Mutagenic Metabolites Research Articles

Gastrointestinal fate of proteins from commercial plant-based meat analogs: Silent passage through the stomach, oxidative stress in intestine, and gut dysbiosis in Wistar rats.

Plant-based meat analogs (PBMAs) are common ultra-processed foods (UPFs) included in the vegan/vegetarian diets as presumed healthy alternatives to meat and meat products. However, such health claims need to be supported by scientific evidence. To gain further insight into this topic, two commercial UPFs typically sold as meat analogs, namely, seitan (S) and tofu (T), were included in a cereal-based chow and provided to Wistar rats for 10weeks. A group of animals had, simultaneously, an isocaloric and isoprotein experimental diet formulated with cooked beef (B). In all cases, experimental chows (∼4kcal/g feed) had their basal protein concentration increased from 14% to 30% using proteins from S, T, or B. Upon slaughter, in vivo protein digestibility was assessed, and the entire gastrointestinal tract (digests and tissues) was analyzed for markers of oxidative stress and untargeted metabolomics. Metagenomics was also applied to assess the variation of microbiota composition as affected by dietary protein. Diets based on PBMAs showed lower protein digestibility than those containing meat and promoted an intense luminal glycoxidative stress and an inflammatory intestinal response. The fermentation of undigested oxidized proteins from T in the colon of Wistar rats likely led to formation of mutagenic metabolites such as p-cresol. The presence of these compounds in the animal models raises concerns about the potential effects of full replacement of meat by certain PBMAs in the diet. Therefore, future research might target on translational human studies to shed light on these findings.

When less is more: The association between the expression of polymorphic CYPs and AFB1-induced HCC.

An individual's genetic fingerprint is emerging as a pivotal predictor of numerous disease- and treatment-related factors. Single nucleotide polymorphisms (SNPs) in drug-metabolizing enzymes play key roles in an individual's exposure to a malignancy-associated risk, such as Aflatoxin B1 (AFB1)-induced hepatocellular carcinoma (HCC). This study aimed at reviewing literature on the polymorphisms that exist in CYP enzymes and their possible link with susceptibility to AFB1-induced HCC. A set of keywords associated with the study subject of interest was used to search the Google Scholar and the PubMed database. The last ten years' worth of research projects were included in the results filter. The research involved HCC patients and any connection between polymorphic forms of CYP enzymes and their susceptibility to AFB1-induced HCC, including older but significant data. Variations in CYP1A2 and CYP3A4 were reported to impact the rate and magnitude of AFB1 bio-activation, thus influencing an individual's vulnerability to develop HCC. In HCC patients, the activity of CYP isoforms varies, where increased activity has been reported with CYP2C9, CYP2D6, and CYP2E1, while CYP1A2, CYP2C8, and CYP2C19 exhibit decreased activity. CYP2D6*10 frequency has been discovered to differ considerably in HCC patients. Rs2740574 (an upstream polymorphism in CYP3A4 as detected in CYP3A4*1B) and rs776746 (which affects CYP3A5 RNA splicing), both of which influence CYP3A expression, thus impacting the variability of AFB1-epoxide adducts in HCC patients. CYP1A2 is the primary enzyme accountable for the formation of harmful AFBO globally. CYP3A4, CYP3A5, CYP3A7, CYP2B7, and CYP3A3 are also implicated in the bio-activation of AFB1 to mutagenic metabolites. It is thought that CYP3A4 is the protein that interacts with AFB1 metabolism the most. Polymorphic variants of CYP enzymes have a functional impact on the susceptibility to AFB1-induced HCC. Outlining such variation and their implications may provide deeper insights into approaching HCC in a more personalized manner for guiding future risk-assessment, diagnosis, and treatment.

