The in vitro Bacterial Reverse Mutation (Ames) Test is crucial for evaluating the mutagenicity of pharmaceutical impurities. For N-nitrosamines (NAs) historical data indicated that for certain members of this chemical class the outcomes of the Ames Test did not correlate with their associated rodent carcinogenicity outcomes. This has resulted in negative outcomes in an OECD aligned Ames Test alone (standard or enhanced) no longer being considered sufficient by regulatory authorities to assess potential carcinogenic risk of NAs if present as impurities in drug products. Consequently, extensive follow-up in vivo testing can be required to characterise the potential mutagenicity and genotoxic carcinogenicity of NA impurities (i.e., beyond that defined in the ICH M7 guideline for non-NA impurities). We previously demonstrated that the mutagenicity of alkyl-nitrosamines can be detected by the appropriately designed, OECD aligned Ames Test and identified those conditions that contributed most to assay sensitivity. This OECD aligned Ames Test design was used to assess seven NAs, i.e. (methyl(neopentyl)nitrosamine, N-methyl-N-nitroso-2-propanamine, N-nitrosodiisopropylamine, bis(2-methoxyethyl)nitrosoamine, N-nitroso-N-methyl-4-fluoroaniline, dinitrosoethambutol, (R,R)- and mononitrosocaffeidine) that were reported to be negative in historical Ames Tests but positive in rodent carcinogenicity studies. All seven of the NAs were demonstrated to be mutagenic in the OECD aligned Ames test and therefore these compounds should no longer be considered as discordant (false negatives) with respect to the correlation of the Ames Test and rodent carcinogenicity. These results confirm the sensitivity of the OECD aligned Ames Test for the detection of NA mutagenicity and provides further support of its pivotal placement within the ICH M7 framework for the assessment of mutagenic impurities in pharmaceuticals to limit potential carcinogenic risk. In addition, we present data for 1-cyclopentyl-4-nitrosopiperazine, that indicates it could serve as a suitable positive control to provide further confidence in the sensitivity of the Ames Test for the NA chemical class.

Systemic oxidative stress stemming from increased free radical production and reduced antioxidant capacity are common characteristics of obese individuals. Using hydrogen peroxide (H2O2) to induce DNA damage in vitro, in peripheral blood mononuclear cells (PBMCs) from obese subjects and controls, the DNA protective ability of dihidroqercetin (DHQ) and biochaga (B) alone or in combination, were evaluated. The effects of DHQ and B were estimated under two experimental conditions: pre-treatment, where cells were pre-incubated with the substances prior to H2O2 exposure; and post-treatment when cells were first exposed to H2 H2O2, and further treated with the compounds. DNA damage was evaluated using the comet assay. The results of pre- and post-treatment showed a significant decrease in DNA damage produced by H2O2 in the obese group. This decrease was not significant in control group probably due to a small number of subjects in this pilot study. More prominent attenuation was noted in the pre-treatment with DHQ (250 μg/ml). Analysis of antioxidant properties revealed that DHQ's remarkable reducing power, 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, and potent∙OH scavenging properties may contribute to strong attenuation of H2O2-induced DNA damage. Also, B showed strong reducing power, DPPH, and ∙OH scavenging ability, while reducing power and DPPH scavenger effects were increased in the presence of DHQ. Conclusively, DHQ and B may reduce H2O2-induced DNA damage in PBMCs from obese subjects when challenged in vitro, and could be valuable tools in future research against oxidative damage-related conditions.

An ethanol extract of Piper auritum leaves (PAEE) inhibits the mutagenic effect of three food-borne aromatic amines (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx); 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx)) in the TA98 Salmonella typhimurium strain. Preincubation with MeIQx demonstrated in mutagenesis experiments that inhibition of Cytochrome P450 (CYP), as well as direct interaction between component(s) of the plant extract with mutagens, might account for the antimutagenic observed effect. Gas chromatography/mass spectrometry analysis revealed that safrole (50.7%), α-copaene (7.7%), caryophyllene (7.2%), β-pinene (4.2%), γ-terpinene (4.1%), and pentadecane (4.1%) as the main components (PAEE). Piper extract and safrole were able to inhibit the rat liver microsomal CYP1A1 activity that participates in the amines metabolism, leading to the formation of the ultimate mutagenic/ molecules. According to this, safrole and PAEE-inhibited MeIQx mutagenicity but not that of the direct mutagen 2-nitrofluorene. No mutagenicity of plant extract or safrole was detected. This study shows that PAEE and its main component safrole are associated with the inhibition of heterocyclic amines activation due in part to the inhibition of CYP1A subfamily activity.

