DNA Adducts Research Articles

Bulky Adducts in Clustered DNA Lesions: Causes of Resistance to the NER System.

The nucleotide excision repair (NER) system removes a wide range of bulky DNA lesions that cause significant distortions of the regular double helix structure. These lesions, mainly bulky covalent DNA adducts, are induced by ultraviolet and ionizing radiation or the interaction between exogenous/endogenous chemically active substances and nitrogenous DNA bases. As the number of DNA lesions increases, e.g., due to intensive chemotherapy and combination therapy of various diseases or DNA repair impairment, clustered lesions containing bulky adducts may occur. Clustered lesions are two or more lesions located within one or two turns of the DNA helix. Despite the fact that repair of single DNA lesions by the NER system in eukaryotic cells has been studied quite thoroughly, the repair mechanism of these lesions in clusters remains obscure. Identification of the structural features of the DNA regions containing irreparable clustered lesions is of considerable interest, in particular due to a relationship between the efficiency of some antitumor drugs and the activity of cellular repair systems. In this review, we analyzed data on the induction of clustered lesions containing bulky adducts, the potential biological significance of these lesions, and methods for quantification of DNA lesions and considered the causes for the inhibition of NER-catalyzed excision of clustered bulky lesions.

Modeling and optimization of malondialdehyde (MDA) absorbance behavior through response surface methodology (RSM) and artificial intelligence network (AIN): An endeavor to estimate lipid peroxidation by determination of MDA

AbstractOxidative stress (OS) associated with reactive oxygen species (ROS) attacks many biomolecules, leading to cell death, lipid peroxidation (LP), and physiological mechanism damage in the human body because of its reaction with nucleotides, membrane lipids, and proteins. Malondialdehyde (MDA) is one of the end products of polyunsaturated fatty acids peroxidation. The elevated level of MDA is widely known as a biomarker for the indirect measurement of OS during LP. Although several derivatizations' approaches and successful commercial kits are available, their analytical validity and reliability in clinical studies require further attention to obtain robust quantitation and high‐quality data. Also, re‐evaluation and development of new analytical methods with high sensitivity and selectivity in response to free MDA and total MDA, DNA and protein adducts are not well‐characterized. Here, response surface methodology (RSM) and artificial neural network (ANN) were proposed to model, predict, and optimize MDA detection. This paper introduced a novel, simple, and fast modeling method using a new reagent (para methoxy aniline or PMA) for the first time to convert MDA to an adduct which could be detectable by UV‐Vis spectroscopy. Based on the interactions between MDA and PMA at times and temperatures, RSM and artificial intelligence network (AIN) were then applied to forecast the absorbance rate of MDA‐PMA. The optimum mode for the highest absorbance rate of MDA‐PMA is at the temperature of 74.99°C with a PMA concentration of 499.87 μM and MDA concentration of 49.98 μM. The best structure with five hidden layers, including 5,5,5,9,4 neurons with the transfer function logsig for each layer, was selected for the MLP‐ANN model. The maximum error rate of the MLP‐ANN model was 2.08%.Our new approach is an improvement over existing assay kits, overcoming limitations such as time, temperature, and reliability with a bright future to target increased MDA level in human samples.

Abstract A015: Age-associated molecular changes may predispose the ovary to malignant transformation leading to ovarian cancer (OVCA)