Apiole, an important constituent of parsley, is a mixed-type inhibitor of the CYP1A subfamily

Apiole (1-allyl-2,5-dimethoxy-3,4-methylenedioxybenzene) and parsley leaves ethanolic extract containing it inhibit the rat liver microsomal ethoxy- and methoxyresorufin-O-deacetylase activities associated with cytochrome P450 (CYP) 1A1 and 1A2, respectively. Cytochrome P4501A subfamily metabolizes environmental mutagens and several drugs, leading to the formation of mutagenic metabolites. Docking analysis showed that residue Phe123 within the active site of the CYP1A1 enzyme is bound to apiole through a π/π stacking of its benzene ring. In the case of 1A2, its Phe226 interacts with the dioxolane ring of apiole. Furthermore, apiole behaves as a mixed-type inhibitor of bacterial human recombinant CYP1A1. To explore one of the possible biological implications of this inhibitory effect, we tested the capacity of apiole and the parsley ethanolic extract to interfere with the mutagenicity of the promutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) metabolized by CYP1A subfamily. As expected, both apiole and the plant extract reduced the number of revertant colonies of Salmonella typhimurium TA98 Ames strain after exposure to MeIQx, reaching a 78 % and 100 % reduction, respectively. Neither apiol nor parsley extract were mutagenic to the TA98 strain. We speculate that consuming apiole, a constituent of edible herbs, in conjunction with the utilization of pharmaceuticals metabolized by the CYP1A subfamily, may result in herb-drug interactions. Furthermore, the consumption of apiole by individuals who regularly ingest fresh vegetables may contribute to the low incidence of cancer observed in those who adhere to such a dietary regimen.

Induction of AHR Signaling in Response to the Indolimine Class of Microbial Stress Metabolites.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that plays an important role in gastrointestinal barrier function, tumorigenesis, and is an emerging drug target. The resident microbiota is capable of metabolizing tryptophan to metabolites that are AHR ligands (e.g., indole-3-acetate). Recently, a novel set of mutagenic tryptophan metabolites named indolimines have been identified that are produced by M. morganii in the gastrointestinal tract. Here, we determined that indolimine-200, -214, and -248 are direct AHR ligands that can induce Cyp1a1 transcription and subsequent CYP1A1 enzymatic activity capable of metabolizing the carcinogen benzo(a)pyrene in microsomal assays. In addition, indolimines enhance IL6 expression in a colonic tumor cell line in combination with cytokine treatment. The concentration of indolimine-248 that induces AHR transcriptional activity failed to increase DNA damage. These observations reveal an additional aspect of how indolimines may alter colonic tumorigenesis beyond mutagenic activity.

Application of systematic evidence mapping to identify available data on the potential human health hazards of selected market-relevant azo dyes

BackgroundAzo dyes are used in textiles and leather clothing. Human exposure can occur from wearing textiles containing azo dyes. Since the body’s enzymes and microbiome can cleave azo dyes, potentially resulting in mutagenic or carcinogenic metabolites, there is also an indirect health concern on the parent compounds. While several hazardous azo dyes are banned, many more are still in use that have not been evaluated systematically for potential health concerns. This systematic evidence map (SEM) aims to compile and categorize the available toxicological evidence on the potential human health risks of a set of 30 market-relevant azo dyes. MethodsPeer-reviewed and gray literature was searched and over 20,000 studies were identified. These were filtered using Sciome Workbench for Interactive computer-Facilitated Text-mining (SWIFT) Review software with evidence stream tags (human, animal, in vitro) yielding 12,800 unique records. SWIFT Active (a machine-learning software) further facilitated title/abstract screening. DistillerSR software was used for additional title/abstract, full-text screening, and data extraction. Results187 studies were identified that met populations, exposures, comparators, and outcomes (PECO) criteria. From this pool, 54 human, 78 animal, and 61 genotoxicity studies were extracted into a literature inventory. Toxicological evidence was abundant for three azo dyes (also used as food additives) and sparse for five of the remaining 27 compounds. Complementary search in ECHA’s REACH database for summaries of unpublished study reports revealed evidence for all 30 dyes. The question arose of how this information can be fed into an SEM process. Proper identification of prioritized dyes from various databases (including U.S. EPA’s CompTox Chemicals Dashboard) turned out to be a challenge. Evidence compiled by this SEM project can be evaluated for subsequent use in problem formulation efforts to inform potential regulatory needs and prepare for a more efficient and targeted evaluation in the future for human health assessments.

