Ying Fu

Detection of acrolein-derived cyclic DNA adducts in human cells by monoclonal antibodies

Acrolein (Acr) is a ubiquitous environmental pollutant found in cigarette smoke and automobile exhaust. It can also be produced endogenously by oxidation of polyunsaturated fatty acids. The Acr-derived 1,N(2)-propanodeoxyguanosine (Acr-dG) adducts in DNA are mutagenic lesions that are potentially involved in human cancers. In this study, monoclonal antibodies were raised against Acr-dG adducts and characterized using ELISA. They showed strong reactivity and specificity toward Acr-dG, weaker reactivity toward crotonaldehyde- and trans-4-hydroxy-2-nonenal-derived 1,N(2)-propanodeoxyguanosines, and weak or no reactivity toward 1,N(6)-ethenodeoxyadenosine and 8-oxo-deoxyguanosine. Using these antibodies, we developed assays to detect Acr-dG in vivo: first, a simple and quick FACS-based assay for detecting these adducts directly in cells; second, a highly sensitive direct ELISA assay for measuring Acr-dG in cells and tissues using only 1 μg of DNA without DNA digestion and sample enrichment; and third, a competitive ELISA for better quantitative measurement of Acr-dG levels in DNA samples. The assays were validated using Acr-treated HT29 cell DNA samples or calf thymus DNA, and the results were confirmed by LC-MS/MS-MRM. An immunohistochemical assay was also developed to detect and visualize Acr-dG in HT29 cells as well as in human oral cells. These antibody-based methods provide useful tools for the studies of Acr-dG as a cancer biomarker and of the molecular mechanisms by which cells respond to Acr-dG as a ubiquitous DNA lesion.

In vivo detection of a novel endogenous etheno-DNA adduct derived from arachidonic acid and the effects of antioxidants on its formation.

Previous studies showed that 7-(1,2-dihydroxyheptyl)-substituted etheno DNA adducts are products of reactions with the epoxide of (E)-4-hydroxy-2-nonenal, an oxidation product of ω-6 polyunsaturated fatty acids (PUFAs). In this work, we report the detection of 7-(1,2-dihydroxyheptyl)-1,N(6)-ethenodeoxyadenosine (DHHedA) in rodent and human tissues by two independent methods: a (32)P-postlabeling/HPLC method and an isotope dilution liquid chromatography-electrospray ionization-tandem mass spectrometry method, demonstrating for the first time that DHHedA is a background DNA lesion in vivo. We showed that DHHedA can be formed upon incubation of arachidonic acid with deoxyadenosine, supporting the notion that ω-6 PUFAs are the endogenous source of DHHedA formation. Because cyclic adducts are derived from the oxidation of PUFAs, we subsequently examined the effects of antioxidants, α-lipoic acid, Polyphenon E, and vitamin E, on the formation of DHHedA and γ-hydroxy-1,N(2)-propanodeoxyguanosine (γ-OHPdG), a widely studied acrolein-derived adduct arising from oxidized PUFAs, in the livers of Long Evans Cinnamon (LEC) rats. LEC rats are afflicted with elevated lipid peroxidation and prone to the development of hepatocellular carcinomas. The results showed that although the survival of LEC rats was increased significantly by α-lipoic acid, none of the antioxidants inhibited the formation of DHHedA, and only Polyphenon E decreased the formation of γ-OHPdG. In contrast, vitamin E caused a significant increase in the formation of both γ-OHPdG and DHHedA in the livers of LEC rats.

Nucleotide excision repair deficiency increases levels of acrolein-derived cyclic DNA adduct and sensitizes cells to apoptosis induced by docosahexaenoic acid and acrolein.