Abstract Background: Ovarian cancer (OVCA) is a fatal malignancy of women which in most cases is a disease of postmenopausal women. Thus, age-associated changes in women may predispose them to malignancy. Persistent high levels of follicle stimulating hormone (FSH) is a feature of aging and we have shown that aging was associated with increased expression of inflammatory cytokine interleukin 16 (IL-16) and glucose regulatory protein (GRP78), a marker of cellular stress. It is possible that age-associated increased expression of IL-16 and GRP78 might be involved in production of mutagenic 8-OXO-2dG or its cognate enzyme OGG1 and malondialdehyde (MDA) as well as markers of DNA repair mechanisms. The goal of this study was to determine the mechanism of age-associated malignant transformation in the ovary. Materials and Methods: Exploratory study: Archived tissue specimens were selected for immunohistochemical, immunoblotting, genomic studies and immunoassays based on the review of their hematoxylin and eosin staining and final Pathological reports. Specimens were grouped as normal premenopausal women (30-50 years old), normal postmenopausal women (55-85 years old) and ovarian high grade serous carcinoma (HGSC). In vitro study: human ovarian surface epithelium (HOSE) cells were treated with or without FSH for 24 hours, and their cytoplasmic and nuclear fractionations together with that of OVCAR3 (HGSC cell line) cells were extracted. Expression of markers of inflammation (IL-16), oxidative stress (SOD2, GRP78), DNA adducts (8-OXO-2dG/OGG1, MDA) and epigenetics (HDAC1, gH2AX) and were examined. Significant differences in expression of different markers among different age groups and ovarian HGSC, and treated or control OVCAR3 cells were determined by ANOVA and t-tests. Results: Compared with premenopausal women, expression of markers of inflammation and oxidative stress including IL-16, GRP78, SOD2, 8-OXO-2dG/OGG1, MDA as well as markers of epigenetic changes including HDAC1 and gH2AX was significantly higher (<0.0001) in a subset of postmenopausal women. Similar patterns of expression were also observed in patients with ovarian HGSC. Nuclear expression of mutagenic DNA adducts (OGG1, MDA) and inhibitor of DNA damage repair mechanism (GRP78) enhanced during aging. Thus, age-associated inflammation and oxidative stress are predispositions to malignant transformation. This assumption is based on the observation from the treatment of HOSE cells with FSH which showed increased expression of the above markers as also observed in OVCAR3 cells. Conclusion: Expression of IL-16, GRP78, SOD2, 8-OXO-2dG/OGG1, MDA and HDAC1 increased in ovary during aging. These results suggest that aging is associated with persistent inflammation and oxidative stress in ovarian tissues, which predisposes these tissues to malignant transformation leading to OVCA development. Support: NIH/NCI: CA210370 Citation Format: Jessica Ramirez, Elizabeth Paris, Sanjib Basu, Animesh Barua. Age-associated molecular changes may predispose the ovary to malignant transformation leading to ovarian cancer (OVCA) [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A015.

Nuclear Fructose-1,6-Bisphosphate Inhibits Tumor Growth and Sensitizes Chemotherapy by Targeting HMGB1.

Metabolites are important for cell fate determination. Fructose-1,6-bisphosphate (F1,6P) is the rate-limiting product in glycolysis and the rate-limiting substrate in gluconeogenesis. Here, it is discovered that the nuclear-accumulated F1,6P impairs cancer cell viability by directly binding to high mobility group box 1 (HMGB1), the most abundant non-histone chromosome structural protein with paradoxical roles in tumor development. F1,6P disrupts the association between the HMGB1 A-box and C-tail by targeting K43/K44 residues, inhibits HMGB1 oligomerization, and stabilizes P53 protein by increasing P53-HMGB1 interaction. Moreover, F1,6P lowers the affinity of HMGB1 for DNA and DNA adducts, which sensitizes cancer cells to chemotherapeutic drug(s)-induced DNA replication stress and DNA damage. Concordantly, F1,6P resensitizes cancer cells with chemotherapy resistance, impairs tumor growth and enhances chemosensitivity in mice, and impedes the growth of human tumor organoids. These findings reveal a novel role for nuclear-accumulated F1,6P and underscore the potential utility of F1,6P as a drug for cancer therapy.

Mass Spectrometry Analysis of DNA and Protein Adducts as Biomarkers in Human Exposure to Cigarette Smoking: Acrolein as an Example.

Acrolein is a major component in cigarette smoke and a product of endogenous lipid peroxidation. It is difficult to distinguish human exposure to acrolein from exogenous sources versus endogenous causes, as components in cigarette smoke can stimulate lipid peroxidation in vivo. Therefore, analysis of acrolein-induced DNA and protein adducts by the highly accurate, sensitive, and specific mass spectrometry-based methods is vital to estimate the degree of damage by this IARC Group 2A carcinogen. This Perspective reviews the analyses of acrolein-induced DNA and protein adducts in humans by mass spectrometry focusing on samples accessible for biomonitoring, including DNA from leukocytes and oral cells and abundant proteins from blood, i.e., hemoglobin and serum albumin.