The Degradation Product of Ramipril Is Potentially Carcinogenic, Genotoxic and Mutagenic

(1) Background: The aim of this study was to identify the degradation product of ramipril (RAM) formed under dry air and to verify its potential modes of carcinogenicity. We intended to check whether its formation and presence in final dosage forms could pose a cancer risk to humans who are treated with RAM due to cardiological indications. The carcinogenicity of this compound was evaluated with respect to two mechanisms: a potential direct DNA-damage and indirect toxicity, secondary to forming mutagenic N-nitroso metabolites. (2) Methods: Firstly, a forced ageing test under dry air was conducted for pure RAM in order to induce its degradation. The validated HPLC system was used to describe the kinetic order of this reaction. The emerging degradation impurity was identified by HPLC-MS. In the second stage, the cancer risk of the identified RAM degradant was predicted using a structure-based assessment by in silico QSAR model, employing three endpoints: carcinogenicity, genotoxicity and mutagenicity. In the third stage, the obtained QSAR results were experimentally verified. To verify genotoxicity prediction, in vitro micronucleus assay was employed. It enabled us to assess the potential direct DNA-damaging properties of RAM degradant at high concentrations (as screening series) and at concentrations usually observed in human blood (to mimic the clinical scenario). To verify the QSAR mutagenicity prediction, an in vitro Ames test was carried out. It was designed so as to detect two mechanisms of mutagenicity: a direct one (for pure degradant) and an indirect one (via N-nitroso-metabolites formation). N-nitroso-metabolites for mutagenicity assessment were obtained using NAP test. (3) Results: The kinetic mechanism of RAM degradation was first-order, the degradation rate constant was k = 1.396 ± 0.133 × 10−5 s−1 (T = 373 K), thus the formation of impurity was rapid. Energy of activation was 174.12 ± 46.2 kJ/mol, entropy was positive, thus reaction was bimolecular and favored; enthalpy was 171.65 ± 48.7 kJ/mol, thus reaction was endothermic. Only one degradation impurity was formed, and it was identified as RAM diketopiperazine derivative (DKP). QSAR simulation predicted that DKP could be carcinogenic and genotoxic, but this result had only moderate reliability. DKP was also predicted to be non-mutagenic and this prediction was strong (endpoint score 0.2). The confirmatory micronucleus experiment for genotoxicity prediction suggested that DKP was cytotoxic and it could be also aneugenic at a high concentration (0.22 mg/mL), evidenced by a three-fold increase in micronuclei relative to the control (11.86:33.33%, p = 0.0184). At physiologic concentrations, its cytotoxicity and genotoxicity did not occur. This means that the genotoxicity of DKP was limited by a threshold mechanism. In the mutagenicity in vitro assessment, pure DKP was not mutagenic, but its nitrosation product induced base substitutions mutations in test bacteria TA100 following metabolic activation at a concentration of 4.5 mg/mL, confirming its mutagenicity. (4) Conclusions: RAM rapidly cyclizes to diketopiperazine derivative under dry air. This impurity resides in drugs administered to patients. DKP is potentially aneugenic and cytotoxic at high concentrations, yet at concentrations typically occurring in human blood, this effect is unlikely. The exposure of patients to high concentrations of DKP, exceeding the typical blood level and standard RAM dosing, could lead to cancer development, thus the safe threshold for human exposure to DKP must be verified in follow-up in vivo experiments. Based on our results, it is impossible to establish the maximum safe dose of pure DKP to humans. Furthermore, DKP itself is not mutagenic, but it is liable to the formation of mutagenic nitroso-metabolites in vivo. Nitroso-derivatives of DKP are in vitro mutagens and their real-life impact on humans must be further evaluated in in vivo studies. Until this is carried out, RAM should not be formulated by manufacturers using dry procedures to minimize DKP formation and reduce risk of human carcinogenesis, since DKP could cause cancer via two independent mechanisms: direct genotoxicity when the exposure over standard RAM dosing occurs, and indirect mutagenicity via in vivo N-nitrosamine formation.

Acrylamide and Glycidamide Hemoglobin Adduct Levels and Breast Cancer Risk in Japanese Women: A Nested Case-Control Study in the JPHC.