DNA methylation is an important mechanism in the regulation of gene expression and maintenance of genomic integrity. Aberrant DNA methylation is an early event in carcinogenesis. DNA methyltransferase inhibitors are used to restore aberrant DNA methylation and inhibit tumor growth. Evaluation of DNA methylation level is important for an effective anti-cancer therapy. In the present study, the determination of global DNA methylation levels in patients with urinary bladder cancer was proposed. The methylation-sensitive comet assay determined the global DNA methylation level at the level of single cells. McrBC enzyme, a methylation-sensitive restriction endonuclease, was used for enzymatic digestion to generate additional breaks at methylated sites. % DNA methylation level was significantly higher in patients with bladder cancer compared to the control group. The clinical performance of % DNA methylation analysis by methylation-sensitive comet assay was evaluated by ROC curve. Using the cutoff value of 6.5% DNA methylation, 92% sensitivity, and 42% specificity were obtained. In conclusion, global DNA methylation measured by methylation-sensitive comet assay may be a promising noninvasive biomarker that reduces interventional tests required in the diagnosis and follow-up of urinary bladder cancer.

River water in Kosovo is exposed to various discharges from industrial and agricultural activities as well as to urban wastewater. Rivers Sitnica and Drenica are among the most affected ones and water samples drawn from these rivers show the presence of various toxic substances. Genotoxic effects are seen in fish living in these rivers indicating a cytotoxic and mutagenic potential of the river water. Aiming at substantiating these observations, we assessed the cyto- and genotoxic effects of water samples collected at different locations from the Drenica and Sitnica rivers. Samples drawn from Lake Badovc served for comparison. To address seasonal effects, samples were collected at different seasons/time points during the period of summer 2016-spring 2018. The water samples were analyzed employing primary rat hepatocytes as a reliable in vitro cell model for the assessment of cytotoxic effects (mitotic arrest and cell death) and DNA damage/genotoxicity (micronucleus assay and Comet assay). The results do not account for significant effects associated with specific locations but demonstrate seasonal differences of the genotoxic potential of the water samples collected along both rivers, which are accompanied by a limited cytotoxic potential. Our data provide substantial support to earlier observations and strongly warrant the need for continuous chemical as well as biological monitoring of the river water in Kosovo, focusing on improved toxicant profiling of the river water and investigations addressing the observed seasonal variations.

The COVID-19 pandemic has led to the emergence of acute and chronic post-COVID syndromes, which present diverse clinical manifestations. The underlying pathophysiology of these conditions is not yet fully understood, but genetic instability has been proposed as a potential contributing factor. This study aimed to explore the differential impact of physical and psychological health factors on genetic instability in individuals with acute and chronic post-COVID syndromes. In this study, three groups of subjects were analyzed: a control group, an acute post-COVID group, and a chronic post-COVID group, with a total of 231 participants. The participants were assessed using a questionnaire for long-COVID-19COVID, and female participants reported more symptoms than male participants in areas related to fatigue, memory, mental health, and well-being during the chronic phase. Genetic instability was assessed using the comet assay, and participants' physical and psychological profiles were evaluated. The overall results showed no significant differences in DNA damage, as measured by the comet assay, among the three groups, suggesting that genetic instability, as assessed by this method, may not be a primary driver of the distinct clinical presentations observed in post-COVID syndromes. However, when gender was considered, male participants in the acute long COVID group exhibited higher levels of genetic instability compared to females. Multiple linear regression analysis revealed that gender, age, and waist circumference were significant predictors of DNA damage. Among females in the acute group, sexual health, and eye-related symptoms significantly influenced the increase in DNA damage. These findings indicate the need for further investigation on the gender-specific differences in genetic instability and their potential implications for the pathophysiology of post-COVID syndromes. Exploring alternative markers of genetic instability and the interplay between genetic, inflammatory, and cellular processes could provide valuable insights for the management of these debilitating post-viral sequelae.

