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).

Association of body burden of mercury with liver function test status in the U.S. population.

The majority of mercury (Hg) exposure in the US population is from consumption of fish contaminated with methylmercury (MeHg). Since inorganic Hg is the predominant form excreted in the feces and urine, hepatic biotransformation is a critical step in its normal clearance. This study was set to test the hypothesis that compromised liver function is associated with body burden of Hg as indirectly reflected by Hg sampled in blood and urine. From the National Health and Nutrition Examination Survey (NHANES, 2003-2008), 3769 adults aged 20 years and above were selected for analysis. Hepatic function was inferred from the three standard serum liver-related enzyme activities, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and γ-glutamyltransferase (GGT). Multivariate regression models were used to examine the associations of interest. Although urinary Hg was significantly correlated with serum Hg, the blood-urinary Hg relationship was influenced by liver function, which is also a function of demographic and lifestyle factors (e.g., gender). Although the results were only marginally significant for examined enzymes (p=0.06-0.08), urinary Hg tended to be lower among subjects with elevated liver enzymes, as compared to those with normal enzyme measurements. Conversely, MeHg generally represents a higher fraction of the total circulating Hg among those with elevated liver enzyme levels, especially among participants with elevations in all three enzymes (p=0.01). In conclusion, this population-based study identified an association between liver function, serum Hg and urinary Hg. Urinalysis may not be the optimal approach to monitor Hg elimination toxicokinetics or Hg exposure, since the majority of Hg excretion is fecal and the fidelity of urinary excretion may depend on healthy liver function. Future prospective studies are warranted to expand these findings.

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.

Regioselective formation of acrolein-derived cyclic 1,N(2)-propanodeoxyguanosine adducts mediated by amino acids, proteins, and cell lysates.

Acrolein (Acr) is a major component in cigarette smoke and a ubiquitous environmental pollutant. It is also formed as a product of lipid peroxidation. Following ring closure via the Michael addition, Acr modifies deoxyguanosine (dG) in DNA by forming cyclic 1,N(2)-propanodeoxyguanosine adducts (OHPdG). The reactions of Acr with dG yield, depending on the direction of ring closure, two regioisomers, α- and γ-OHPdG, in approximately equal amounts. However, previous (32)P-postlabeling studies showed that the γ isomers were detected predominantly in the DNA of rodent and human tissues. Because of the potential differential biological activity of the isomeric OHPdG adducts, it is important to confirm and study the chemical basis of the regioselective formation of γ isomers in vivo. In this study, it is confirmed that γ-OHPdG adducts are indeed the major isomers formed in vivo as evidenced by a LC-MS/MS method specifically developed for Acr-derived dG adducts. Furthermore, we have shown that the formation of γ-isomers is increased in the presence of amino-containing compounds, including amino acids, proteins, and cell lysates. A product of Acr and arginine that appears to mediate the regioselective formation of γ isomers was identified, but its structure was not fully characterized due to its instability. This study demonstrates that intracellular amino-containing compounds may influence the regiochemistry of the formation of OHPdG adducts and reveals a mechanism for the preferential formation of γ-OHPdG by Acr in vivo.

Detoxification: a novel function of BRCA1 in tumor suppression?

Our studies found that BRCA1 levels negatively correlate with DNA adducts induced by Benzo(a)pyrene (BaP). Pulse-chase experiments showed that the increase in BaP-induced DNA adducts in BRCA1 knockdown cells may not be associated with BRCA1's function in nucleotide excision repair activity; rather, it may be associated with its function in modulating transcriptional regulation. BRCA1 knockdown in MCF-10A cells significantly attenuated the induction of CYP1A1 following BaP treatment indicating that the increase in BaP-induced adducts in BRCA1 knockdown cells is not CYP1A1 dependent. However, our study shows that BRCA1 defective cells may still be able to biotransform BaP by regulating other CYP enzymes, including CYP1B1. Knockdown of BRCA1 also severely affected the expression levels of two types of uridine diphosphate glucorunyltransferase (UGT1A1 and UGT1A9) and NRF2. Both UGTs are known as BaP-specific detoxification enzymes, and NRF2 is a master regulator of antioxidant and detoxification genes. Thus, we concluded that the increased amount of BaP-induced DNA adducts in BRCA1 knockdown cells is strongly associated with its loss of functional detoxification. Chromatin immunoprecipitation assay revealed that BRCA1 is recruited to the promoter/enhancer sequences of UGT1A1, UGT1A9, and NRF2. Regulation of UGT1A1 and UGT1A9 expression showed that the induction of DNA adducts by BaP is directly affected by their expression levels. Finally, overexpression of UGTs, NRF2, or ARNT significantly decreased the amount of BaP-induced adducts in BRCA1-deficient cells. Overall, our results suggest that BRCA1 protects cells by reducing the amount of BaP-induced DNA adducts possibly via transcriptional activation of detoxification gene expression.