Acrylamide (AA) is classified as "probably carcinogenic to humans (class 2A)" by the International Agency for Research on Cancer. AA causes cancer owing to its mutagenic and genotoxic metabolite, glycidamide (GA), and its effects on sex hormones. Both AA and GA can interact with hemoglobin to hemoglobin adducts (HbAA and HbGA, respectively), which are considered appropriate biomarkers of internal exposure of AA. However, few epidemiologic studies reported an association of HbAA and HbGA with breast cancer. We conducted a nested case-control study within the Japan Public Health Center-based Prospective Study cohort (125 cases and 250 controls). Cases and controls were categorized into tertiles (lowest, middle, and highest) using the distribution of HbAA or HbGA levels in the control group and estimated ORs and 95% confidence intervals (CI) using conditional logistic regression, adjusting for potential confounders. No association was observed between HbAA (ORHighestvs.Lowest, 1.34; 95% CI, 0.69-2.59), HbGA (ORHighest vs. Lowest, 1.46; 95% CI, 0.79-2.69), their sum HbAA+HbGA (ORHighest vs. Lowest, 1.36; 95% CI, 0.72-2.58) and breast cancer; however, some evidence of positive association was observed between their ratio, HbGA/HbAA, and breast cancer (ORHighest vs. Lowest, 2.19; 95% CI, 1.11-4.31). There was no association between biomarkers of AA and breast cancer. It is unlikely that AA increases breast cancer risk; however, the association of AA with breast cancer may need to be evaluated, with a focus not only on the absolute amount of HbAA or HbGA but also on HbGA/HbAA and the activity of metabolic genes.

Antifungal Activity of Essential Oil and Plant-Derived Natural Compounds against Aspergillus flavus.

Aspergillus flavus is a facultative parasite that contaminates several important food crops at both the pre- and post-harvest stages. Moreover, it is an opportunistic animal and human pathogen that causes aspergillosis diseases. A. flavus also produces the polyketide-derived carcinogenic and mutagenic secondary metabolite aflatoxin, which negatively impacts global food security and threatens human and livestock health. Recently, plant-derived natural compounds and essential oils (EOs) have shown great potential in combatting A. flavus spoilage and aflatoxin contamination. In this review, the in situ antifungal and antiaflatoxigenic properties of EOs are discussed. The mechanisms through which EOs affect A. flavus growth and aflatoxin biosynthesis are then reviewed. Indeed, several involve physical, chemical, or biochemical changes to the cell wall, cell membrane, mitochondria, and related metabolic enzymes and genes. Finally, the future perspectives towards the application of plant-derived natural compounds and EOs in food protection and novel antifungal agent development are discussed. The present review highlights the great potential of plant-derived natural compounds and EOs to protect agricultural commodities and food items from A. flavus spoilage and aflatoxin contamination, along with reducing the threat of aspergillosis diseases.

Quercetin and Isorhamnetin Reduce Benzo[a]pyrene-Induced Genotoxicity by Inducing RAD51 Expression through Downregulation of miR−34a

Benzo[a]pyrene (B[a]P) is metabolized in the liver into highly reactive mutagenic and genotoxic metabolites, which induce carcinogenesis. The mutagenic factors, including B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE) and reactive oxygen species, generated during B[a]P metabolism can cause DNA damage, such as BPDE-DNA adducts, 8-oxo-dG, and double-strand breaks (DSBs). In this study, we mechanistically investigated the effects of quercetin and its major metabolite isorhamnetin on the repair of B[a]P-induced DNA DSBs. Whole-transcriptome analysis showed that quercetin and isorhamnetin each modulate the expression levels of genes involved in DNA repair, especially those in homologous recombination. RAD51 was identified as a key gene whose expression level was decreased in B[a]P-treated cells and increased by quercetin or isorhamnetin treatment. Furthermore, the number of γH2AX foci induced by B[a]P was significantly decreased by quercetin or isorhamnetin, whereas RAD51 mRNA and protein levels were increased. Additionally, among the five microRNAs (miRs) known to downregulate RAD51, miR-34a level was significantly downregulated by quercetin or isorhamnetin. The protective effect of quercetin or isorhamnetin was lower in cells transfected with a miR-34a mimic than in non-transfected cells, and the B[a]P-induced DNA DSBs remained unrepaired. Our results show that quercetin and isorhamnetin each upregulates RAD51 by downregulating miR-34a and thereby suppresses B[a]P-induced DNA damage.