A new toxicity mechanism of N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine quinone: Formation of DNA adducts in mammalian cells and aqueous organisms

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPDQ), one of the oxidation products of rubber antioxidant 6PPD, has been identified as a novel toxicant to many organisms. However, an understanding of its underlying toxicity mechanisms remained elusive. In this study, we reported that 6PPDQ could react with deoxyguanosine to form one isomer of 3-hydroxy-1, N2-6PPD-etheno-2′-deoxyguanosine (6PPDQ-dG). Next, by employing an ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method, we found that 6PPDQ-dG could be detected in genomic DNA from 6PPDQ-treated mammalian cells and Chlamydomonas reinhardtii. We observed positive correlations between concentrations of exogenous 6PPDQ and the amounts of 6PPDQ-dG, and a recovery period after removal of 6PPDQ also led to decreased levels of the adduct in both organisms, which suggested potential repair pathways for this adduct in mammalian cells and unicellular algae. Additionally, we extracted the genomic DNA from tissues of frozen capelin and observed substantial amounts of the adduct in roe and gills, as well as livers at a relatively lower level. These results provided insights into the target organs and tissues that 6PPDQ might accumulate or harm fish. Overall, our study provides a new understanding of the mechanisms of toxicity of 6PPDQ in mammalian cells and aqueous organisms.

Identification of differentially expressed genes and pathways in BEAS-2B cells upon long-term exposure to particulate matter (PM2.5) from biomass combustion using bioinformatics analysis.

Long-term exposure to PM2.5 from burning domestic substances has been linked to an increased risk of lung disease, but the underlying mechanisms are unclear. This study is to explore the hub genes and pathways involved in PM2.5 toxicity in human bronchial epithelial BEAS-2B cells. The GSE158954 dataset is downloaded from the GEO database. Differentially expressed genes (DEGs) were screened using the limma package in RStudio (version 4.2.1). In addition, DEGs analysis was performed by Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. A protein-protein interaction (PPI) network was constructed, MCODE plug-in and the cytoHubba plug-in in Cytoscape software was used to identify the hub genes. Finally, CytoHubba and DEGs were used to integrate the hub genes, and preliminary validation was performed by comparing the toxicology genomics database (CTD). Differential immune cell infiltration was investigated using the CIBERSORT algorithm. A total of 135 DEGs were identified, of which 57 were up-regulated and 78 were down-regulated. Functional enrichment analyses in the GO and KEGG indicated the potential involvement of DEGs was mainly enriched in the regulation of endopeptidase activity and influenza A. Gene Set Enrichment Analysis revealed that Chemical Carcinogenesis - DNA adducts were remarkably enriched in PM2.5 groups. 53 nodes and 198 edges composed the PPI network. Besides, 5 direct-acting genes were filtered at the intersection of cytohubba plug-in, MCODE plug-in and CTD database. There is a decreasing trend of dendritic cells resting after BEAS-2B cells long-term exposure to PM2.5. The identified DEGs, modules, pathways, and hub genes provide clues and shed light on the potential molecular mechanisms of BEAS-2B cells upon long-term exposure to PM2.5.

Chemical mechanisms of DNA damage by carcinogenic chromium(VI).

Hexavalent chromium is a firmly established human carcinogen with documented exposures in many professional groups. Environmental exposure to Cr(VI) is also a significant public health concern. Cr(VI) exists in aqueous solutions as chromate anion that is unreactive with DNA and requires reductive activation inside the cells to produce genotoxic and mutagenic effects. Reduction of Cr(VI) in cells is nonenzymatic and in vivo principally driven by ascorbate with a secondary contribution from nonprotein thiols glutathione and cysteine. In addition to its much faster rate of reduction, ascorbate-driven metabolism avoids the formation of Cr(V) which is the first intermediate in Cr(VI) reduction by thiols. The end-product of Cr(VI) reduction is Cr(III) which forms several types of Cr-DNA adducts that are collectively responsible for all mutagenic and genotoxic effects in Cr(VI) reactions with ascorbate and thiols. Some Cr(V) forms can react with H2O2 to produce DNA-oxidizing peroxo species although this genotoxic pathway is suppressed in cells with physiological levels of ascorbate. Chemical reactions of Cr(VI) with ascorbate or thiols lack directly DNA-oxidizing metabolites. The formation of oxidative DNA breaks in early studies of these reactions was caused by iron contamination. Production of Cr(III)-DNA adducts in cells showed linear dose-dependence irrespective of the predominant reduction pathway and their processing by mismatch repair generated more toxic secondary genetic lesions in euchromatin. Overall, Cr(III)-DNA adduction is the dominant pathway for the formation of genotoxic and mutagenic DNA damage by carcinogenic Cr(VI).