A novel in vitro pancreatic carcinogenesis model.

Environmental factors (e.g., BaP) have been pointed out as one of the etiologies of pancreatic cancer. However, very limited experimental assays are available to identify pancreatic specific environmental mutagens or susceptibility genes. In this study, we have developed a simple in vitro cell culture model system that can be used to study the molecular and biochemical aspects of carcinogenesis in a near-normal immortalized pancreatic ductal epithelial cell lines. In order to demonstrate that xenobiotic stress response is intact in these cells, we employed standard molecular biology techniques. For examples, luciferase reporter and/or real-time quantitative PCR assays were used to determine stress-induced CYP1A1 and CYP1B1 gene expression. Western blotting and immunocytochemistry assays were used to demonstrate that TCDD or BaP could activate AhR signaling. For exploring the carcinogenesis mechanism, we incubated cells with [³H]BaP and determined BaP-DNA binding activity by measuring its radioactivity. BaP-DNA adduct formation was further confirmed by [³²P]-postlabeling assay. Finally, we demonstrated the effects of endogenous AhR or BRCA1 in BaP-DNA adduct accumulation in our cell system. As results, no apparent BaP-DNA adduct accumulation by [³²P]-postlabeling assay was found in either control-siRNA or AhR-siRNA pretreated cells. On the other hand, a significant increase of BaP-DNA adduct accumulation was found in BRCA1 knockdown cells. In conclusion, we suggest that this in vitro model may provide the feasibility for future studies on the molecular basis of pancreatic ductal cell carcinogenesis caused by dietary mutagens.

Effects of epigallocatechin gallate, L-ascorbic acid, alpha-tocopherol, and dihydrolipoic acid on the formation of deoxyguanosine adducts derived from lipid peroxidation.

Oxidation of polyunsaturated fatty acids (PUFAs) releases alpha,beta-unsaturated aldehydes that modify deoxyguanosine (dG) to form cyclic 1,N(2)-propanodeoxyguanosine adducts. One of the major adducts detected in vivo is acrolein (Acr)-derived 1,N(2)-propanodeoxyguanosine (Acr-dG). We used a chemical model system to examine the effects of 4 antioxidants known to inhibit fatty acid oxidation on the formation of Acr-dG and 8-oxodeoxyguaonsine (8-oxodG) from the PUFA docosahexaenoic acid (DHA) under oxidative conditions. We found that epigallocatechin gallate (EGCG) and dihydrolipoic acid (DHLA) inhibit both Acr-dG and 8-oxodG formation. In contrast, ascorbic acid and alpha-tocopherol actually increase Acr-dG at high concentrations and do not show a concentration-dependant inhibition of 8-oxodG. We also studied their effects on blocking Acr-dG formation directly from Acr. EGCG and DHLA can both effectively block Acr-dG formation, but ascorbic acid and alpha-tocopherol show weak or little effect. These results highlight the complexity of antioxidant mechanisms and also reveal that EGCG and DHLA are effective at suppressing lipid peroxidation-induced Acr-dG and 8-oxodG formation as well as blocking the reaction of dG with Acr.

Detection of the acrolein-derived cyclic DNA adduct by a quantitative 32P-postlabeling/solid-phase extraction/HPLC method: blocking its artifact formation with glutathione.