Genotoxicity of pyrrolizidine alkaloids in metabolically inactive human cervical cancer HeLa cells co-cultured with human hepatoma HepG2 cells

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites, which can be found as contaminant in various foods and herbal products. Several PAs can cause hepatotoxicity and liver cancer via damaging hepatic sinusoidal endothelial cells (HSECs) after hepatic metabolization. HSECs themselves do not express the required metabolic enzymes for activation of PAs. Here we applied a co-culture model to mimic the in vivo hepatic environment and to study PA-induced effects on not metabolically active neighbour cells. In this co-culture model, bioactivation of PA was enabled by metabolically capable human hepatoma cells HepG2, which excrete the toxic and mutagenic pyrrole metabolites. The human cervical epithelial HeLa cells tagged with H2B-GFP were utilized as non-metabolically active neighbours because they can be identified easily based on their green fluorescence in the co-culture. The PAs europine, riddelliine and lasiocarpine induced micronuclei in HepG2 cells, and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Metabolic inhibition of cytochrome P450 enzymes with ketoconazole abrogated micronucleus formation. The efflux transporter inhibitors verapamil and benzbromarone reduced micronucleus formation in the co-culture model. Furthermore, mitotic disturbances as an additional genotoxic mechanism of action were observed in HepG2 cells and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Overall, we were able to show that PAs were activated by HepG2 cells and the metabolites induced genomic damage in co-cultured HeLa cells.

Plant-based natural flavonoids show strong inhibition of aflatoxin production and related gene expressions correlated with chemical structure

Aflatoxins are strong carcinogenic and mutagenic fungal metabolites, and aflatoxin contamination is a critical issue in agriculture and food production. Natural flavonoids can suppress aflatoxin biosynthesis; however, the structure–activity relationship remains unclear. In the present study, a total of 36 structurally related natural flavonoids were tested against the aflatoxigenic Aspergillus flavus, both in-vitro and in-situ (on maize kernels), to investigate their structure–activity relationship and biological activity. Aflatoxin production (IC50 values: 10.85–20.09 μg/mL) and the expression of related genes (aflD, aflK, aflQ, and aflR) were found to be strongly inhibited. Structure–activity relationship studies revealed that the [–OH] or [–O–CH3] groups at position 6 of ring A and position 4′ of ring B were closely associated with antifungal and antiaflatoxigenic activities. These findings provide valuable information for the development of clean and safe methods to prevent aflatoxin contamination in food.

Use of Biomarker Data and Relative Potencies of Mutagenic Metabolites to Support Derivation of Cancer Unit Risk Values for 1,3-Butadiene from Rodent Tumor Data.

Unit Risk (UR) values were derived for 1,3-butadiene (BD) based upon its ability to cause tumors in laboratory mice and rats. Metabolism has been established as the significant molecular initiating event of BD’s carcinogenicity. The large quantitative species differences in the metabolism of BD and potency of critical BD epoxide metabolites must be accounted for when rodent toxicity responses are extrapolated to humans. Previously published methods were extended and applied to cancer risk assessments to account for species differences in metabolism, as well as differences in mutagenic potency of BD metabolites within the context of data-derived adjustment factors (DDEFs). This approach made use of biomarker data (hemoglobin adducts) to quantify species differences in the internal doses of BD metabolites experienced in mice, rats, and humans. Using these methods, the dose–response relationships in mice and rats exhibit improved concordance, and result in upper bound UR values ranging from 2.1 × 10−5 to 1.2 × 10−3 ppm−1 for BD. Confidence in these UR values was considered high based on high confidence in the key studies, medium-to-high confidence in the toxicity database, high confidence in the estimates of internal dose, and high confidence in the dose–response modeling.