Quasi-intrinsic fluorescent probes for detecting the DNA adduct (ABPdG) based on an excited-state intermolecular charge transfer mechanism.

N'-(2'-Deoxyguanosin-8-yl)-4-aminobiphenyl (ABPdG) is one of the most representative carcinogenic DNA adducts formed by human exposure to 4-aminobiphenyl (4-ABP) during dye production, rubber-manufacturing processes and cigarette smoke. Accordingly, the ultrasensitive detection of ABP-derived adducts in DNA with minimal interference to the native structures becomes key for elucidating carcinogenesis mechanisms and mitigating the risk of cancer. In view of the lack of efficient optical emission in ABPG, we report a theoretical study on the photophysical properties of a set of quasi-intrinsic fluorescent C-analogues, which can form stable W-C base pairs with ABPG. It is found that fluorophore replacement and ring-expansion can bring a red-shifted absorption and bright photoluminescence due to additional π-conjugation. In particular, because the tricyclic cytosine analogue 1,3-diaza-2-oxophenoxazine (tCO) possesses distinct optical properties, it is proposed as a biosensor to identify ABPG. The TDDFT-calculated absorption maximum of tCO is red-shifted by 97 nm in comparison with that of the native C base, which contributes to selective excitation after incorporating into the nucleic acids. Although the fluorescence is insensitive to base pairing with natural guanine, the excited state intermolecular charge transfer (ESICT)-governed "OFF-ON" signal can be observed in the presence and absence of ABPG. Moreover, to evaluate the direct availability of the bright C-analogues with high selectivity for the deoxyguanosine adduct ABPG in DNA, we further investigated thoroughly the effects of its linking to deoxyribose on its absorption and emission, which shows little difference from that of experiment.

Genetic and epigenetic instability induced by betel quid associated chemicals.

Over the years, betel quid chewing and tobacco use have attracted considerable interest as they are implicated as the most likely causative risk factors of oral and esophageal cancers. Although areca nut use and betel quid chewing may lead to apoptosis, chronic exposure to areca nut and slaked lime may promote pre-malignant and malignant transformation of oral cells. The putative mutagenic and carcinogenic mechanisms may involve endogenous nitrosation of areca and tobacco alkaloids as well as the presence of direct alkylating agents in betel quid and smokeless tobacco. Metabolic activation of carcinogenic N-nitrosamines by phase-I enzymes is required not only to elicit the genotoxicity via the reactive intermediates but also to potentiate the mutagenicity with the sporadic alkylations of nucleotide bases, resulting in the formation of diverse DNA adducts. Persistent DNA adducts provides the impetus for genetic and epigenetic lesions. The genetic and epigenetic factors cumulatively influence the development and progression of disorders such as cancer. Accumulation of numerous genetic and epigenetic aberrations due to long-term betel quid (with or without tobacco) chewing and tobacco use culminates into the development of head and neck cancers. We review recent evidence that supports putative mechanisms for mutagenicity and carcinogenicity of betel quid chewing along with tobacco (smoking and smokeless) use. The detailed molecular mechanisms of the extent of accumulation and patterns of genetic alterations, indicative of the prior exposure to carcinogens and alkylating agents because of BQ chewing and tobacco use, have not yet been elucidated.

Abstract C030: Relationship between the oral microbiome and tobacco-induced DNA damage in African American and White persons who smoke

