E-cigarette smoke damages DNA and reduces repair activity in mouse lung, heart, and bladder as well as in human lung and bladder cells.

E-cigarette smoke delivers stimulant nicotine as aerosol without tobacco or the burning process. It contains neither carcinogenic incomplete combustion byproducts nor tobacco nitrosamines, the nicotine nitrosation products. E-cigarettes are promoted as safe and have gained significant popularity. In this study, instead of detecting nitrosamines, we directly measured DNA damage induced by nitrosamines in different organs of E-cigarette smoke-exposed mice. We found mutagenic O6-methyldeoxyguanosines and γ-hydroxy-1,N2 -propano-deoxyguanosines in the lung, bladder, and heart. DNA-repair activity and repair proteins XPC and OGG1/2 are significantly reduced in the lung. We found that nicotine and its metabolite, nicotine-derived nitrosamine ketone, can induce the same effects and enhance mutational susceptibility and tumorigenic transformation of cultured human bronchial epithelial and urothelial cells. These results indicate that nicotine nitrosation occurs in vivo in mice and that E-cigarette smoke is carcinogenic to the murine lung and bladder and harmful to the murine heart. It is therefore possible that E-cigarette smoke may contribute to lung and bladder cancer, as well as heart disease, in humans.

Aldehydes are the predominant forces inducing DNA damage and inhibiting DNA repair in tobacco smoke carcinogenesis.

Tobacco smoke (TS) contains numerous cancer-causing agents, with polycyclic aromatic hydrocarbons (PAHs) and nitrosamines being most frequently cited as the major TS human cancer agents. Many lines of evidence seriously question this conclusion. To resolve this issue, we determined DNA adducts induced by the three major TS carcinogens: benzo(a)pyrene (BP), 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanoe (NNK), and aldehydes in humans and mice. In mice, TS induces abundant aldehyde-induced γ-hydroxy-propano-deoxyguanosine (γ-OH-PdG) and α-methyl-γ-OH-PdG adducts in the lung and bladder, but not in the heart and liver. TS does not induce the BP- and NNK-DNA adducts in lung, heart, liver, and bladder. TS also reduces DNA repair activity and the abundance of repair proteins, XPC and OGG1/2, in lung tissues. These TS effects were greatly reduced by diet with polyphenols. We found that γ-OH-PdG and α-methyl-γ-OH-PdG are the major adducts formed in tobacco smokers' buccal cells as well as the normal lung tissues of tobacco-smoking lung cancer patients, but not in lung tissues of nonsmokers. However, the levels of BP- and NNK-DNA adducts are the same in lung tissues of smokers and nonsmokers. We found that while BP and NNK can induce BPDE-dG and O6-methyl-dG adducts in human lung and bladder epithelial cells, these inductions can be inhibited by acrolein. Acrolein also can reduce DNA repair activity and repair proteins. We propose a TS carcinogenesis paradigm. Aldehydes are major TS carcinogens exerting dominant effect: Aldehydes induce mutagenic PdG adducts, impair DNA repair functions, and inhibit many procarcinogens in TS from becoming DNA-damaging agents.

Effect of CpG methylation at different sequence context on acrolein- and BPDE-DNA binding and mutagenesis.

Acrolein (Acr), an α,ß-unsaturated aldehyde, is abundant in tobacco smoke and cooking and exhaust fumes. Acr induces mutagenic α- and γ- hydroxy-1,N(2)-cyclic propano-deoxyguanosine adducts in normal human bronchial epithelial cells. Our earlier work has found that Acr-induced DNA damage preferentially occurs at lung cancer p53 mutational hotspots that contain CpG sites and that methylation at CpG sites enhances Acr-DNA binding at these sites. Based on these results, we hypothesized that this enhancement of Acr-DNA binding leads to p53 mutational hotspots in lung cancer. In this study, using a shuttle vector supF system, we tested this hypothesis by determining the effect of CpG methylation on Acr-DNA binding and the mutations in human lung fibroblasts. We found that CpG methylation enhances Acr-induced mutations significantly. Although CpG methylation enhances Acr-DNA binging at all CpG sites, it enhances mutations at selective--TCGA--sites. Similarly, we found that CpG methylation enhances benzo(a)pyrene diol epoxide binding at all -CpG- sites. However, the methylated CpG sequences in which benzo(a)pyrene diol epoxide-induced mutations are enhanced are different from the CpG sequences in which Acr-induced mutations are enhanced. CpG methylation greatly increases Acr-induced G to T and G to A mutation frequency to levels similar to these types of mutations found in the CpG sites in the p53 gene in tobacco smoke-related lung cancer. These results indicate that both CpG sequence context and the chemical nature of the carcinogens are crucial factors for determining the effect of CpG methylation on mutagenesis.

