Raghu G. Nath

Glutathione depletion enhances the formation of endogenous cyclic DNA adducts derived from t-4-hydroxy-2-nonenal in rat liver.

Earlier, we detected the cyclic adducts of deoxyguanosine (dG) derived from t-4-hydroxy-2-nonenal (HNE), a long chain alpha,beta-unsaturated aldehyde (enal) product from oxidation of omega-6 polyunsaturated fatty acids, in tissue DNA of rats and humans as endogenous DNA damage. Recent evidence implicates the cyclic HNE adducts in human liver carcinogenesis. Because glutathione (GSH) protects against oxidative stress, we undertook a study to examine the effect of GSH depletion on the HNE-derived cyclic adducts in vivo. Four F344 rats were administered L-buthionine-(S,R)-sulfoximine (BSO), a potent inhibitor of GSH biosynthesis, at 10 mM in drinking water for 2 weeks. Rats in the control group were given water only. Livers were harvested, and each liver was divided into portions for GSH and DNA adduct analyses. The BSO treatment depleted hepatic GSH by 77%; the GSH levels were reduced from 6.3 +/- 0.3 in the control rats to 1.5 +/- 0.1 micromol/g tissues in the treated group. The formation of HNE-dG adducts, analyzed by an HPLC-based 32P-postlabeling assay, was increased by 4-fold, from 6.2 +/- 2.2 nmol/mol dG in liver DNA of control rats to 28.5 +/- 16.1 nmol/mol dG in the rats treated with BSO (p <0.05). The formation of 8-oxodG in liver DNA was also increased as a result of BSO treatment, although the increase was not statistically significant. These results further support the endogenous origin of HNE-dG adducts and, more importantly, indicate a critical role that GSH plays in protecting against in vivo formation of the promutagenic cyclic DNA adducts derived from HNE.

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.

Effects of Epigallocatechin Gallate, L-Ascorbic Acid, α-Tocopherol, and Dihydrolipoic Acid on the Formation of Deoxyguanosine Adducts Derived From Lipid Peroxidation

Oxidation of polyunsaturated fatty acids (PUFAs) releases α,β-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 α-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 α-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.

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.

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).

Abstract 2738: Mechanisms of cadmium carcinogenesis

Cadmium is a transition metal and an ubiquitous environmental and industrial pollutant. Laboratory animal studies and epidemiological studies have shown that exposure to cadmium is associated with various organ toxicities and carcinogenic effects. Several national and international regulatory agencies have classified cadmium compounds as carcinogenic to humans. In 2012, International Agency for Research on Cancer (IARC) reviewed the relevant information on cadmium exposure and concluded that there is sufficient evidence in humans for the carcinogenicity of cadmium and its compounds. However, the underlying molecular mechanism(s), especially for the carcinogenicity, are unknown. The mechanisms proposed for carcinogenicity include aberrant gene expression, induction of oxidative DNA damage, stimulation of cell proliferation, malignant cell transformation, inhibition of DNA damage repair, inhibition of apoptosis and epigenetic mechanisms including alteration of DNA methylation. Studies have shown that due to the slow elimination, cadmium is a cumulative toxicant and past exposures could result in toxic effect of the residual metal. To better understand the mechanisms of cadmium carcinogenicity we have reviewed in vitro experimental and in vivo animal studies focused on the proposed mechanisms of carcinogenicity as well as epidemiological evidence on carcinogenicity in lung and kidney following cadmium exposure. Disclaimer: The views expressed are those of the authors and do not necessarily represent the views and/or policies of the U.S. EPA Citation Format: Raghu G. Nath, BR Sonawane, SV Vulimiri, YS Lin. Mechanisms of cadmium carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2738. doi:10.1158/1538-7445.AM2015-2738