Abstract Exposure to tobacco smoke chemicals and toxicants may cause unique changes to an individual’s oral microbiome, impacting immuno-inflammatory responses, the metabolism of tobacco smoke carcinogens, and overall health status. In our previous study, HPB-releasing DNA adducts (DNA damage derived from tobacco-specific nitrosamines) were higher in oral cells of African American (AA, n=75) when compared to White (WH, n=71) persons who smoke, even after adjusting for key confounders such as smoking dose. In this study, we examined the relationship between the levels of HPB-releasing DNA adducts and microbiome composition in these individuals. 16S rRNA gene sequencing, targeting the V4 hypervariable region, was performed on DNA isolated from mouthwash samples. Sequences were clustered into Amplicon Sequence Variants (ASV) using QIIME, and metagenomic content was inferred using PICRUSt. Our results showed a significant difference in alpha-diversity metrics: observed richness and Shannon indexes (Kruskal-Wallis test, H = 8.42, p <0.01 and H = 6.85, p <0.03, respectively) across levels of DNA damage (low, intermediate, and high). Post hoc tests with pairwise comparisons showed that, for both indexes, individuals with high levels of HPB-releasing DNA adducts had significantly lower alpha diversity than those with intermediate levels (p <0.05). However, there were no differences in beta diversity as measured by Bray-Curtis (R2 = 1.15, p = 0.2) and Jaccard (R2 = 1.1, p = 0.1) indices across individuals with different levels of HPB-releasing DNA adducts. Multivariable association tests revealed that high levels of the DNA adducts were positively associated with Enterobacteriaceae, while intermediate levels were associated with Porphyromonas endodontalis, Prevotella spp, Neisseria spp, Actinomyces spp, and Fusobacterium. In addition, predicted functional profiling showed that intermediate levels of HPB-releasing DNA adducts were positively associated with amino acid, carbohydrate, and hem biosynthesis, along with reductive tricarboxylic acid cycle and formaldehyde degradation pathways. Comparisons between AA and WH persons who smoke showed that AA had significantly higher alpha diversity as assessed by observed bacterial richness and The Shannon index (Wilcoxon rank-sum tests, q-value=0.03 and 0.004, respectively). Differences between AA and WH were also found for beta diversity (Bray-Curtis: R2=2.4, p=0.01, Jaccard: R2=1.9, p=0.01, weighted Unifrac R2=0.02 p=0.007). The abundance of taxa associated with intermediate levels of DNA damage (Prevotella spp, Neisseria spp, Actinomyces spp, Fusobacterium) was also higher in AA persons who smoke. Together, our findings suggest that certain compositional and functional characteristics of the oral microbiome may serve as indicators of macromolecular responses to harmful chemical exposures from tobacco. The potential mechanistic contribution of the oral microbiome to such responses and its potential role in the observed racial/ethnic differences in cancer risk due to smoking should be further investigated. Citation Format: Aleksandra Alcheva. Relationship between the oral microbiome and tobacco-induced DNA damage in African American and White persons who smoke [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C030.

Reaction of human telomeric unit TTAGGG and a photoactivatable Pt(IV) anticancer prodrug.

The interaction of a photoactivatable diazidodihydroxido Pt(IV) prodrug, trans,trans,trans-[Pt(N3)2(OH)2(py)2] (py = pyridine; 1), with a hexamer straight human telomeric DNA unit sequence (5'-T1T2A3G4G5G6-3', I) upon light irradiation was investigated by electrospray ionization mass spectroscopy (ESI-MS). In the primary mass spectrum, two major mono-platinated I adducts with the bound Pt moieties, trans-[PtII(N3)(py)2]+ (1') and trans-[PtII(py)2]2+ (1''), respectively, were detected. It is rare to observe such high abundance and nearly equal intensity platinated DNA adducts formed by these two PtII species because 1' is usually the only major reduced Pt(II) species produced by the photodecomposition of complex 1 in the presence of DNA while 1'' was rarely detected as the major reduced PtII species reported previously. Subsequent tandem mass spectrometric analysis by collision-induced dissociation (CID) showed that in the former adduct {I + 1'}2+, G6 and A3 were the platination sites. While in the latter adduct {I + 1''}2+, a potential intrastrand crosslink was speculated after G4 and G6 sites were identified. Additionally, other minor platinated adducts like di-platinated I adduct by 1' with platination sites at G4 and G6 and mono-platinated I adducts containing base oxidation were also detected by mass spectrometry. Due to the rich guanines and their sensitivity to oxidation, the oxidation induced by 1 most probably occurred at guanine. The oxidation adducts were proposed as 8-hydroxyl guanine, spiroiminodihydantoin (Sp), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 5-guanidinohydantoin (Gh), and/or dehydroguanidinohydantoin (DGh) referring to previous reports. The obtained results provide useful chemical information about the photoreaction between photoactivatable Pt(IV) anticancer prodrugs and human telomeric DNA. Such special damages of Pt(IV) prodrugs on human telomeric DNA implicate its active role in the mechanism of Pt(IV) prodrugs and further support the unique sequence-dependent photointeraction profile of complex 1 reacting with DNA.