Chromium (VI) induces both bulky DNA adducts and oxidative DNA damage at adenines and guanines in the p53 gene of human lung cells.

Chromium (VI) [Cr(VI)], a ubiquitous environmental carcinogen, is generally believed to induce mainly mutagenic binary and ternary Cr(III)-deoxyguanosine (dG)-DNA adducts in human cells. However, both adenine (A) and guanine (G) mutations are found in the p53 gene in Cr exposure-related lung cancer. Using UvrABC nuclease and formamidopyrimidine glycosylase (Fpg), and ligation-mediated PCR methods, we mapped the distribution of bulky DNA adducts (BDA) and oxidative DNA damage (ODD) in the p53 gene in Cr(VI)-treated human lung cells. We found that both BDA and ODD formed at 2'-deoxyadenosine (dA) and dG bases. To understand the causes for these Cr-induced DNA damages, we mapped the distribution of BDA adducts and ODD in the p53 gene DNA fragments induced by Cr(III), Cr(VI) and Cr(V), the three major cellular Cr forms. We found that (i) dA at -CA- is a major Cr(VI) binding site followed by -GG- and -G-. Cr(VI) does not bind to -GGG-, (ii) Cr(VI)-DNA binding specificity is distinctly different from the Cr(III)-DNA binding in which -GGG- and -GG- are preferential sites, (iii) Cr(V) binding sites include all of Cr(VI) and Cr(III)-DNA binding sites and (iv) Cr(VI) and Cr(V) induce Fpg-sensitive sites at -G-. Together, these results suggest that Cr(VI) induction of BDA and ODD at dA and dG residues is through Cr(V) intermediate. We propose that these Cr(VI)-induced BDA and ODD contribute to mutagenesis of the p53 gene that leads to lung carcinogenesis.

Repair of mitomycin C mono- and interstrand cross-linked DNA adducts by UvrABC: a new model.

Mitomycin C induces both MC-mono-dG and cross-linked dG-adducts in vivo. Interstrand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation. In Escherichia coli cells, UvrABC repairs ICL lesions that cause DNA bending. The mechanisms and consequences of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. Using DNA fragments containing a MC-mono-dG or an ICL dG-MC-dG adduct, we found (i) UvrABC incises only at the strand containing MC-mono-dG adducts; (ii) UvrABC makes three types of incisions on an ICL dG-MC-dG adduct: type 1, a single 5' incision on 1 strand and a 3' incision on the other; type 2, dual incisions on 1 strand and a single incision on the other; and type 3, dual incisions on both strands; and (iii) the cutting kinetics of type 3 is significantly faster than type 1 and type 2, and all of 3 types of cutting result in producing DSB. We found that UvrA, UvrA+UvrB and UvrA+UvrB+UvrC bind to MC-modified DNA specifically, and we did not detect any UvrB- and UvrB+UvrC-DNA complexes. Our findings challenge the current UvrABC incision model. We propose that DSBs resulted from NER of ICL dG-MC-dG adducts contribute to MC antitumor activity and mutations.

DNA damage, DNA repair and carcinogenicity: Tobacco smoke versus electronic cigarette aerosol.

The allure of tobacco smoking is linked to the instant gratification provided by inhaled nicotine. Unfortunately, tobacco curing and burning generates many mutagens including more than 70 carcinogens. There are two types of mutagens and carcinogens in tobacco smoke (TS): direct DNA damaging carcinogens and procarcinogens, which require metabolic activation to become DNA damaging. Recent studies provide three new insights on TS-induced DNA damage. First, two major types of TS DNA damage are induced by direct carcinogen aldehydes, cyclic-1,N2-hydroxy-deoxyguanosine (γ-OH-PdG) and α-methyl-1, N2-γ-OH-PdG, rather than by the procarcinogens, polycyclic aromatic hydrocarbons and aromatic amines. Second, TS reduces DNA repair proteins and activity levels. TS aldehydes also prevent procarcinogen activation. Based on these findings, we propose that aldehydes are major sources of TS induce DNA damage and a driving force for carcinogenesis. E-cigarettes (E-cigs) are designed to deliver nicotine in an aerosol state, without burning tobacco. E-cigarette aerosols (ECAs) contain nicotine, propylene glycol and vegetable glycerin. ECAs induce O6-methyl-deoxyguanosines (O6-medG) and cyclic γ-hydroxy-1,N2--propano-dG (γ-OH-PdG) in mouse lung, heart and bladder tissues and causes a reduction of DNA repair proteins and activity in lungs. Nicotine and nicotine-derived nitrosamine ketone (NNK) induce the same types of DNA adducts and cause DNA repair inhibition in human cells. After long-term exposure, ECAs induce lung adenocarcinoma and bladder urothelial hyperplasia in mice. We propose that E-cig nicotine can be nitrosated in mouse and human cells becoming nitrosamines, thereby causing two carcinogenic effects, induction of DNA damage and inhibition of DNA repair, and that ECA is carcinogenic in mice. Thus, this article reviews the newest literature on DNA adducts and DNA repair inhibition induced by nicotine and ECAs in mice and cultured human cells, and provides insights into ECA carcinogenicity in mice.