STUDY THE EFFECTS OF (8-OXO-GUANINE DNA GLYCOSYLASE 1 RS1052133) POLYMORPHISM ON GENE EXPRESSION AS A PREDISPOSING FACTOR FOR DNA MUTATION IN MIDLAND REFINERIES COMPANY-DURA REFINERY WORKERS

Background: Refineries are exposed to different types of air pollutants, among them, polycyclic aromatic hydrocarbons (PAHs), they are a group of over hundred different kinds of hazardous organic chemicals (pollutants), which are formed primarily during the incomplete burning of oil, gas and coal. PAHs are metabolized in different organs of human body especially in the liver leading to generation of mutagenic metabolites, of them, the reactive oxygen species, which damage DNA by binding with guanine base leading to formation of 8-hydroxyguanine. The 8-oxo-guanine DNA glycosylase (hOGG1 rs1052133) gene, is a regulatory gene binds with 8-oxo-DG in the DNA to initiate base excision repair (BER) pathway. Objective: To investigate the role of (hOGG1 rs1052133) gene polymorphism in single nucleotide polymorphism repair. Methods: One hundred sixty-eight (168) subjects were participated in this study. They were they divided into three groups (country side, office workers and field workers). PAHs were analyzed in all participant by Gas Chromatography Coupled with a Mass Spectrophotometry GC/MS. SNP for hOGG1 and gene expression were detecting by real time polymerase chain reaction and quantitative polymerase chain reaction for each subject respectively. Results: PAH were detected in blood serum of refineries workers and not detected in country side participants. The level of hOGG1 gene expression was higher in GG genotype than GC and CC genotype. Conclusion: Refineries worker are at high risk of developing malignant transformation and serious disease due to high level of PAH compared to other population. hOGG1 plays an important role in DNA repair following DNA mutation. Keywords: Poly cyclic aromatic hydrocarbons, HOGGI, gene expression Citation: Al-Ani RH, Al-Wasiti EA, Shukur AS. Study the Effects of (8-oxo-Guanine DNA Glycosylase 1 rs1052133) Polymorphism on Gene Expression as a Predisposing Factor for DNA Mutation in Midland Refineries Company-Dura Refinery Workers. Iraqi JMS. 2022; 20(1): 90-97. doi: 10.22578/IJMS.20.1.12

Efficient Prevention of Aspergillus flavus Spores Spread in Air Using Plasmonic Ag-AgCl/α-Fe2O3 under Visible Light Irradiation.

Aspergillus flavus is a kind of widespread fungi that can produce carcinogenic, teratogenic, and mutagenic secondary metabolites known as aflatoxins. Aspergillus flavus mainly spread through the means of fungal spores in air, thus preventing the spores spread is an effective strategy to control aflatoxins contamination from source. Herein, a rapid and efficient control way to prevent the spread of Aspergillus flavus spores in air was demonstrated. Ag-AgCl nanoparticles were combined with tetrahedral α-Fe2O3 to form plasmonic composites that presented 93.65 ± 1.53% prevention rate of Aspergillus flavus spores under 50 min visible light irradiation. The efficient activity was attributed to the synergy effect of Ag including intrinsic disinfection, electron sink, and localized surface plasmon resonance effect, which were proven by photoelectric characterization, density functional theory, and finite difference time domain methods. The calculated work functions of α-Fe2O3, Ag, and AgCl were 3.71, 4.52, and 5.38 eV, respectively, which could accelerate photoinduced carrier transfer through Ag during photoreaction. Moreover, it was found that the intrinsic disinfection of Ag and hydroxyl radical from photocatalytic reaction were the main factors to the prevention of Aspergillus flavus spores, which resulted in the destruction of spore structure and the leakage of intracellular protein with 62.15 ± 2.63 μg mL-1. Most important, it was proven that the composites also showed high activity (90.52 ± 1.26%) to prevent Aspergillus flavus spore spread in the storage process of peanuts. These findings not only provided useful information for an efficient and potential strategy to prevent Aspergillus flavus contamination but also could be as a reference in toxic fungi control.

Quantitative NanoLC/NSI+-HRMS Method for 1,3-Butadiene Induced bis-N7-guanine DNA-DNA Cross-Links in Urine.

