Endogenous Adducts Research Articles

Abstract 6235: Detection of γ-OHPdG in circulating liver cells and its potential use as a prognostic biomarker in patients with hepatocellular carcinoma recurrence

Abstract Background: Primary liver cancer is the fourth most common cause of cancer-related death. Resection is the most widely used potentially curative treatment for patients with early-stage hepatocellular carcinoma (HCC). Recurrence within 2 years occurs in 30-50% of patients and is a major cause of mortality. A biomarker to predict the risk of HCC and its recurrence is urgently needed. Our studies showed that hepatic levels of γ-OHPdG, an endogenous adduct from lipid peroxidation, closely correlate with hepatocarcinogenesis in various independent animal models, including an obesity model. Importantly, we observed that higher levels of γ-OHPdG in liver specimens from HCC patients who underwent surgical resection were strongly associated with poor survival (p < 0.0001) as well as with poor recurrence-free survival (RFS) (p = 0.007). These finding suggest that γ-OHPdG may serve as a prognostic biomarker of HCC and its recurrence. A non-invasive method to detect γ-OHPdG is highly desirable. There is a surge of interest in using circulating liver cells (CLCs) from patient blood to diagnose cancer stages and predict treatment outcomes, metastatic potential, and recurrence. We developed a non-invasive method for detecting and quantifying γ-OHPdG using CLCs from patient blood. Method: The method was developed by following steps: (i) isolate CLCs from blood samples using a RosetteSep™ Human CD45 Depletion Cocktail and Ficoll Paque, (ii) detect γ-OHPdG by immunocytochemistry, (iii) quantify the images of γ-OHPdG-positive stained CLCs, and (iv) compare with that found in liver biopsies by immunohistochemistry. Results: To determine the specificity of the anti-γ-OHPdG antibody developed previously in our laboratory, we showed that the proportion of γ-OHPdG-positively stained cells and staining intensity increased in HepG2 liver cancer cells upon acrolein treatment. Liver cells were co-stained with asialoglycoprotein receptor 1 (ASGPR1), a cell surface protein expressed solely on the surface of hepatic cells. CLC γ-OHPdG was quantified using Metamorph (BioVision)-based journal software, developed to quantify the percentage of ASGPR1-stained cells that also stain positively, and with what level of intensity, for γ-OHPdG. We then applied this method to 35 HCC patients’ blood samples. This method detects the number of ASGPR1 stained CLC and determines the cellular localization of γ-OHPdG, allowing the quantification of γ-OHPdG in these cells. While the nuclear γ-OHPdG reflects DNA damage, adducts in both the nucleus and the cytosol will be evaluated against IHC staining using biopsies and other clinical endpoints, such as disease stage and recurrence. Conclusion: The CLC method descried above for detecting and quantifying γ-OHPdG can be potentially used to validate this adduct as a prognostic biomarker for predicting HCC risk and recurrence in clinical trials. Citation Format: Monika Aggarwal, Zizhao Zhu, Sophie Gould, Peter Johnson, Samira Beheshtian, Hongbin Fang, Bhaskar Kallakury, Susette C. Mueller, Aiwu Ruth He, Fung-Lung Chung. Detection of γ-OHPdG in circulating liver cells and its potential use as a prognostic biomarker in patients with hepatocellular carcinoma recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6235.

Molecular Dosimetry of DNA Adducts in Rats Exposed to Vinyl Acetate Monomer.