The role of TAp63γ and P53 point mutations in regulating DNA repair, mutational susceptibility and invasion of bladder cancer cells.

It has long been recognized that non-muscle-invasive bladder cancer (NMIBC) has a low propensity (20%) of becoming muscle-invasive (MIBC), and that MIBC carry many more p53 point mutations (p53m) than NMIBC (50% vs 10%). MIBC also has a higher mutation burden than NMIBC. These results suggest that DNA repair capacities, mutational susceptibility and p53m are crucial for MIBC development. We found MIBC cells are hypermutable, deficient in DNA repair and have markedly downregulated DNA repair genes, XPC, hOGG1/2 and Ref1, and the tumor suppressor, TAp63γ. In contrast, NMIBC cells are hyperactive in DNA repair and exhibit upregulated DNA repair genes and TAp63γ. A parallel exists in human tumors, as MIBC tissues have markedly lower DNA repair activity, and lower expression of DNA repair genes and TAp63γ compared to NMIBC tissues. Forced TAp63γ expression in MIBC significantly mitigates DNA repair deficiencies and reduces mutational susceptibility. Knockdown of TAp63γ in NMIBC greatly reduces DNA repair capacity and enhances mutational susceptibility. Manipulated TAp63γ expression or knockdown of p53m reduce the invasion of MIBC by 40-60%. However, the combination of p53m knockdown with forced TAp63γ expression reduce the invasion ability to nil suggesting that p53m contributes to invasion phenotype independent from TAp63γ. These results indicate that in BC, TAp63γ regulates DNA repair capacities, mutational susceptibility and invasion, and that p53m contribute to the invasion phenotype. We conclude that concurrent TAp63γ suppression and acquisition of p53m are a major cause for MIBC development.

AFB1 hepatocarcinogenesis is via lipid peroxidation that inhibits DNA repair, sensitizes mutation susceptibility and induces aldehyde-DNA adducts at p53 mutational hotspot codon 249.

Aflatoxin B1 (AFB1) contamination in the food chain is a major cause of hepatocellular carcinoma (HCC). More than 60% of AFB1 related HCC carry p53 codon 249 mutations but the causal mechanism remains unclear. We found that 1) AFB1 induces two types of DNA adducts in human hepatocytes, AFB1-8,9-epoxide-deoxyguanosine (AFB1-E-dG) induced by AFB1-E and cyclic α-methyl-γ-hydroxy-1,N2-propano-dG (meth-OH-PdG) induced by lipid peroxidation generated acetaldehyde (Acet) and crotonaldehyde (Cro); 2) the level of meth-OH-PdG is >30 fold higher than the level of AFB1-E-dG; 3) AFB1, Acet, and Cro, but not AFB1-E, preferentially induce DNA damage at codon 249; 4) methylation at -CpG- sites enhances meth-OH-PdG formation at codon 249; and 5) repair of meth-OH-PdG at codon 249 is poor. AFB1, Acet, and Cro can also inhibit DNA repair and enhance hepatocyte mutational sensitivity. We propose that AFB1-induced lipid peroxidation generated aldehydes contribute greatly to hepatocarcinogenesis and that sequence specificity of meth-OH-PdG formation and repair shape the codon 249 mutational hotspot.

Alleviation of benzo[a]pyrene-diolepoxide-DNA damage in human lung carcinoma by glutathione S-transferase M2.