1,3-Butadiene (BD) is a common environmental and industrial chemical widely used in plastic and rubber manufacturing and also present in cigarette smoke and automobile exhaust. BD is classified as a known human carcinogen based on evidence of carcinogenicity in laboratory animals treated with BD by inhalation and epidemiological studies revealing an increased risk of leukemia and lymphohematopoietic cancers in workers occupationally exposed to BD. Upon exposure via inhalation, BD is bioactivated to several toxic epoxides including 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB); these are conjugated with glutathione and excreted as 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-L-cystein-S-yl)-2-hydroxybut-3-ene (MHBMA), 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (DHBMA), and 1,4-bis-(N-acetyl-L-cystein-S-yl)butane-2,3-diol (bis-BDMA). Exposure to DEB generates monoalkylated DNA adducts, DNA-DNA crosslinks, and DNA-protein crosslinks, which can cause base substitutions, genomic rearrangements, and large genomic deletions. In this study, we developed a quantitative nanoLC/NSI+-HRMS methodology for 1,4-bis-(gua-7-yl)-2,3-butanediol (bis-N7G-BD) adducts in urine (LOD: 0.1 fmol/mL urine, LOQ: 1.0 fmol/mL urine). This novel method was used to quantify bis-N7G-BD in urine of mice treated with 590 ± 150 ppm BD for 2 weeks (6 h/day, 5 days/week). Bis-N7G-BD was detected in urine of male and female BD-exposed mice (574.6 ± 206.0 and 571.1 ± 163.4 pg/mg of creatinine, respectively). In addition, major urinary metabolites of BD, bis-BDMA, MHBMA and DHBMA, were measured in the same samples. Urinary bis-N7G-BD adduct levels correlated with DEB-derived metabolite bis-BDMA (r = 0.80, Pearson correlation), but not with the EB-derived DNA adducts (EB-GII) or EB-derived metabolites MHBMA and DHBMA (r = 0.24, r = 0.14, r = 0.18, respectively, Pearson correlations). Urinary bis-N7G-BD could be employed as a novel non-invasive biomarker of exposure to BD and bioactivation to its most mutagenic metabolite, DEB. This method will be useful for future studies of 1,3-butadiene exposure and metabolism.

Towards an advanced testing strategy for genotoxicity using image-based 2D and 3D HepG2 DNA damage response fluorescent protein reporters.

In vitro assessment of mutagenicity is an essential component in the chemical risk assessment. Given the diverse modes of action by which chemicals can induce DNA damage, it is essential that these in vitro assays are carefully evaluated for their possibilities and limitations. In this study, we used a fluorescent protein HepG2 reporter test system in combination with high content imaging. To measure induction of the DNA damage response (DDR), we used three different green fluorescent protein reporters for p53 pathway activation. These allowed for accurate quantification of p53, p21 and BTG2 (BTG anti-proliferation factor 2) protein expression and cell viability parameters at a single cell or spheroid resolution. The reporter lines were cultured as 2D monolayers and as 3D spheroids. Furthermore, liver maturity and cytochrome P450 enzyme expression were increased by culturing in an amino acid-rich (AAGLY) medium. We found that culture conditions that support a sustained proliferative state (2D culturing with normal DMEM medium) give superior sensitivity when genotoxic compounds are tested that do not require metabolisation and of which the mutagenic mode of action is dependent on replication. For compounds, which are metabolically converted to mutagenic metabolites, more differentiated HepG2 DDR reporters (e.g. 3D cultures) showed a higher sensitivity. This study stratifies how different culture methods of HepG2 DDR reporter cells can influence the sensitivity towards diverse genotoxicants and how this provides opportunities for a tiered genotoxicity testing strategy.

A Metabolic Model of Intestinal Secretions: The Link between Human Microbiota and Colorectal Cancer Progression.

The human gut microbiota plays a dual key role in maintaining human health or inducing disorders, for example, obesity, type 2 diabetes, and cancers such as colorectal cancer (CRC). High-throughput data analysis, such as metagenomics and metabolomics, have shown the diverse effects of alterations in dynamic bacterial populations on the initiation and progression of colorectal cancer. However, it is well established that microbiome and human cells constantly influence each other, so it is not appropriate to study them independently. Genome-scale metabolic modeling is a well-established mathematical framework that describes the dynamic behavior of these two axes at the system level. In this study, we created community microbiome models of three conditions during colorectal cancer progression, including carcinoma, adenoma and health status, and showed how changes in the microbial population influence intestinal secretions. Conclusively, our findings showed that alterations in the gut microbiome might provoke mutations and transform adenomas into carcinomas. These alterations include the secretion of mutagenic metabolites such as H2S, NO compounds, spermidine and TMA (trimethylamine), as well as the reduction of butyrate. Furthermore, we found that the colorectal cancer microbiome can promote inflammation, cancer progression (e.g., angiogenesis) and cancer prevention (e.g., apoptosis) by increasing and decreasing certain metabolites such as histamine, glutamine and pyruvate. Thus, modulating the gut microbiome could be a promising strategy for the prevention and treatment of CRC.