Vinyl acetate monomer (VAM) is heavily used to synthesize polymers. Previous studies have shown that inhaled VAM, being metabolized to acetaldehyde, may form DNA adducts including N2-ethylidene-deoxyguanosine (N2-EtD-dG), which may subsequently cause mutations and contribute to its carcinogenesis. Currently, there is little knowledge on the molecular dosimetry between VAM exposure and DNA adducts under dosages relevant to human exposure. In this study, 0.02, 0.1, 1, 10, 50, 200, and 600 ppm VAM were exposed to rats by inhalation for 14 days (6 h/day). The use of [13C2]-VAM allows unambiguous differentiation and quantification of the exogenous and endogenous N2-EtD-dG by highly sensitive LC-MS/MS. Our data indicate that VAM-induced exogenous DNA adducts were formed in a non-linear manner. Exogenous DNA adducts were only detected in the nasal epithelium of rats exposed to 10, 50, 200, and 600 ppm VAM, whereas endogenous adducts were found in all nasal and other tissues analyzed. In addition, ratios of exogenous/endogenous DNA adducts were less than 1 with the dose up to 50 ppm, indicating that endogenous DNA adducts are predominant at low VAM concentrations. Moreover, differential dose-response in terms of exogenous DNA adduct formation were observed between nasal respiratory and olfactory epithelium. Furthermore, the lack of exogenous DNA adducts in distant tissues, including peripheral blood mononuclear cells, liver, brain, and bone marrow, indicates that VAM and/or its metabolite do not distribute systemically to cause DNA damage in distant tissues. Together, these results provided new molecular dosimetry to improve science-based cancer risk assessments of VAM.

N-retinylidene-N-retinylethanolamine adduct induces expression of chronic inflammation cytokines in retinal pigment epithelium cells

Blindness due to photoreceptor degeneration is observed in both genetic and acquired eye disorders. Long blue light exposure can contribute to increase levels of oxidative compounds within the retinal pigment epithelium (RPE), enhancing risk of retinal damage. In retina, reactive oxygen species contribute to the activation of inflammatory cascade. If chronic, this inflammatory response can result in photoreceptor death. Therefore, we investigated the effects of the endogenous adduct N-retinylidene-N-retinylethanolamine (A2E) on RPE cells, in order to identify the most dysregulated cytokines and their related inflammatory pathways. RPE cells were exposed to A2E and blue light for 3h and 6h. By transcriptome analysis, we identified differentially expressed genes in A2E-treated cells, when compared to untreated ones. Expression values were quantified by the Limma R package. Enrichment analysis was performed according to the “Reactome” and the Gene Ontology databases. Expression of pro-inflammatory cytokines increased after 3h of A2E treatment and pathways related to IL-6 and IL-1 signaling resulted enriched. Also the up-regulation of genes having a protective role against inflammation was observed. Moreover, our results show that ferroptosis could contribute to RPE degeneration induced by A2E and blue light. Dysregulated genes related to retinal degeneration triggered by oxidative damage and inflammatory response activation identified in this study can be considered as potential biomarkers for targeted therapies.

Expression of Pro-Angiogenic Markers Is Enhanced by Blue Light in Human RPE Cells.

Inherited retinal dystrophies are characterized by photoreceptor death. Oxidative stress usually occurs, increasing vision loss, and oxidative damage is often reported in retinitis pigmentosa (RP). More than 300 genes have been reported as RP causing. In contrast, choroidal neovascularization (CNV) only occasionally develops in the late stages of RP. We herein study the regulation of RP causative genes that are likely linked to CNV onset under oxidative conditions. We studied how the endogenous adduct N-retinylidene-N-retinylethanolamine (A2E) affects the expression of angiogenic markers in human retinal pigment epithelium (H-RPE) cells and a possible correlation with RP-causing genes. H-RPE cells were exposed to A2E and blue light for 3 and 6h. By transcriptome analysis, genes differentially expressed between A2E-treated cells and untreated ones were detected. The quantification of differential gene expression was performed by the Limma R package. Enrichment pathway analysis by the FunRich tool and gene prioritization by ToppGene allowed us to identify dysregulated genes involved in angiogenesis and linked to RP development. Two RP causative genes, AHR and ROM1, can be associated with an increased risk of CNV development. Genetic analysis of RP patients affected by CNV will confirm this hypothesis.