DNA ligase (LIG) I and IIIα finalize base excision repair (BER) by sealing a nick product after nucleotide insertion by DNA polymerase (pol) β at the downstream steps. We previously demonstrated that a functional interplay between polβ and BER ligases is critical for efficient repair, and polβ mismatch or oxidized nucleotide insertions confound the final ligation step. Yet, how targeting downstream enzymes with small molecule inhibitors could affect this coordination remains unknown. Here, we report that DNA ligase inhibitors, L67 and L82-G17, slightly enhance hypersensitivity to oxidative stress-inducing agent, KBrO3, in polβ+/+ cells more than polβ-/- null cells. We showed less efficient ligation after polβ nucleotide insertions in the presence of the DNA ligase inhibitors. Furthermore, the mutations at the ligase inhibitor binding sites (G448, R451, A455) of LIG1 significantly affect nick DNA binding affinity and nick sealing efficiency. Finally, our results demonstrated that the BER ligases seal a gap repair intermediate by the effect of polβ inhibitor that diminishes gap filling activity. Overall, our results contribute to understand how the BER inhibitors against downstream enzymes, polβ, LIG1, and LIGIIIα, could impact the efficiency of gap filling and subsequent nick sealing at the final steps leading to the formation of deleterious repair intermediates.

Doxorubicin, a well-known and widely used antineoplastic agent with direct ROS-accumulating activity, has proven effective in treating various cancer types. However, its non-specific cytotoxicity towards non-cancerous cells prompts concerns regarding potential adverse effects. Azithromycin is an antibiotic for treating bacterial infections and an anti-inflammatory agent, particularly beneficial in managing respiratory conditions like bronchitis and sinusitis. Despite azithromycin's well-documented antibacterial properties, its potential cellular/genomic protective effects remain unexplored. As an in vitro model, BEAS-2B cells (normal human bronchial epithelium cells) were employed in the present study to assess whether azithromycin possesses any protective properties against doxorubicin-induced cellular toxicity. Cells in pre-treatment culture were treated to various amounts of azithromycin (3.125, 6.25, 12.5, 25, and 50 μg/mL) in combination with doxorubicin at IC50 (0.08 μg/mL). Doxorubicin at 0.08 μg/mL highlighted cytotoxicity, oxidative stress, and genotoxicity. Azithromycin at 25 and 50 μg/mL markedly modulated oxidative stress and genomic damage by decreasing the ROS and LPO amounts, and suppressing DNA fragmentation in the comet assay parameters. Consequently, azithromycin may be regarded as a cytomodulating, antigenotoxic, and antioxidant agent.

The proceedings of the 36th annual meeting of the Industrial Genotoxicology Group (IGG) are shared here. The meeting held at Lhasa Limited, Leeds, UK on 28th November 2023, focussed two aspects; New Approach Methodologies (NAM's), including those for the assessment of non-standard modalities such as gas-vapour assessments and nanomaterials, and addressing the regulatory challenges associated with understanding the genotoxic and carcinogenic potential of N-nitrosamines and N-nitrosamine impurities. New approach methodologies, such as error-corrected sequencing and enhanced Ames tests that may help address these challenges were also discussed.

Colorectal cancer is a global killer that causes approximately 940 thousand deaths annually. Terminalia ivorensis (TI) is a tropical tree, the bark of which is used in African traditional medicine for the treatment of diabetes, malaria and ulcer. This study investigated TI as a potential anticancer agent in human colon cells in vitro. TI was extracted sequentially with petroleum ether, chloroform, ethyl acetate and ethanol. Antioxidant activity was assessed by DPPH and FRAP, and differential effects on cell viability, growth, DNA damage, DNA repair, and migration were measured in human colon cancer cells (CaCo-2) and/or non-cancerous human colonocytes (NCM460). The TI phytochemicals most strongly associated with these effects were identified by partial least-squares discriminant analysis. DPPH and FRAP activity were highest in TI ethyl acetate and ethanol extracts (p=0.001). All TI extracts significantly inhibited cell viability and growth and induced DNA damage and inhibited DNA repair in both cell models. The majority of TI extracts were significantly (p=0.01) more toxic to cancer cells than non-cancerous colonocytes. DNA repair was significantly (p=0.001) inhibited in CaCo-2 cells by ethyl acetate extract compared with NCM460 cells. Migration was also significantly inhibited (p<0.001) in CaCo-2 by ethyl acetate (80%) and ethanol extracts (75%). Specific benzoic acids, flavonoids and phenols were identified to be strongly associated with these effects. TI displayed strong antioxidant activity and specific anticancer effects by inducing cell death and DNA damage, and by inhibiting DNA repair, cell proliferation and migration.