In Vitro Reconstitutive Base Excision Repair (BER) Assay.

The mammalian cell genome is continuously exposed to endogenous and exogenous insults that modify its DNA. These modifications can be single-base lesions, bulky DNA adducts, base dimers, base alkylation, cytosine deamination, nitrosation, or other types of base alteration which interfere with DNA replication. Mammalian cells have evolved with a robust defense mechanism to repair these base modifications (damages) to preserve genomic stability. Base excision repair (BER) is the major defense mechanism for cells to remove these oxidative or alkylated single-base modifications. The base excision repair process involves replacement of a single-nucleotide residue by two sub-pathways, the single-nucleotide (SN) and the multi-nucleotide or long-patch (LP) base excision repair pathways. These reactions have been reproduced in vitro using cell free extracts or purified recombinant proteins involved in the base excision repair pathway. In the present chapter, we describe the detailed methodology to reconstitute base excision repair assay systems. These reconstitutive BER assay systems use artificially synthesized and modified DNA. These reconstitutive assay system will be a true representation of biologically occurring damages and their repair.

POLIMORFISME GEN ENZIME EPOXIDE HIYDROLASE DAN RISIKO TERJADINYA KANKER PARU

Approximately 80-90% of lung cancer patients are smokers. Smoking is the activity of burning cigarettes which produces smoke. Cigarette smoke contains 4000 harmful chemical compounds. Among these chemical compounds, Polycyclic Aromatic Hydrocarbons (PaH) are inhaled and persist in the bronchial epithelium. PaH will be metabolized to Benzo A Pyrene (BaP) catalyzed by the enzyme epoxide hydrolase (EPHX). BaP is lipophilic and carcinogenic. The carcinogenic properties of BaP are very reactive so that they bind to DNA to form DNA adducts. If this occurs in the cell cycle regulatory gene, then this will initiate the initiation process of lung cancer. But of all smokers only 10-15% are known to suffer from lung cancer. This shows that there is a genetic role that causes a smoker to suffer from lung cancer. Several studies have stated that the EPHX gene polymorphism can increase or decrease the risk of a smoker suffering from lung cancer. There are two EPHX gene polymorphisms that are thought to be significantly related to the incidence of lung cancer. The two polymorphisms are EPHX Tyr113His and EPHX His139Arg. The EPHX Tyr113His polymorphism in vitro causes the EPHX enzyme activity to decrease so that DNA mutations decrease. Individuals with the EPHX Tyr113His genetic variation have a lower risk of developing lung cancer. The EPHX His139Arg polymorphism increases EPHX enzyme activity thereby increasing DNA mutations. Individuals with the EPHX His139Arg genetic variation have a higher risk of developing lung cancer.

Metabolic Activation of Benzo[a]pyrene by Human Tissue Organoid Cultures.

Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[a]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, CYP1A1 and NQO1, were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP-t-7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.

Cancer Cells Upregulate Tau to Gain Resistance to DNA Damaging Agents

Recent reports suggested a role for microtubules in double-strand-DNA break repair. We herein investigated the role of the microtubule-associated protein Tau in radio- and chemotherapy. Noticeably, a lowered expression of Tau in breast cancer cell lines resulted in a significant decrease in mouse-xenograft breast tumor volume after doxorubicin or X-ray treatments. Furthermore, the knockdown of Tau impaired the classical nonhomologous end-joining pathway and led to an improved cellular response to both bleomycin and X-rays. Investigating the mechanism of Tau's protective effect, we found that one of the main mediators of response to double-stranded breaks in DNA, the tumor suppressor p53-binding protein 1 (53BP1), is sequestered in the cytoplasm as a consequence of Tau downregulation. We demonstrated that Tau allows 53BP1 to translocate to the nucleus in response to DNA damage by chaperoning microtubule protein trafficking. Moreover, Tau knockdown chemo-sensitized cancer cells to drugs forming DNA adducts, such as cisplatin and oxaliplatin, and further suggested a general role of Tau in regulating the nuclear trafficking of DNA repair proteins. Altogether, these results suggest that Tau expression in cancer cells may be of interest as a molecular marker for response to DNA-damaging anti-cancer agents. Clinically targeting Tau could sensitize tumors to DNA-damaging treatments.