A Kinetic Analysis of DNA-Deoxy Guanine Adducts in the Nasal Epithelium Produced by Inhaled Formaldehyde in Rats-Assessing Contributions to Adduct Production From Both Endogenous and Exogenous Sources of Formaldehyde.

Although formaldehyde is a normal constituent of tissues, lifetime inhalation exposures at 6 h/day, 5 days/week at concentrations ≥6 ppm caused a nonlinear increase in nasal tumors in rats with incidence reaching close to 50% at 15 ppm. Studies with heavy isotope labeled [13CD2]-formaldehyde permit quantification of both the mass-labeled exogenous and endogenous DNA-formaldehyde reaction products. An existing pharmacokinetic model developed initially to describe 14C-DNA-protein crosslinks (DPX) provided a template for describing the time course of mass-labeled adducts. Published datasets included both DPX and N2-HO13CD2-dG adducts measured after a single 6-h exposure to 0.7, 2, 6, 9, 10, or 15 ppm formaldehyde, after multi-day exposures to 2 ppm for 6 h/day, 7 days/week with interim sacrifices up to 28 days, and after 28-day exposures for 6 h/day, 7 days/week to 0.3, 0.03, or 0.001 ppm. The existing kinetic model overpredicted endogenous adducts in the nasal epithelium after 1-day [13CD2]-formaldehyde exposure, requiring adjustment of parameters for rates of tissue metabolism and background formaldehyde. After refining tissue formaldehyde parameters, we fit the model to both forms of adducts by varying key parameters and optimizing against all 3 studies. Fitting to all these studies required 2 nonlinear pathways—one for high-exposure saturation of clearance in the nasal epithelial tissues and another for extracellular clearance that restricts uptake into the epithelial tissue for inhaled concentrations below 0.7 ppm. This refined pharmacokinetic model for endogenous and exogenous formaldehyde acetal adducts can assist in updating biologically based dose-response models for formaldehyde carcinogenicity.

Targeted High Resolution LC/MS3 Adductomics Method for the Characterization of Endogenous DNA Damage.

DNA can be damaged through covalent modifications of the nucleobases by endogenous processes. These modifications, commonly referred to as DNA adducts, can persist and may lead to mutations, and ultimately to the initiation of cancer. A screening methodology for the majority of known endogenous DNA adducts would be a powerful tool for investigating the etiology of cancer and for the identification of individuals at high-risk to the detrimental effects of DNA damage. This idea led to the development of a DNA adductomic approach using high resolution data-dependent scanning, an extensive MS2 fragmentation inclusion list of known endogenous adducts, and neutral loss MS3 triggering to profile all DNA modifications. In this method, the detection of endogenous DNA adducts is performed by observation of their corresponding MS3 neutral loss triggered events and their relative quantitation using the corresponding full scan extracted ion chromatograms. The method's inclusion list consists of the majority of known endogenous DNA adducts, compiled, and reported here, as well as adducts specific to tobacco exposure included to compare the performance of the method with previously developed targeted approaches. The sensitivity of the method was maximized by reduction of extraneous background signal through the purification and minimization of the amount of commercially obtained enzymes used for the DNA hydrolysis. In addition, post-hydrolysis sample purification was performed using off-line HPLC fraction collection to eliminate the highly abundant unmodified bases, and to avoid introduction of plasticizers found in solid-phase extraction cartridges. Also, several instrument parameters were evaluated to optimize the ion signal intensities and fragmentation spectra quality. The method was tested on an animal model of lung carcinogenesis where A/J mice were exposed to the tobacco specific lung carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) with its effects enhanced by co-exposure to the pro-inflammatory agent lipopolysaccharide (LPS). Lung DNA were screened for endogenous DNA adducts known to result from oxidative stress and LPS-induced lipid peroxidation, as well as for adducts due to NNK exposure. The relative quantitation of the detected DNA adducts was performed using parallel reaction monitoring MS2 analysis, demonstrating a general workflow for analysis of endogenous DNA adducts.