The purposes of this review were to investigate the application of the comet assay in Allium cepa root cells to assess the genotoxicity of environmental samples and to analyse the experimental procedures employed. A literature search was performed selecting articles published between January 2000 and October 2023 from online databases using the combined search terms 'comet assay' and 'A. cepa'. Only 18 papers met the inclusion criteria. None of these were published in the first eight years (2000-2007), highlighting the increasing interest in using the comet assay on A. cepa to analyse environmental samples over the last decade. The majority of the selected studies (15/18, 83%) were performed on samples belonging to the water compartment on onion bulbs. Half of the selected studies (9/18) were conducted to demonstrate the DNA damaging effect of the sample, while the other half of the studies not only recognized the presence of genotoxic agents but also addressed possible remediation measures. Detailed analysis of the experimental procedures revealed heterogeneity in many key steps, such as exposure time, test controls, nuclei isolation solutions, duration of electrophoresis, and number of nuclei scored. This literature review has shown that the comet assay on A. cepa, although recognized as an appropriate tool, is underutilized in environmental toxicology. Greater standardization could lead to its more widespread use, providing valuable information on the genotoxicity of environmental samples and the ability of different processes to mitigate their negative effects on plants.

Chemoresistance represents a major issue affecting cancer therapy efficacy. Because microRNAs (miRNAs) regulate gene expression on multiple levels, their role in chemoresistance development is reasonably certain. In our previous study, miR-122-5p and miR-142-5p were identified as diagnostic, prognostic, and predictive biomarkers for primary and metastatic rectal cancer. The aim of the present study was to investigate whether these miRNAs can also reflect the disease course of colon cancer (CC) patients. Further, we focused on a deeper understanding of their involvement in 5-fluorouracil (5-FU) chemoresistance development. The expression analysis of both miRNAs was analysed in repeated whole plasma samplings (n=3, approximately every 6 months) of CC patients (n=49) by RT-qPCR. Expression levels of both miRNAs were determined in the 5-FU sensitive and resistant CC cell lines. From RNA-seq profiles of both sensitive and 5-FU resistant DLD-1 cell lines, the expression levels of miR-122-5p and miR-142-5p validated target genes were detected and compared. Significant differences in the expression levels of both miRNAs between T0 and T1 or T2 samplings were observed. Further, an association between the occurrence of relapse and miR-122-5p expression levels was noticed. Patients who did not relapse had higher expression of miR-122-5p at T1 (p=0.01; 3.16-fold change) and T2 (p=0.04; 2.79-fold change) samplings in comparison with T0 sampling. Out of all miR-122-5p validated targets (n=102), 25 genes were significantly differentially expressed between sensitive and 5-FU-resistant cell lines. Our data suggest that miR-122-5p may represent a predictive marker of tumour relapse in CC patients. In vitro data suggests that this aspect may be linked to the potential therapeutic targets of miR-122-5p related to 5-FU-based chemoresistance. However, deeper mechanistic studies are still needed for progress toward personalized medicine.

Diagnostic performance of molecular markers in surrogate tissues like stool may be affected by colorectal cancer (CRC) morphological heterogeneity. The mucinous histotype represents a subgroup of CRC with a peculiar molecular program and unfavorable disease progression. However, the percentage of mucinous morphology necessary to define this subtype is still a matter of debate. In this study, we investigated whether stool miRNA profiles of CRC patients differ in patients with mucinous histopathological subtypes compared to non-mucinous cancers. In this respect, we also explored how the stool miRNA signature reported in our previous multicentric study (Pardini et al., Gastroenterology 2023) behave in this histotype. Small-RNA sequencing was performed in fecal and tissue samples of an Italian cohort (n=172), including 27 CRC with mucinous morphology (mucinous cancers with >50% mucinous morphology and those with mucinous component >5% but <50%), 58 non-mucinous CRC, and 87 colonoscopy-negative controls. Results were compared with fecal miRNA profiles of a cohort from the Czech Republic (n=98). Most of the differentially expressed (DE) stool miRNAs (n=324) were in common between CRC with mucinous morphology and non-mucinous histopathological subtypes in comparison with healthy controls. Interestingly, the altered levels of 25 fecal miRNAs previously identified distinguishing CRC cases from controls in both cohorts were also confirmed after stratification for mucinous morphology. Forty-nine miRNAs were DE exclusively in CRC with mucinous morphology and 61 in non-mucinous CRC. Mucinous cancers and those with mucinous component showed fairly similar profiles that were comparable in the Czech cohort. Among the stool DE miRNAs observed in CRC with mucinous morphology, 20 were also altered in the comparison between tumor and adjacent mucosa tissue. This study highlights miRNAs specifically altered in CRC with mucinous morphology. Nevertheless, the performance of our stool miRNA signature in accurately distinguishing CRC cases from controls was not significantly affected by this histological subtype. This aspect further supports the use of stool miRNAs for noninvasive diagnosis and screening strategies.

Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are mycotoxins widely distributed in maize and maized-based products, often occurring together. The implications of co-exposure to aflatoxin and fumonsin for human health are numerous, but a particular concern is the potential of FB1 to modulate AFB1 hepatotoxicity. This study evaluated the toxicity of these mycotoxins, alone or combined, in a human non-tumorigenic liver cell line, HHL-16 cells, and assessed the effects of AFB1 and FB1 on expression of genes involved in immune and growth factor pathways. The results demonstrated that in HHL-16 cells, both AFB1 and FB1 had dose-dependent and time-dependent toxicity, and the combination of them showed a synergistic toxicity in the cells. Moreover, AFB1 caused upregulation of IL6, CCL20, and BMP2, and downregulation of NDP. In combination of AFB1 with FB1, gene expression levels of IL6 and BMP2 were significantly higher compared to individual FB1 treatment, and had a tendency to be higher than individual AFB1 treatment. This study shows that FB1 may increase the hepatoxicity of AFB1 through increasing the inflammatory response and disrupting cell growth pathways.

The current Organisation for Economic Co-Operation and Development test guideline number 487 (OECD TG No. 487) provides instruction on how to conduct the in vitro micronucleus assay. This assay is one of the gold standard approaches for measuring the mutagenicity of test items; however, it is directed at testing low molecular weight molecules and may not be appropriate for particulate materials (e.g. engineered nanoparticles [ENPs]). This study aimed to adapt the in vitro micronucleus assay for ENP testing and underpins the development of an OECD guidance document. A harmonized, nano-specific protocol was generated and evaluated by two independent laboratories. Cell lines utilized were human lymphoblastoid (TK6) cells, human liver hepatocytes (HepG2) cells, Chinese hamster lung fibroblast (V79) cells, whole blood, and buffy coat cells from healthy human volunteers. These cells were exposed to reference ENPs from the Joint Research Council (JRC): SiO2 (RLS-0102), Au5nm and Au30nm (RLS-03, RLS-010), CeO2 (NM212), and BaSO4 (NM220). Tungsten carbide-cobalt (WC/Co) was used as a trial particulate positive control. The chemical controls were positive in all cell cultures, but WC/Co was only positive in TK6 and buffy coat cells. In TK6 cells, mutagenicity was observed for SiO2- and both Au types. In HepG2 cells, Au5nm and SiO2 showed sub-two-fold increases in micronuclei. In V79 cells, whole blood, and buffy coat cells, no genotoxicity was detected with the test materials. The data confirmed that ENPs could be tested with the harmonized protocol, additionally, concordant data were observed across the two laboratories with V79 cells. WC/Co may be a suitable particulate positive control in the in vitro micronucleus assay when using TK6 and buffy coat cells. Detailed recommendations are therefore provided to adapt OECD TG No. 487 for testing ENP.

Pleiotropic variants (i.e., genetic polymorphisms influencing more than one phenotype) are often associated with cancer risk. A scan of pleiotropic variants was successfully conducted ten years ago in relation to pancreatic ductal adenocarcinoma susceptibility. However, in the last decade, genetic association studies performed on several human traits have greatly increased the number of known pleiotropic variants. Based on the hypothesis that variants already associated with a least one trait have a higher probability of association with other traits, 61,052 variants reported to be associated by at least one genome wide association study (GWAS) with at least one human trait were tested in the present study consisting of two phases (discovery and validation), comprising a total of 16,055 pancreatic ductal adenocarcinoma (PDAC) cases and 212,149 controls. The meta-analysis of the two phases showed two loci (10q21.1-rs4948550 (P=6.52×10-5) and 7q36.3-rs288762 (P=3.03×10-5) potentially associated with PDAC risk. 10q21.1-rs4948550 shows a high degree of pleiotropy and it is also associated with colorectal cancer risk while 7q36.3-rs288762 is situated 28,558 base pairs upstream of the Sonic Hedgehog (SHH) gene, which is involved in the cell differentiation process and PDAC etiopathogenesis. In conclusion, none of the single nucleotide polymorphisms (SNPs) showed a formally statistically significant association after correction for multiple testing. However, given their pleiotropic nature and association with various human traits including colorectal cancer, the two SNPs showing the best associations with PDAC risk merit further investigation through fine mapping and ad hoc functional studies.