Incidence of IgA antibodies specific to benzo[a]pyrene and steroid hormones in women with colorectal cancer and breast cancer

Formation of DNA adducts of chemical carcinogens is a trigger for carcinogenesis. Adducts of benzo[a]pyrene metabolites and estradiol metabolites with DNA have been found in normal and tumor cells in healthy women and patients with breast and colorectal cancer. These low-weight compounds in macromolecular complexes induce the synthesis of specific antibodies. Previously, the presence of specific antibodies against benzo[a]pyrene (IgA-Bp), estradiol (IgA-Es) and progesterone (IgA-Pg) was revealed in breast cancer patients. The aim of this study is to identify the putative features of the IgA-Bp, IgA-Es, and IgA-Pg formation in postmenopausal women with colorectal cancer, in comparison with healthy and breast cancer patients. Using a noncompetitive enzyme-linked immunosorbent assay, the content of these antibodies was studied in the blood serum of healthy women (n = 401), patients with colorectal cancer (n = 219) and breast cancer (n = 1469) using conjugates of Bp, Es, and Pg with bovine serum albumin as adsorbed antigens. When compared with healthy people, the patients with colorectal cancer exhibited higher incidence of IgA-Bp 3 (75% vs 37%, p 0.0001, OR = 5.0), as well as more common levels of individual antibody ratios: IgA-Bp/IgA-Es 1 (82% vs 41%, p 0.0001, OR = 6.5); IgA-Bp/IgA-Pg 1.5 (77% vs 20%, p 0.0001, OR = 13.4); IgA-Es/IgA-Pg 1 (89% vs 48%, p 0.0001, OR = 8.7). In breast cancer patients, compared with healthy people, high IgA-Bp values ( 3) were more common (45% vs 37%, p 0.004, OR = 1.4), as well as increased IgA-Bp/IgA-Es ratio 1 (57% vs 41%, p 0.0001, OR = 1.9), IgA-Bp/IgA-Pg 1.1 (71% vs 36%, p 0.0001, OR = 4.4) and IgA-Es/IgA-Pg 1.1 (71% vs 41%, p 0.0001, OR = 3.5). In patients with colorectal cancer, compared with patients with breast cancer we have found higher incidence of increased IgA-Bp values ( 3) (75% vs 45%, p 0.0001), IgA-Es 3 (53% vs 39%, p 0, 0001), and of IgA-Pg 2 (52% vs 44%, p = 0.025), as well as IgA-Bp/IgA- Es 1 (82% vs 57%, p 0.0001, OR = 50.8 ); IgA-Bp/IgA-Pg 1.5 (77% vs 49%, p 0.0001); IgA-Es/IgA-Pg 1.1 (85% vs 71%, p 0.0001). The apparently high serum IgA-Bp levels reflect the formation of DNA-Bp adducts at large scale in target cells in colorectal cancer compared with healthy women and breast cancer patients, due to direct exposure of colon epithelium to Bp from food. Immunoassay for IgA-Bp, IgA-Es and IgA-Pg is proposed for assessing individual risk of colorectal cancer in postmenopausal women. The ratios of IgA Bp/IgA-Pg levels 1.5 represent the most informative marker of individual risk for colorectal cancer.

Enrichment of nucleobase adducts from genomic DNA in the cytoplasm by solid-phase extraction

The exact levels of some DNA adducts, like N7-deoxyguanosine (N7-dG), can be under-calculated since these adducts may depurinate due to their chemical instability, leading to corresponding nucleobase adducts being released into the cytoplasm. To accurately quantify the levels of DNA adducts, it is necessary to consider those modified nucleobases. However, high levels and diversity of cytoplasmic small molecule metabolites (SMMs) can strongly interfere with the detection of adducts, and it is almost impossible to remove them with nucleobase adducts being well retained. Therefore, we aimed to establish an optimized enrichment method based on solid-phase extraction (SPE) to reduce the co-elution of SMMs with target analytes. In this vein, we employed three bisphenols (BPA, BPF, and BPAF) as examples, prepared corresponding N7-guanine (N7-Gua) adducts, loaded on an Oasis hydrophilic-lipophilic balance ® (HLB) cartridge, used a series of mobile phases containing different percentage of methanol for elution, and evaluated the levels of these adducts in each eluent. First, we found that neutral samples led to the best retention for all three adducts compared with acidified or basified ones. We next employed normal distribution fitting model to characterize the elution of analytes from H2O/methanol with different pHs and observed that neutral mobile phases resulted in more hydrophobic elution for all three adducts. Besides, N7-BPA-Gua and N7-BPF-Gua obtained narrow fitted peaks at neutral pH, while N7-BPAF-Gua had minimized elution windows at low pH. After optimization, we exposed 293T cells to the aforementioned bisphenols and quantified the N7-Gua adducts in the cytoplasm and the corresponding N7-dG adducts in genomic DNA. The results revealed that with the same levels of BPs exposure, BPAF led to the highest levels of adducts in both cytoplasm and genomic DNA samples, followed by BPA and BPF in order. In summary, our research established an appropriate model to describe the elution of DNA adducts in the SPE, applied it to optimize the loading and elution conditions, and discussed the genotoxicity of bisphenols by accurate quantification of both cleaved and uncleaved N7-dG adducts.