Acrolein (Acr), a hazardous air pollutant, reacts readily with deoxyguanosine (dG) in DNA to produce cyclic 1, N2-propanodeoxyguanosine adducts (Acr-dG). Studies demonstrate that these adducts are detected in vivo and may play a role in mutagenesis and carcinogenesis. In the study described here, a quantitative 32P-postlabeling/solid-phase extraction/HPLC method was developed by optimizing the solid-phase extraction and the 32P-postlabeling conditions for analysis of Acr-dG in DNA samples with a detection limit of 0.1 fmol. It was found that Acr-dG can form as an artifact during the assay. Evidence obtained from mass spectrometry indicates that the Acr in water used in the assay is a likely source of artifact formation of Acr-dG. The formation of Acr-dG as an artifact can be effectively blocked by adding glutathione (GSH) to the DNA sample to be analyzed. In addition, Acr-dG was detected as a contaminant in the commercial dG and dT 3'-monophosphate samples. Finally, this method was used to detect Acr-dG in calf thymus and human colon HT29 cell DNA with an excellent linear quantitative relationship.

A solid-phase extraction/high-performance liquid chromatography-based (32)P-postlabeling method for detection of cyclic 1,N(2)-propanodeoxyguanosine adducts derived from enals.

The cyclic 1,N(2)-propanodeoxyguanosine (PdG) adducts are Michael addition products from reactions of deoxyguanosine (dG) with enals, including acrolein (Acr), crotonaldehyde (Cro), pentenal (Pen), heptenal (Hep), and 4-hydroxy-2-nonenal (HNE). Although this is a general reaction, only the PdG adducts derived from Acr, Cro, and HNE have been detected in vivo as endogenous DNA lesions. Our previous in vitro study demonstrated that PdG adducts of Acr, Cro, and Pen are predominantly derived from oxidation of omega-3 polyunsaturated fatty acids (PUFAs), whereas the long-chain Hep and HNE adducts are from omega-6 PUFAs. PdG adducts are important because they represent a new class of endogenous promutagenic DNA lesions with potential roles in carcinogenesis. Earlier, we developed a (32)P-postlabeling method for detecting PdG adducts from Acr and Cro and a modified method for the long-chain HNE adducts. Both methods require multiple high-performance liquid chromatography steps and, in some cases, time-consuming thin-layer chromatography for purification. There is a lack of a single, versatile, and efficient method for simultaneous detection of all five enal-derived PdG adducts. In this paper, we report an improved (32)P-postlabeling method which permits detection of Acr, Cro, Pen, Hep, and HNE adducts in a single DNA sample. This method relies on solid-phase extraction for adduct enrichment before and after (32)P-labeling; all five PdG adducts were converted to the ring-opened derivatives for confirmation of identities and quantification. The method was validated using the synthetic adducts and enal-modified DNA and was finally applied to rat liver DNA and rat liver DNA samples spiked with different amount of standards. The detection limit was determined to be as low as 0.5 fmol in 80 microg DNA, corresponding to 9 adducts/10(9) dG.

Polyamines stimulate the formation of mutagenic 1,N2-propanodeoxyguanosine adducts from acetaldehyde.

Alcoholic beverage consumption is associated with an increased risk of upper gastrointestinal cancer. Acetaldehyde (AA), the first metabolite of ethanol, is a suspected human carcinogen, but the molecular mechanisms underlying AA carcinogenicity are unclear. In this work, we tested the hypothesis that polyamines could facilitate the formation of mutagenic alpha-methyl-gamma-hydroxy-1,N2-propano-2'-deoxyguanosine (Cr-PdG) adducts from biologically relevant AA concentrations. We found that Cr-PdG adducts could be formed by reacting deoxyguanosine with muM concentrations of AA in the presence of spermidine, but not with either AA or spermidine alone. The identities of the Cr-PdG adducts were confirmed by both liquid and gas chromatography-mass spectrometry. Using a novel isotope-dilution liquid chromatography-mass spectrometry assay, we found that in the presence of 5 mM spermidine, AA concentrations of 100 microM and above resulted in the formation of Cr-PdG in genomic DNA. These AA levels are within the range that occurs in human saliva after alcoholic beverage consumption. We also showed that spermidine directly reacts with AA to generate crotonaldehyde (CrA), most likely via an enamine aldol condensation mechanism. We propose that AA derived from ethanol metabolism is converted to CrA by polyamines in dividing cells, forming Cr-PdG adducts, which may be responsible for the carcinogenicity of alcoholic beverage consumption.