The acrolein derived cyclic 1,N(2)-propanodeoxyguanosine adduct (Acr-dG), formed primarily from ω-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) under oxidative conditions, while proven to be mutagenic, is potentially involved in DHA-induced apoptosis. The latter may contribute to the chemopreventive effects of DHA. Previous studies have shown that the levels of Acr-dG are correlated with apoptosis induction in HT29 cells treated with DHA. Because Acr-dG is shown to be repaired by the nucleotide excision repair (NER) pathway, to further investigate the role of Acr-dG in apoptosis, in this study, NER-deficient XPA and its isogenic NER-proficient XAN1 cells were treated with DHA. The Acr-dG levels and apoptosis were sharply increased in XPA cells, but not in XAN1 cells when treated with 125μM of DHA. Because DHA can induce formation of various DNA damage, to specifically investigate the role of Acr-dG in apoptosis induction, we treated XPA knockdown HCT116+ch3 cells with acrolein. The levels of both Acr-dG and apoptosis induction increased significantly in the XPA knockdown cells. These results clearly demonstrate that NER deficiency induces higher levels of Acr-dG in cells treated with DHA or acrolein and sensitizes cells to undergo apoptosis in a correlative manner. Collectively, these results support that Acr-dG, a ubiquitously formed mutagenic oxidative DNA adduct, plays a role in DHA-induced apoptosis and suggest that it could serve as a biomarker for the cancer preventive effects of DHA.

An endogenous DNA adduct as a prognostic biomarker for hepatocarcinogenesis and its prevention by Theaphenon E in mice

Hepatocellular carcinoma (HCC) is the third leading cause of cancer–related deaths worldwide, mainly because of its poor prognosis. A valid mechanism‐based prognostic biomarker is urgently needed. γ‐hydroxy‐1,N2‐propanodeoxyguanosine (γ‐OHPdG) is an endogenously formed mutagenic DNA adduct derived from lipid peroxidation. We examined the relationship of γ‐OHPdG with hepatocarcinogenesis in two animal models and its potential role as a prognostic biomarker for recurrence in HCC patients. Bioassays were conducted in xeroderma pigmentosum group A knockout mice and diethylnitrosamine‐injected mice, both prone to HCC development. γ‐OHPdG levels in the livers of these animals were determined. The effects of antioxidant treatments on γ‐OHPdG and hepatocarcinogenesis were examined. Using two independent sets of HCC specimens from patients, we examined the relationship between γ‐OHPdG and survival or recurrence‐free survival. γ‐OHPdG levels in liver DNA showed an age‐dependent increase and consistently correlated with HCC development in all three animal models. Theaphenon E treatment significantly decreased γ‐OHPdG levels in the liver DNA of xeroderma pigmentosum group A knockout mice and remarkably reduced HCC incidence in these mice to 14% from 100% in the controls. It also effectively inhibited HCC development in the diethylnitrosamine‐injected mice. Using clinical samples from two groups of patients, our study revealed that higher levels of γ‐OHPdG are strongly associated with low survival (P < 0.0001) and low recurrence‐free survival (P = 0.007). Conclusion: These results support γ‐OHPdG as a mechanism‐based, biologically relevant biomarker for predicting the risk of HCC and its recurrence. (Hepatology 2018;67:159‐170).

Detection of a lipid peroxidation-induced DNA adduct across liver disease stages

BackgroundnOxidative stress and chronic inflammation can increase cellular levels of reactive oxygen species and lipid peroxidation (LPO) when associated with the pathogenesis of hepatocellular carcinoma (HCC), which can develop following the progression of steatosis, fibrosis and cirrhosis. Using a monoclonal antibody for cyclic γ-hydroxy-1, N2 -propanodeoxyguanosine (γ-OHPdG), a promutagenic DNA adduct formed endogenously by LPO, we examined its formation across liver disease stages to understand its potential role in HCC development.nnnMethodsnFormalin-fixed paraffin embedded (FFPE) liver tissue samples from 49 patients representing normal, steatosis, fibrosis, cirrhosis and HCC were stained for γ-OHPdG and 8-hydroxydeoxyguanosine (8-oxo-dG), an oxidative damage biomarker. Quantification of immunohistochemical (IHC) staining was performed using histological scoring of intensity and distribution. Using primary human hepatocytes (HH) and a stellate cell (SC) co-culture, immunocytochemical staining of γ-OHPdG and Nile Red was performed to determine if the formation of γ-OHPdG was consistent between the clinical sample disease stages and the in vitro steatotic and fibrotic conditions.nnnResultsnγ-OHPdG levels varied significantly between the stages of normal and steatosis, steatosis and fibrosis, and steatosis and cirrhosis (P≤0.005). There was a trend, although not significant, of increased levels of γ-OHPdG in HCC compared to the other groups. A strong correlation was observed (Pearsons, R2 =0.85) between levels of γ-OHPdG and 8-oxo-dG across the disease spectrum. The increase of γ-OHPdG in steatosis and decrease in fibrosis was a pattern confirmed in an in vitro model using primary HH co-cultured with human SCs.nnnConclusionsnγ-OHPdG was detected in FFPE liver tissues of patients with different stages of liver disease and in vitro studies, demonstrating that its formation is consistent with LPO in early stages of liver disease and suggesting that it may be a source of mutagenic DNA damage in liver disease progression.