Evaluation of inhaled low-dose formaldehyde-induced DNA adducts and DNA-protein cross-links by liquid chromatography-tandem mass spectrometry.

As a widespread industrial chemical, formaldehyde carcinogenicity has been highly controversial. Meanwhile, formaldehyde is an essential metabolite in all living cells. Previously, we have demonstrated exogenous formaldehyde causes DNA adducts in a nonlinear manner between 0.7 and 15.2ppm using [13CD2]-formaldehyde for exposure coupled with the use of sensitive mass spectrometry. However, the responses from exposure to low doses of formaldehyde are still unknown. In this study, rats were exposed to 1, 30, and 300ppb [13CD2]-formaldehyde for 28days (6h/day) by nose-only inhalation, followed by measuring DNA mono-adduct (N2-HOMe-dG) and DNA-protein crosslinks (dG-Me-Cys) as formaldehyde specific biomarkers. Both exogenous and endogenous DNA mono-adducts and dG-Me-Cys were examined with ultrasensitive nano-liquid chromatography-tandem mass spectrometry. Our data clearly show that endogenous adducts are present in all tissues analyzed, but exogenous adducts were not detectable in any tissue samples, including the most susceptible nasal epithelium. Moreover, formaldehyde exposure at 1, 30 and 300ppb did not alter the levels of endogenous formaldehyde-induced DNA adducts or DNA-protein crosslinks. The novel findings from this study provide new data for risk assessment of exposure to low doses of formaldehyde.

Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage.

Formaldehyde is not only a widely used chemical with well-known carcinogenicity but is also a normal metabolite of living cells. It thus poses unique challenges for understanding risks associated with exposure. N(2-)hydroxymethyl-dG (N(2)-HOMe-dG) is the main formaldehyde-induced DNA mono-adduct, which together with DNA-protein crosslinks (DPCs) and toxicity-induced cell proliferation, play important roles in a mutagenic mode of action for cancer. In this study, N(2)-HOMe-dG was shown to be an excellent biomarker for direct adduction of formaldehyde to DNA and the hydrolysis of DPCs. The use of inhaled [(13)CD2]-formaldehyde exposures of rats and primates coupled with ultrasensitive nano ultra performance liquid chromatography-tandem mass spectrometry permitted accurate determinations of endogenous and exogenous formaldehyde DNA damage. The results show that inhaled formaldehyde only reached rat and monkey noses, but not tissues distant to the site of initial contact. The amounts of exogenous adducts were remarkably lower than those of endogenous adducts in exposed nasal epithelium. Moreover, exogenous adducts accumulated in rat nasal epithelium over the 28-days exposure to reach steady-state concentrations, followed by elimination with a half-life (t1/2) of 7.1 days. Additionally, we examined artifact formation during DNA preparation to ensure the accuracy of nonlabeled N(2)-HOMe-dG measurements. These novel findings provide critical new data for understanding major issues identified by the National Research Council Review of the 2010 Environmental Protection Agency's Draft Integrated Risk Information System Formaldehyde Risk Assessment. They support a data-driven need for reflection on whether risks have been overestimated for inhaled formaldehyde, whereas underappreciating endogenous formaldehyde as the primary source of exposure that results in bone marrow toxicity and leukemia in susceptible humans and rodents deficient in DNA repair.

Dosimetry ofN6-Formyllysine Adducts Following [13C2H2]-Formaldehyde Exposures in Rats

With formaldehyde as the major source of endogenous N6-formyllysine protein adducts, we quantified endogenous and exogenous N6-formyllysine in the nasal epithelium of rats exposed by inhalation to 0.7, 2, 5.8, and 9.1 ppm [13C2H2]-formaldehyde using liquid chromatography-coupled tandem mass spectrometry. Exogenous N6-formyllysine was detected in the nasal epithelium, with concentration-dependent formation in total as well as fractionated (cytoplasmic, membrane, nuclear) proteins, but was not detected in the lung, liver, or bone marrow. Endogenous adducts dominated at all exposure conditions, with a 6 h 9.1 ppm formaldehyde exposure resulting in one-third of the total load of N6-formyllysine being derived from exogenous sources. The results parallel previous studies of formaldehyde-induced DNA adducts.