DNA adducts as link between in vitro and in vivo carcinogenicity – A case study with benzo[a]pyrene

To reduce the need for animal tests, in vitro assays are often used as alternative methods. To derive toxic doses for higher tier organisms from in vitro assay results, quantitative in vitro-in vivo extrapolation (qIVIVE) based on physiological-based toxicokinetic (PBTK) models is typically the preferred approach. Such PBTK models require many input parameters to address the route from dose to target site concentration. However, respective data is very often not available. Hence, our aim is to call attention to an alternative way to build a link between animal (in vivo) and cell-derived (in vitro) toxicity data. To this end, we selected the carcinogenic chemical benzo[a]pyrene (BaP) for our study. Our approach relates both in vitro assay and in vivo data to a main intermediate marker structure for carcinogenicity on the subcellular level – the BaP-DNA adduct BaP-7,8-dihydrodiol-9,10-epoxide-deoxyguanosine. Thus, BaP dose is directly linked to a measure of the toxicity-initiating event. We used Syrian hamster embryo (SHE) and Balb/c 3T3 cell transformation assay as in vitro data and compared these data to outcomes of in vivo carcinogenicity tests in rodents. In vitro and in vivo DNA adduct levels range within three orders of magnitude. Especially metabolic saturation at higher doses and interspecies variabilities are identified and critically discussed as possible sources of errors in our simplified approach. Finally, our study points out possible routes to overcome limitations of the envisaged approach in order to allow for a reliable qIVIVE in the future.

Chemopreventive effects and anti-tumorigenic mechanisms of Actinidia arguta, known as sarunashi in Japan toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- induced lung tumorigenesis in a/J mouse

Previously, we reported the inhibitory effect of Actinidia arguta juice, known as sarunashi juice (sar-j) in Japan, on mutagenesis, inflammation, and mouse skin tumorigenesis. The components of A. arguta responsible for the anti-mutagenic effects were identified to be water-soluble, heat-labile phenolic compounds. We proposed isoquercetin (isoQ) as a candidate anticarcinogenic component. In this study, we sought to investigate the chemopreventive effects of A. arguta juice and isoQ on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice, and identify the possible mechanisms underlying the anti-tumorigenic effects of A. arguta. The number of tumor nodules per mouse lung in the group injected with NNK and administered A. arguta juice orally was significantly lower than that in the group injected with NNK only. Oral administration of isoQ also reduced the number of nodules in the mouse lungs. As expected, the mutagenicity of NNK and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) detected using S. typhimurium TA1535 decreased in the presence of sar-j. However, NNK and MNNG mutagenicity detected using S. typhimurium YG7108, a strain lacking the O6-methylguanine DNA methyltransferases (ogtST and adaST) did not decrease in the presence of sar-j suggesting that sar-j may mediate its antimutagenic effect by enhancing the DNA damage repair by ogtST and adaST. Phosphorylation of Akt, with or without epidermal growth factor stimulation, in A549 cells was significantly decreased following sar-j and isoQ treatment, indicating that components in sar-j including isoQ suppressed the PI3K/AKT signaling pathways. Sar-j and isoQ reduced NNK-induced lung tumorigenesis. Sar-j targets both the initiation and growth/progression steps during carcinogenesis, specifically via anti-mutagenesis, stimulation of alkyl DNA adduct repair, and suppression of Akt-mediated growth signaling. IsoQ might contribute in part to the biological effects of sar-j via suppression of Akt phosphorylation, but it may not be the main active ingredient.