A click chemistry approach to identify protein targets of cancer chemopreventive phenethyl isothiocyanate

Here we report the identification of protein targets of chemopreventive phenethyl isothiocyanate (PEITC) via click chemistry in the A549 human lung cancer cell line, using a novel alkyne-tagged PEITC which was also found to show potent in vitro anticancer activity.

Abstract 120: Detection of acrolein-derived cyclic DNA adducts in human cells by monoclonal antibodies.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnAcrolein (Acr) is a ubiquitous environmental pollutant found in cigarette smoke and automobile exhaust. It can also be produced endogenously by oxidation of polyunsaturated fatty acids. The Acr-derived 1,N2-propanodeoxyguanosine (Acr-dG) adducts in DNA are mutagenic lesions that are potentially involved in human cancers. In this study, monoclonal antibodies were raised against Acr-dG adducts and characterized using ELISA. They showed strong reactivity and specificity towards Acr-dG, weaker reactivity towards crotonaldehyde- and trans-4-hydroxy-2-nonenal-derived 1,N2-propanodeoxyguanosines, and weak or no reactivity towards 1,N6-ethenodeoxyadenosine, 8-oxo-deoxyguanosine, and benzo(a)pyrene- and malondialdehdye-derived adducts. Using these novel antibodies, we developed assays to detect Acr-dG in vivo: First, a simple and quick FACS-based assay for detecting these adducts directly in cells; Second, a highly sensitive direct ELISA assay for measuring Acr-dG in cells and tissues using only one μg DNA; And third, a competitive ELISA for better quantitative measurement of Acr-dG levels in DNA. The assays were validated using Acr-treated HT29 cell DNA samples or calf thymus DNA and the results were confirmed by LC-MS/MS-MRM. An immunohistochemical assay was also developed to detect and visualize Acr-dG in HT29 cells as well as in human oral cells. These antibody-based methods provide useful tools for the studies of Acr-dG as a cancer biomarker and of the molecular mechanisms by which cells respond to Acr-dG as a ubiquitous DNA lesion. (This work was supported by NCI grant CA134892)nnCitation Format: Jishen Pan, Bisola Awoyemi, Zhuoli Xuan, Priya Vohra, Hsiang-Tsui Wang, Marcin Dyba, Emily Greenspan, Ying Fu, Karen Creswell, Lihua Zhang, Deborah Berry, Moon-Shong Tang, Fung-Lung Chung. Detection of acrolein-derived cyclic DNA adducts in human cells by monoclonal antibodies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 120. doi:10.1158/1538-7445.AM2013-120

Oxidative stress and hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. There are two major challenges for HCC, the first being that early detection is generally not applicable, and secondly, it is usually fatal within several months after diagnosis. HCC is an inflammation-induced cancer. It is known that chronic inflammation leads to oxidative/nitrosative stress and lipid peroxidation, generating excess oxidative stress, together with aldehydes which can react with DNA bases to form promutagenic DNA adducts. In this review, the evidence between oxidative stress and liver carcinogenesis is summarized. We focused on the potential of using DNA adducts as oxidative stress biomarkers for liver carcinogenesis.