Biomarkers of exposure and effect in human lymphoblastoid TK6 cells following [13C2]-acetaldehyde exposure.

The dose-response relationship for biomarkers of exposure (N(2)-ethylidene-dG adducts) and effect (cell survival and micronucleus formation) was determined across 4.5 orders of magnitude (50nM-2mM) using [(13)C2]-acetaldehyde exposures to human lymphoblastoid TK6 cells for 12h. There was a clear increase in exogenous N (2)-ethylidene-dG formation at exposure concentrations ≥ 1µM, whereas the endogenous adducts remained nearly constant across all exposure concentrations, with an average of 3.0 adducts/10(7) dG. Exogenous adducts were lower than endogenous adducts at concentrations ≤ 10µM and were greater than endogenous adducts at concentrations ≥ 250µM. When the endogenous and exogenous adducts were summed together, statistically significant increases in total adduct formation over the endogenous background occurred at 50µM. Cell survival and micronucleus formation were monitored across the exposure range and statistically significant decreases in cell survival and increases in micronucleus formation occurred at ≥ 1000µM. This research supports the hypothesis that endogenously produced reactive species, including acetaldehyde, are always present and constitute the majority of the observed biological effects following very low exposures to exogenous acetaldehyde. These data can replace default assumptions of linear extrapolation to very low doses of exogenous acetaldehyde for risk prediction.

Quantitative changes in endogenous DNA adducts correlate with conazole in vivo mutagenicity and tumorigenicity

The mouse liver tumorigenic conazole fungicides triadimefon and propiconazole have previously been shown to be in vivo mouse liver mutagens in the Big Blue™ transgenic mutation assay when administered in feed at tumorigenic doses, whereas the nontumorigenic conazole myclobutanil was not mutagenic. DNA sequencing of the mutants recovered from each treatment group as well as from animals receiving control diet revealed that propiconazole- and triadimefon-induced mutations do not represent general clonal expansion of background mutations, and support the hypothesis that they arise from the accumulation of endogenous reactive metabolic intermediates within the liver in vivo. We therefore measured the spectra of endogenous DNA adducts in the livers of mice from these studies to determine if there were quantitative or qualitative differences between mice receiving tumorigenic or nontumorigenic conazoles compared to concurrent control animals. We resolved and quantitated 16 individual adduct spots by (32)P postlabelling and thin layer chromatography using three solvent systems. Qualitatively, we observed the same DNA adducts in control mice as in mice receiving conazoles. However, the 13 adducts with the highest chromatographic mobility were, as a group, present at significantly higher amounts in the livers of mice treated with propiconazole and triadimefon than in their concurrent controls, whereas this same group of DNA adducts in the myclobutanil-treated mice was not different from controls. This same group of endogenous adducts were significantly correlated with mutant frequency across all treatment groups (P = 0.002), as were total endogenous DNA adduct levels (P = 0.005). We hypothesise that this treatment-related increase in endogenous DNA adducts, together with concomitant increases in cell proliferation previously reported to be induced by conazoles, explain the observed increased in vivo mutation frequencies previously reported to be induced by treatment with propiconazole and triadimefon.