Cellular detoxification is important for the routine removal of environmental and dietary carcinogens. Glutathione S-transferases (GST) are major cellular phase II detoxification enzymes. MRC-5 cells have been found to exhibit significantly higher GST activity than human H1355 cells. This study investigates whether GST-M2 activity acts as a critical determinant of the target dose of carcinogenic benzo[a]pyrene-diolepoxide (BPDE) and whether it has an effect on MDM2 splicing in the two cell lines. We used RT-PCR to clone Mu-class GST cDNA. Two forms of GST coming from the cell lines were characterized as GST-M2 (from MRC-5 cells) and GST-M4 (from H1355 cells). Nested-PCR showed that BPDE-induced MDM2 splicing had occurred in the H1355 cell line but not in normal MRC-5 cells. Furthermore, using nested-PCR and competitive ELISA, we found that in H1355 cells modified to stably overexpress GST-M2, splicing was abolished and BPDE adducts appeared in low abundance. In conclusion, exogenously overexpressed GST-M2 was effective in reducing BPDE-induced DNA damage in H1355 cells. The catalytic activity of GST-M2 may play an important future role in lowering the incidence of BPDE-induced DNA damage.

Alternative splicing of MDM2 mRNA in lung carcinomas and lung cell lines.

The MDM2 gene is overexpressed in several human tumors and its product may be processed into various isoforms. Recently, alternative splicing forms of MDM2 mRNA have been detected in various types of tumors. In this study, lung tissue from human non small cell lung cancers was examined for MDM2 mRNA splicing variants by nested RT-PCR. Of the 117 lung cancer tissue samples analyzed, a total of 31 (26.5%) had splice variants for the MDM2 gene, while 59 (50.4%) had undetectable levels of MDM2 transcript. Further analysis indicated that the predominant variant for 26 of the 31 samples with alternative MDM2 splicing products was MDM2-657, a splice variant lacking exons 3-11. Significant associations were found between the frequency of alternative splicing and the gender and smoking habits of the patients. Approximately 36% of male patients had alternative splicing of MDM2 compared with only 9.5% of female patients (P = 0.008); 44.2% of the smoker patients had alternative MDM2 splice forms versus 16.2% of nonsmokers (P = 0.003). Furthermore, most normal lung cell lines examined possessed only full-length MDM2 mRNA, while among several lung cancer cell lines, only H1355 and CaLu-1 cells lacked alternatively spliced MDM2 transcripts. When H1355 cells were treated in vitro with the cigarette smoke carcinogen benzo[a]pyrene (B[a]P) or the B[a]P metabolite benzo[a]pyrene diolepoxide (BPDE), three MDM2 splicing products were detected by nested RT-PCR. Finally, with the use of several specific inhibitors, we found that BPDE-induced MDM2 mRNA alternative splicing in H1355 cells may occur through the PI3K or MAPK pathway. Overall, our results suggest that carcinogens present in cigarette smoke increase the risk of alternative MDM2 splicing, which is highly associated with lung cancer.

Cigarette side-stream smoke lung and bladder carcinogenesis: inducing mutagenic acrolein-DNA adducts, inhibiting DNA repair and enhancing anchorage-independent-growth cell transformation.

Second-hand smoke (SHS) is associated with 20-30% of cigarette-smoke related diseases, including cancer. Majority of SHS (>80%) originates from side-stream smoke (SSS). Compared to mainstream smoke, SSS contains more tumorigenic polycyclic aromatic hydrocarbons and acrolein (Acr). We assessed SSS-induced benzo(a)pyrene diol epoxide (BPDE)- and cyclic propano-deoxyguanosine (PdG) adducts in bronchoalveolar lavage (BAL), lung, heart, liver, and bladder-mucosa from mice exposed to SSS for 16 weeks. In SSS exposed mice, Acr-dG adducts were the major type of PdG adducts formed in BAL (p < 0.001), lung (p < 0.05), and bladder mucosa (p < 0.001), with no significant accumulation of Acr-dG adducts in heart or liver. SSS exposure did not enhance BPDE-DNA adduct formation in any of these tissues. SSS exposure reduced nucleotide excision repair (p < 0.01) and base excision repair (p < 0.001) in lung tissue. The levels of DNA repair proteins, XPC and hOGG1, in lung tissues of exposed mice were significantly (p < 0.001 and p < 0.05) lower than the levels in lung tissues of control mice. We found that Acr can transform human bronchial epithelial and urothelial cells in vitro. We propose that induction of mutagenic Acr-DNA adducts, inhibition of DNA repair, and induction of cell transformation are three mechanisms by which SHS induces lung and bladder cancers.