Prevention of Lipid Peroxidation–derived Cyclic DNA Adduct and Mutation in High-Fat Diet–induced Hepatocarcinogenesis by Theaphenon E

Obesity is associated with cancer risk and its link with liver cancer is particularly strong. Obesity causes non-alcoholic fatty liver disease (NAFLD) that could progress to hepatocellular carcinoma (HCC). Chronic inflammation likely plays a key role. We carried out a bioassay in the high-fat diet (HFD)-fed C57BL/6J mice to provide insight into the mechanisms of obesity-related HCC by studying γ-OHPdG, a mutagenic DNA adduct derived from lipid peroxidation. In an 80-week bioassay, mice received a low-fat diet (LFD), high-fat diet (HFD), and HFD with 2% Theaphenon E (TE) (HFD+TE). HFD mice developed a 42% incidence of HCC and LFD mice a 16%. Remarkably, TE, a standardized green tea extract formulation, completely blocked HCC in HFD mice with a 0% incidence. γ-OHPdG measured in the hepatic DNA of mice fed HFD and HFD+TE showed its levels increased during the early stages of NAFLD in HFD mice and the increases were significantly suppressed by TE, correlating with the tumor data. Whole-exome sequencing showed an increased mutation load in the liver tumors of HFD mice with G>A and G>T as the predominant mutations, consistent with the report that γ-OHPdG induces G>A and G>T. Furthermore, the mutation loads were significantly reduced in HFD+TE mice, particularly G>T, the most common mutation in human HCC. These results demonstrate in a relevant model of obesity-induced HCC that γ-OHPdG formation during fatty liver disease may be an initiating event for accumulated mutations that leads to HCC and this process can be effectively inhibited by TE. Cancer Prev Res; 11(10); 665–76. ©2018 AACR.

Abstract 4051: Lipid peroxidation-derived DNA adduct formation in obesity-related hepatocarcinogenesis

In this study we are investigating the role and mechanism of formation of Lipid-peroxidation (LPO)-derived DNA adduct formation in obesity-related hepatocarcinogenesis. Obesity has been implicated as a risk factor for many types of cancer, particularly hepatocellular carcinoma (HCC). HCC incidence correlates with the increasing prevalence of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) in the US. The mechanisms underlying this increased risk of liver cancer in the obese population are still unclear. Liver tissue damage from fat accumulation in NAFLD and NASH produce inflammation and increased levels of reactive oxygen species (ROS). ROS induce LPO of polyunsaturated fatty acids (PUFAs) in cell membranes leading to the formation of reactive aldehydes, such as acrolein and 4-hydroxy-2-nonenal (HNE), which react with DNA forming the DNA adducts γ-OHPdG and DHH-edA, respectively. γ-OHPdG is derived from ω-6 and ω-3 PUFAs, while DHH-edA is specifically from ω-6 fatty acids. Previous data has indicated that mutational hotspots targeted by γ-OHPdG and DHH-edA may be within key cancer driver genes, such as p53. Research has shown that B-6 mice fed a high fat diet (HFD) develop fatty liver disease and eventually HCC. The livers of B-6 mice fed a HFD showed an increased ω-6/ω-3 PUFAs ratio. We have detected and quantified γ-OHPdG and DHH-edA by LC-MS/MS in livers from six healthy individuals and six NAFLD patients and found that levels of DHH-edA in the DNA of the fatty liver samples were nearly three-fold higher than that in normal liver samples. Preliminary in vitro data using primary human hepatocytes has indicated that treatment with oleic and palmitic acid promote the formation of γ-OHPdG. Similarly, treatment of these cells with the epoxide of HNE, 2,3-epoxy-4-hydroxynonanal (EH), and the omega-6 fatty acid, arachidonic acid, induce the formation of DHH-edA, supporting the proposed mechanism of adduct formation. In an 80-week tumor bioassay using C57Bl/6J mice on a HFD, we have observed through live-animal MRI imaging and immunohistochemistry, an increase in pro-inflammatory white adipose tissue accumulation, infiltration of fat into the liver and an increase in overall body weight compared to a low fat diet control. In addition, in mice fed a HFD combined with the green-tea derived antioxidant Theaphenon E, we observed through MRI, reduction in body weight gain, white adipose tissue, and lipid accumulation in liver. Theaphenon E has the potential to decrease fat accumulation and inflammation within the liver that lead to decreases in LPO-derived adduct formation and consequently, the mutations critical for HCC development. Citation Format: Heidi Coia, Hongyi Guan, Ying Fu, Marcin Dyba, Fung-Lung Chung. Lipid peroxidation-derived DNA adduct formation in obesity-related hepatocarcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4051.