Disruption of mitochondrial DNA polymerase γ replication by the aldehyde adduct of DNA, M1dG

M1dG is a mutagenic adduct generated in human nuclear and mitochondrial DNA (mtDNA) by reaction of the cellular peroxidation products, malondialdehyde and base propenal, with guanine. Since M1dG is known to interfere with DNA synthesis by replicative and Y‐family DNA polymerases, we have utilized recombinant exonuclease‐deficient human DNA polymerase γ (exo− pol γ) with or without its accessory subunit p55 to investigate the effects of the adduct on mtDNA synthesis. Using primer‐templates that required exo−pol γ to extend the primer by insertion opposite the adduct position, we found that M1dG decreased exo− pol γ primer extension by 50% and prevented synthesis of a fully‐extended 35‐mer primer. A dAMP was inserted preferentially opposite M1dG, but extension of the primer past the adduct was supported only by dCMP incorporation. The insertion efficiency for exo− pol γ with either dCTP or dATP as substrates was substantially reduced by M1dG, and p55 had no effect on exo−pol γ nucleotide selection or insertion kinetics at the adduct. Since human mitochondria have no known mechanism for repair of M1dG, which is a target of nucleotide excision repair in the nucleus, our data indicate that the endogenous adduct poses a significant threat to mtDNA stability by disrupting DNA pol γ replication. (Supported by National Institutes of Health Grants GM087681 to S.D.C. and CA087819 to L.J.M, and NIH intramural funding to W.C.C.)

Determination of N2-Hydroxymethyl-dG Adducts in the Nasal Epithelium and Bone Marrow of Nonhuman Primates Following 13CD2-Formaldehyde Inhalation Exposure

The presence of endogenous and exogenous N(2)-hydroxymethyl-dG adducts in DNA from the nasal mucosa and bone marrow of cynomolgus macaques exposed to 1.9 and 6.1 ppm of [(13)CD(2)]-formaldehyde for 6 h a day for 2 consecutive days was investigated using a highly sensitive nano-UPLC-MS/MS method with a limit of detection of 20 amol. Both exogenous and endogenous adducts were readily detected and quantified in the nasal tissues of both exposure groups, with an exposure dependent increase in exogenous adducts observed. In contrast, only endogenous adducts were detectable in the bone marrow, even though ∼10 times more DNA was analyzed.

Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment.

There is a strong need for science-based risk assessment that utilizes known data from diverse sources to arrive at accurate assessments of human health risk. Such assessments will protect the public health without mandating unreasonable regulation. This paper utilizes 30 years of research on three "known human carcinogens": formaldehyde, vinyl chloride (VC), and ethylene oxide (EO), each of which forms DNA adducts identical to endogenous DNA adducts in all individuals. It outlines quantitative data on endogenous adducts, mutagenicity, and relationships between endogenous and exogenous adducts. Formaldehyde has the richest data set, with quantitative data on endogenous and exogenous DNA adducts from the same samples. The review elaborates on how such data can be used to inform the current risk assessment on formaldehyde, including both the biological plausibility and accuracy of projected risks. Finally, it extends the thought process to VC, EO, and additional areas of potential research, pointing out needs, nuances, and potential paths forward to improved understanding that will lead to strong science-based risk assessment.

Secondary oxidation of cyclic 1,N 2-propano and 1,N 2-etheno-2′-deoxyguanosine DNA adducts. Consequences in oxidative stress biomarker development

This work is an attempt to investigate the chemical stability of 1,N 2-propano-2′-deoxyguanosine (pdG-HNE) and 1,N 2-etheno-2′-deoxyguanosine (εdG) DNA adducts against hydrolysis and upon oxidation reactions. It includes both kinetic issues together with proposed degradation pathways. While both chemicals are stable in the 3.5–9 pH range, the results suggest that pdG-HNE adduct is less prone to in vitro oxidative transformation than εdG adduct. εdG and pdG-HNE behave differently upon hydroxyl radical and one electron oxidation reactions. The exocyclic ring of εdG is mainly affected by oxidative processes leading to the regeneration of 2′-deoxyguanosine (dG) while the integrity of the exocyclic ring is preserved for pdG-HNE. Consequently, pdG-HNE might be a better biomarker than εdG for monitoring oxidative stress during environmental or occupational exposures to chemicals. Understanding the in vitro routes of etheno and propano DNA adduct degradation would probably help to guide the development of analytical methodologies for the reliable detection of these endogenous adducts.

