbons and oxidative agents. We thus consider the possibility that these sequences form a new class of biomarkers for carcinogen exposure. In order to validate this hypothesis, we designed a sensitive PCR-based assay able to detect specific mutations induced by a common food-borne carcinogen in the colon epithelium of rats exposed for a short period to this carcinogen. This assay is sensitive enough to allow early detection of induced mutations and therefore allows to differentiate between unexposed animal and those exposed for a period as short as 1 week.

Ellagic acid induces transcription of the rat glutathione S-transferase-Ya gene.

Induction of glutathione S-transferase (GST) enzymes can increase detoxification of carcinogens and reduce carcinogen-induced mutagenesis and tumorigenesis. To determine if the anticarcinogen ellagic acid induces cellular enzymes which detoxify carcinogens, we examined the effect of ellagic acid on the expression of glutathione S-transferase-Ya. Rats fed ellagic acid demonstrated significant increases in total hepatic GST activity, hepatic GST-Ya activity and hepatic GST-Ya mRNA. To determine if the observed increase in GST-Ya mRNA was due to ellagic acid inducing transcription of the GST-Ya gene, transfection studies were performed with plasmid constructs containing various portions of the 5' regulatory region of the rat GST-Ya gene. The transfection studies demonstrated that ellagic acid increased GST-Ya mRNA by inducing transcription of the GST-Ya gene and demonstrated that this induction is mediated through the antioxidant responsive element of the GST-Ya gene.

Ellagic acid induces NAD(P)H:quinone reductase through activation of the antioxidant responsive element of the rat NAD(P)H:quinone reductase gene.

Induction of cellular detoxification enzymes can increase detoxification of carcinogens and reduce carcinogen-induced mutagenesis and tumorigenesis. To determine if the dietary anticarcinogen ellagic acid induced enzymes which detoxify xenobiotics and carcinogens, we examined the effect of ellagic acid on the expression of the phase II detoxification enzyme NAD(P)H:quinone reductase (QR). QR is induced by xenobiotics and antioxidants interacting with the xenobiotic responsive and antioxidant responsive elements of the 5' regulatory region of the QR gene. Ellagic acid is structurally related to the antioxidants which induce QR and we proposed that ellagic acid would induce QR expression through activation of the antioxidant responsive element of the QR gene. Rats fed ellagic acid demonstrated a 9-fold increase in hepatic and a 2-fold increase in pulmonary QR activity, associated with an 8-fold increase in hepatic QR mRNA. To determine if this increase in QR mRNA was due to activation of the antioxidant responsive element, transient transfection studies were performed with plasmid constructs containing various portions of the 5' regulatory region of the rat QR gene. These transfection studies confirmed that ellagic acid induces transcription of the QR gene and demonstrated that this induction is mediated through the antioxidant responsive element of the QR gene.

Effect of retinoids on carcinogen-induced mutagenesis in Salmonella tester strains

The ability of retinol (Rol) in altering mutation frequencies induced by 7 carcinogens was studied in Salmonella/microsome assay using 4 tester strains namely TA98, TA100, TA102 and TA1535. The 7 carcinogens used were aflatoxin B 1 (AFB), cyclophosphamide (CPP), 3-methylcholanthrene (MCA), benzo[ a]pyrene (BP), benz[ a]anthracene (BA), 9,10-dimethyl-1,2-benz[ a]anthracene (DMBA) and mitomycin C (MMC). As reported previously, Rol significantly reduced the number of His + revertants induced by AFB. It also reduced mutations induced by CPP or MCA but not that by BP, BA, DMBA or MMC. The abilities of Rol, retinoic acid, retinyl acetate and a known inhibitor for certain P-450 isozymes, 7,8-benzo-flavone (BF) in inhibiting mutations caused by AFB and BP were studied and compared. All the 3 retinoids caused significant reduction of AFB-induced His + revertants in a dose-dependent manner, but there was no effect on BP-induced mutation. BF strongly inhibited both AFB- and BP-induced revertants. The possibility of retinoids in exerting their effects on mutagenesis of precarcinogens by inhibiting only certain forms of cytochrome P-450 enzymes is discussed.

Enhancement of recovery of chemical carcinogen-induced ouabain-resistant mutants in chinese hamster cells by the tumor-promoting agent, 12- O-tetradecanoyl-phorbol-13-acetate

The effects of a tumor-promoting agent on the frequency of mutation to ouabain resistance and survival of Chinese hamster cells treated with a chemical carcinogen have been investigated. 12- O-Tetradecanoyl-phorbol-13-acetate (TPA) significantly enhanced the mutation frequency induced by the carcinogen, methylazoxymethanol acetate (MAM), without having similar effects on cytotoxicity, at concentrations of 2 μg/ml or less. The observed degree of enhancement of mutagenesis increased with promoter concentration up to the point where the lateter exhibited frank toxicity. Exposure of the cells to the promoter for a period of 2 or 6 h was found ineffective, but subsequently a significant enhancement was found after a 27-h exposure time. The maximum effect occurred after a 5-day exposure, with a increase in the mutation frequency of 250%. Treatment of cells with TPA alone resulted in no enhancement of spontaneous mutation rates, nor did treatment of cells prior to addition of carcinogen-induced mutagenesis. In contrast, TPA was found to be effective when applied as late as 6 weeks following carcinogen treatment. These results are consistent with the hypothesis that TPA owes its promoting activity towards chemically-induced mutagenesis and carciogenesis to its ability to enhance expression of latent somatic genetic modifications by epigenetic mechanisms. They do not support mechanisms involving TPA-induced inhibition of DNA-repair replication, or mutagenic activity of TPA per se. The notably similar qualitative response to TPA of several parameters in mouse-skin tumorigenesis and Chinese hamster cell mutagenesis suggest that the mechanism of action of the promoter is imiklar in the 2 diverse biolgical systems.

Analyses of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogens during the cell cycle: Part V. Radiation- and chemical carcinogen-induced mutagenesis

8-Azaguanine (8AG)-resistant mutations induced by X-rays, ultraviolet radiation (UV) and a chemical carcinogen, 4-hydroxyaminoquinoline 1-oxide (4-HAQO) were examined during the cell cycle of synchronized HeLa S3 cells. Mutants induced by 400 R of X-rays occurred in a higher frequency in the X-ray sensitive G 1-S boundary phase than in the X-ray-resistant G 2, S and early g 2 phases. 8AG-resistant mutants induced by treatment with 10 −5 M 4-HAQO for 20 min appeared in a higher frequency in the early to middle S phases than in the other phases. In the case of UV, however, we found no significant difference in the induced mutation frequencies the cell cyle, because the mutation frequencies induced by the UV doses (0–20 Jm 2) used were too low for detection of the difference. These results suggest that there is a close correlation between the critical damage induced in DNA molecule(s) at the DNA-synthetic phase in the cell cycle and mutagenesis, because mitotic cells have a low mutability in spite of their high radio-sensitivity.