Gut barrier dysfunction and related inflammation are known to be associated with the development and progression of colorectal cancer (CRC). We investigated associations of 292 single-nucleotide polymorphisms (SNPs) from 27 genes related to endotoxins/lipopolysaccharide (LPS) sensing and tolerance, mucin synthesis, inflammation, and Crohn's disease with colon and rectal cancer risks. Incident CRC cases (N=1,374; colon=871, rectum=503) were matched 1:1 to controls nested within the European Prospective Investigation into Cancer and Nutrition cohort. Previously measured serum concentrations of gut barrier function and inflammation biomarkers (flagellin/LPS-specific immunoglobulins and C-reactive protein [CRP]) were available for a sub-set of participants (Ncases=1,001; Ncontrols=667). Forty-two unique SNPs from 19 different genes were associated with serum biomarkers at Punadjusted≤0.05 among controls. Among SNPs associated with a gut permeability score, 24 SNPs were in genes related to LPS sensing and mucin synthesis. Nine out of 12 SNPs associated with CRP were in genes related to inflammation or Crohn's disease. TLR4 was associated with colon cancer at the SNP level (nine SNPs, all Punadjusted≤0.04) and at the gene level (Punadjusted≤0.01). TLR4 rs10759934 was associated with rectal cancer but not colon cancer. Similarly, IL10 was associated with rectal cancer risk at a SNP and gene level (both Punadjusted ≤ 0.01), but not colon cancer. Genes and SNPs were selected a priori therefore we present unadjusted P-values. However, no association was statistically significant after multiple testing correction. This large and comprehensive study has identified gut barrier function and inflammation-related genes possibly contributing to CRC risk in European populations and is consistent with potential etiological links between host genetic background, gut barrier permeability, microbial endotoxemia and CRC development.

The Commentary on Special Issue- Current Understanding of Colorectal and Pancreatic Cancers provides the reasoning for the selection of the contributions on pancreatic and colorectal cancer, and summarizes in brief the individual topics and comments upon the main outcomes. The current knowledge, contribution of the individual articles within this Special Issue, and arising priorities in the research on pancreatic ductal adenocarcinoma and colorectal cancer are highlighted.

Nonspecific structural chromosomal aberrations (CAs) are found in around 1% of circulating lymphocytes from healthy individuals but the frequency may be higher after exposure to carcinogenic chemicals or radiation. CAs have been used in the monitoring of persons exposed to genotoxic agents and radiation. Previous studies on occupationally exposed individuals have shown associations between the frequency of CAs in peripheral blood lymphocytes and subsequent cancer risk. The cause for CA formation are believed to be unrepaired or insufficiently repaired DNA double-strand breaks or other DNA damage, and additionally telomere shortening. CAs include chromosome (CSAs) and chromatid type aberrations (CTAs). In the present review, we first describe the types of CAs, the conventional techniques used for their detection and some aspects of interpreting the results. We then focus on germline genetic variation in the frequency and type of CAs measured in a genome-wide association study (GWAS) in healthy individuals in relation to occupational and smoking-related exposure compared to non-exposed referents. The associations (at p<10-5) on 1473 healthy individuals were broadly classified in candidate genes from functional pathways related to DNA damage response/repair, including PSMA1, UBR5, RRM2B, PMS2P4, STAG3L4, BOD1, COPRS and FTO; another group included genes related to apoptosis, cell proliferation, angiogenesis and tumorigenesis, COPB1, NR2C1, COPRS, RHOT1, ITGB3, SYK, and SEMA6A; a third small group mapped to genes KLF7, SEMA5A and ITGB3 which were related to autistic traits, known to manifest frequent CAs. Dedicated studies on 153 DNA repair genes showed associations for some 30 genes, expression of which could be modified by the implicated variants. We finally point out that monitoring of CAs is so far the only method of assessing cancer risk in healthy human populations, and the use of the technology should be made more attractive by developing automated performance steps and incorporating artificial intelligence methods into the scoring.