Dose-Response Relationships for N7-(2-Hydroxyethyl)Guanine Induced by Low-Dose [14C]Ethylene Oxide: Evidence for a Novel Mechanism of Endogenous Adduct Formation

Ethylene oxide (EO) is widely used in the chemical industry and is also formed in humans through the metabolic oxidation of ethylene, generated during physiologic processes. EO is classified as a human carcinogen and is a direct acting alkylating agent, primarily forming N7-(2-hydroxyethyl)guanine (N7-HEG). To conduct accurate human risk assessments, it is vital to ascertain the relative contribution of endogenously versus exogenously derived DNA damage and identify the sources of background lesions. We have therefore defined in vivo dose-response relationships over a concentration range relevant to human EO exposures using a dual-isotope approach. By combining liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography-accelerator mass spectrometry analysis, both the endogenous and exogenous N7-HEG adducts were quantified in tissues of [(14)C]EO-treated rats. Levels of [(14)C]N7-HEG induced in spleen, liver, and stomach DNA increased in a linear manner from 0.002 to 4 adducts/10(8) nucleotides. More importantly, the extent of damage arising through this route was insignificant compared with the background abundance of N7-HEG naturally present. However, at the two highest doses, [(14)C]EO exposure caused a significant increase in endogenous N7-HEG formation in liver and spleen, suggesting that EO can induce physiologic pathways responsible for ethylene generation in vivo and thereby indirectly promote N7-HEG production. We present evidence for a novel mechanism of adduct formation to explain this phenomenon, involving oxidative stress and 1-aminocyclopropane-1-carboxylic acid as a potential biosynthetic precursor to ethylene in mammalian cells. Based on the proposed pathway, N7-HEG may have potential as a biomarker of cellular oxidative stress.

Endogenous Lipid Hydroperoxide-mediated DNA-adduct Formation in Min Mice

Despite intensive research over the last two decades, there are still no specific markers of endogenous lipid hydroperoxide-mediated DNA damage. We recently demonstrated that heptanone-etheno-2'-deoxyguanosine adducts are formed in the DNA of rat intestinal epithelial cells that stably express cyclooxygenase-2. Heptanone-etheno adducts can only arise from the reaction of lipid hydroperoxide-derived 4-oxo-2(E)-nonenal with DNA. This raised the possibility that similar adducts would be formed in vivo in settings where cyclooxygenase-2 expression is increased. Therefore, DNA-adduct formation was studied in C57BL/6JAPC(min) mice, a colorectal cancer mouse model in which cyclooxygenase-2 is up-regulated. 15(S)-Hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid is the major lipid hydroperoxide produced endogenously by cyclooxygenase-2. It undergoes homolytic decomposition to the DNA-reactive bifunctional electrophile 4-oxo-2(E)-nonenal, which forms heptanone-etheno adducts with DNA. A quantitative comparison was made of the heptanone-etheno-DNA adducts present in C57BL/6J and C57BL/6JAPC(min) mice. Using highly specific and sensitive methodology based on stable isotope dilution liquid chromatography/tandem mass spectrometry, we have detected the endogenous formation of heptanone-etheno adducts in mammalian tissue DNA for the first time. In addition, we found that there were statistically significant increased levels of the heptanone-etheno-2'-deoxyguanosine and heptanone-etheno-2'-deoxycytidine adducts in the C57BL/6JAPC(min) mice when compared with the control C57BL/6J mice.