Benzo[a]pyrene diol epoxide up-regulates COX-2 expression through NF-kappaB in rat astrocytes.

Cyclooxygenase may be important in the pathogenesis of smoking-related cancer because it activates carcinogens and is highly inducible in inflammation. Benzo[a]pyrene (B[a]P) is one of the most common ingredients of cigarette smoke and benzo[a]pyrene diol epoxide (BPDE) is a metabolic product of B[a]P. Cigarette smoking-induced inflammation has been found in several tissues and in association with cyclooxygenase-2 (COX-2) expression. The contribution of COX-2 to peripheral inflammation is well documented, however, little is known about its role in brain inflammation. We studied COX-2 expression following treatment with BPDE in the cortical cells of Sprague-Dawley rats in vivo, as well as in DI TNC1 rat astrocytes and rat pheochromocytoma PC-12 cells (neurons) cultured in vitro. Our data showed that BPDE increases levels of COX-2 mRNA and protein in cortical cells of Sprague-Dawley rats. BPDE also increases levels of COX-2 mRNA in PC-12 and DI TNC1 cells. Induction of COX-2 protein was only found in DI TNC1 cells. Gel shift assay and western blot revealed increased NF-kappaB binding activity and protein level after treatment with BPDE. Experiments were performed to define the signaling mechanism by which BPDE induces COX-2, and suggested that BPDE-mediated COX-2 induction increases the risk of brain inflammation.

Acrolein- and 4-Aminobiphenyl-DNA adducts in human bladder mucosa and tumor tissue and their mutagenicity in human urothelial cells.

Tobacco smoke (TS) is a major cause of human bladder cancer (BC). Two components in TS, 4-aminobiphenyl (4-ABP) and acrolein, which also are environmental contaminants, can cause bladder tumor in rat models. Their role in TS related BC has not been forthcoming. To establish the relationship between acrolein and 4-ABP exposure and BC, we analyzed acrolein-deoxyguanosine (dG) and 4-ABP-DNA adducts in normal human urothelial mucosa (NHUM) and bladder tumor tissues (BTT), and measured their mutagenicity in human urothelial cells. We found that the acrolein-dG levels in NHUM and BTT are 10-30 fold higher than 4-ABP-DNA adduct levels and that the acrolein-dG levels in BTT are 2 fold higher than in NHUM. Both acrolein-dG and 4-ABP-DNA adducts are mutagenic; however, the former are 5 fold more mutagenic than the latter. These two types of DNA adducts induce different mutational signatures and spectra. We found that acrolein inhibits nucleotide excision and base excision repair and induces repair protein degradation in urothelial cells. Since acrolein is abundant in TS, inhaled acrolein is excreted into urine and accumulates in the bladder and because acrolein inhibits DNA repair and acrolein-dG DNA adducts are mutagenic, we propose that acrolein is a major bladder carcinogen in TS.

Expression of glutathione S-transferase M2 in stage I/II non-small cell lung cancer and alleviation of DNA damage exposure to benzo[a]pyrene.

Glutathione S-transferases (GSTs) are a family of inducible enzymes that are important in carcinogen detoxification. GST-Mu class is showing the high activity towards most polycyclic aromatic hydrocarbon (PAH) epoxide. Our objective is to clarify the expression of GST-M2 in non-small-cell lung carcinoma (NSCLC) patients and to determine the role of GST-M2 in protecting against DNA damage. We detected changes in GST-M2 expression at mRNA levels with a panel of lung cell lines and clinical samples of malignant and paired adjacent non-malignant tissues from 50 patients with stage I or II non-small-cell lung carcinoma using real-time RT-PCR. Comet assay and gamma-H2AX were used to clarify whether DNA damaged was protected by GST-M2. Our data demonstrate that the expression of GST-M2 in tumor tissues is significantly lower than in paired adjacent non-malignant tissues (p=0.016). Loss of GST-M2 is closely associated with age, gender, T value, N value and cell differentiation (p<0.05) in early stage I/II patients. Downregulation of GST-M2 is mediated through aberrant hypermethylation in lung cancer cell lines. Protection against B[a]P-induced DNA damage by GST-M2 in lung cancer cells was detected by Comet assay and gamma-H2AX. In conclusion, DNA hypermethylation altered and reduced GST-M2 expression that resulted in susceptible to benzo[a]pyrene (B[a]P) induced DNA damage. It implies that GST-M2 reduction occurs prior to tumorigenesis.