Genotoxic potential of Microcystin‐LR and nodularin in vitro in primary cultured rat hepatocytes and in vivo in rat liver

Microcystin-LR (MCYST-LR) and nodularin (NOD) are known as tumor promoters in experimental animals and so present potential health threats for humans. Although their hepatotoxic mechanisms have been very well documented, many other effects of these toxins are relatively undescribed, indeed controversial, notably those related to their genotoxicity. In the present investigation, we examined how these toxins could induce DNA damage using a combination of in vitro and in vivo approaches. We first used the (32)P-postlabeling assay to test hydrophobic adduct formation on DNA from primary cultured rat hepatocytes treated with noncytotoxic concentrations of MCYST-LR and NOD (2 and 10 ng/mL). Analysis of the autoradiograms of DNA digests isolated from the hepatocytes did not show any hydrophobic DNA adduct formation. However, these toxins significantly decreased the amount of hydrophobic endogenous adducts, termed I compounds. We next investigated oxidative DNA damage by using the (32)P-postlabeling assay to analyze 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) content as a biomarker of possible DNA lesions. Both MCYST-LR and NOD significantly enhanced 8-oxo-dG in time- and dose-dependent manner in vitro in primary cultured hepatocytes and in vivo in rat liver cells. Thus, it appears that the depletion of endogenous DNA adducts (I compounds) and/or the increase of 8-oxo-dG levels by MCYST-LR and NOD could be involved in the formation of hepatic tumors during long-term exposure to these cyanobacterial hepatotoxins.

Exocyclic malondialdehyde and aromatic DNA adducts in larynx tissues

Cigarette smoking and alcohol consumption, known to cause free radical generation and lipid peroxidation, are established risk factors for larynx cancer. Malondialdehyde (MDA) is a naturally occurring product of lipid peroxidation, capable of interacting with DNA to form exocyclic MDA–DNA adducts. In the present study, we investigated if the production of MDA–DNA adducts was increased in larynx cancer patients with respect to controls using 32P-DNA postlabeling techniques. Moreover, we examined the potential effects of cigarette smoking and alcohol consumption on endogenous DNA adducts. We then analyzed the same set of larynx tissues for the presence of 32P-postlabeled aromatic DNA adducts to determine more about the levels and types of adducts formed in the larynx. We observed that cancer patients tended to have increased levels of MDA and aromatic DNA adducts with respect to controls. In addition, smoking and alcohol were found to influence the formation of endogenous adducts in the larynx tissues. Finally, the amounts of endogenous adducts were found to be comparable to those observed for aromatic DNA adducts in the same set of larynx tissues. These findings imply that endogenous lesions, if not repaired, may contribute to larynx cancer development.

Ascorbic acid and glutathione modulate the biological activity of S-nitrosoglutathione.

Ascorbic acid and glutathione (GSH) are important determinants of the intracellular redox state, and both are known to accelerate the decomposition of S-nitrosoglutathione (GSNO), an endogenous adduct of nitric oxide (NO). The implications of these observations for GSNO bioactivity are not yet clear. We investigated the effect of ascorbic acid and GSH on GSNO bioactivity by using a bioassay with isolated segments of guinea pig aorta suspended in organ chambers. Arterial segments demonstrated relaxation to GSNO (0.1 micromol/L) that was significantly enhanced by 300 micromol/L ascorbic acid (71+/-6% versus 53+/-6%, P<0.05) but not GSH. Both ascorbic acid and GSH significantly shortened the duration of arterial relaxation in response to 0.1 micromol/L GSNO (from >120 minutes to 22.5+/-3.5 and 36.3+/-4.3 minutes, respectively; P<0.05), consistent with accelerated decomposition of GSNO that was confirmed spectrophotometrically. The effect of ascorbic acid was abrogated by either DTPA or the copper(I)-specific agent bathocuproine but not deferoxamine, indicating a dependence on the availability of redox-active copper. Consistent with this notion, the action of ascorbic acid on GSNO bioactivity was also supported by copper-zinc superoxide dismutase, a physiologically relevant source of copper. In contrast, the effect of GSH on GSNO degradation and GSNO-mediated arterial relaxation was independent of transition metal ions, because DTPA had no effect. These data indicate that both ascorbic acid and GSH modulate GSNO bioactivity and suggest a distinction between the mechanism of GSNO degradation by ascorbic acid or GSH. Whereas both ascorbic acid and GSH accelerate the degradation of GSNO, only ascorbic acid is dependent on the presence of transition metal ions.