Highly Sensitive Detection and Rapid Removal of Picric Acid Using Fe2O3-CdSe Nanocomposite As Fluorescent Probe

Detection of the nitro-fragrant compound at trace level is critical in combating terrorism, for keeping up homeland security, accompanying environmental and clinical safety. Among various nitroaromatics compounds (NACs), the importance of picric acid (PA), also known as 2,4,6-Trinitrophenol (TNP), due to its extensive usage in the production of deadly explosives, fireworks, rocket fuels, leather processing and sensitizers in photographic emulsions. Moreover, it is an odorless, colorless pale yellow solid which profoundly contaminant to soil due to high solvency in an aqueous medium, ends up in adverse effects. The ecological contamination caused by this results in respiratory problems, intense irritation to the skin and eyes, high doses may cause prospective harms in organs and causes cancer, sensitization, destruction, kidney damage, acute hepatitis and diarrhea, etc.[1] The metabolism of these explosive nitro-aromatic compounds end up in generation of reactive nitrogen oxide species, that promptly react with biological macromolecules and cause formation of potent genotoxic and mutagenic metabolites.[2] Therefore, detection and quantitative estimation of those hazardous chemicals are highly indispensable.In the past, there are a number of reports on multifunctional sensors for the detection of NACs, given the appreciable sensitivity and selectivity. As of now, many fluorescence chemosensors/probes have been effectively applied for the detection of PA with functional specificity based on its characteristic properties, particularly resonance energy transfer and electron-deficient nature of the nitro group (-NO2), yet only a few appeared closer to the practical applications.[3] Recent reports on nanoparticle and quantum dot (QD) probes exhibited remarkable chemosensory response towards NACs compared with the conventional organic small molecules, polymers and metal-organic frameworks (MOFs) because of their unique optical properties and thanks to the confinement of electronic states of quantum dots.[4] Recently, thiols capped semiconductor NPs, synthesize through water treatment, has given a fascinating and alternate methodology to synthesize semiconductor NPs. Among these, CdSe nanoparticles are the extensively examined semiconductor NPs primarily attributable to their well-tuned emissive properties process and moderately mature synthetic methods. Late reports on CdSe nanoparticles and expedient to surface modification showed its fate in the detection of NACs.[3] Quantum dots have a clear advantage over molecule-based emitters; however, these multi-model sensors are inexpedient for the evacuation of the earmark species, which might be a source of secondary contamination. From this perspective, the fuse of a fluorescent and magnetic functionality in a solitary nanocomposite molecule would be a promising alternative. This study aimed to focus on the development of this kind nanocomposite, serving a challenge that magnetic NPs possess a strong photoluminescence quenching in the merely fluorescent moiety. Various reports reveal that this undesirable photoluminescence quenching in heterodimer structures comprised of immediate contact between the semiconductor and magnetic domain can be caused by strong electronic coupling.[5] Reasoning from this fact, extra care must be taken to suppress undesirable preventive interactions within the nanocomposite that would revoke the unwelcome properties.In this work, we reported a hybrid nanostructure, comprises a magnetic core of Fe2O3 NPs encapsulated inside a thin silica shell, electrostatically adsorbed with a positively charged spacer arm of florescent Cysteamine-capped CdSe quantum dots (QDs). This promising novel hybrid nanostructure, namely magneto-fluorescent nanosensor (particle size ≈ 12.7 nm), stabilized through strong electrostatic attraction between 3-((Triethoxylsilyl)propylcarbamoyl) butyric acid-anchored Fe2O3@SiO2 NPs and Cysteamine-capped CdSe QDs, electrostatically attached to the free anionic carboxyl group in the spacer arm of Fe2O3@SiO2 NPs. This multimodal nanosensor probe showed a strong specific response to Picric Acid over a number of other explosive (NACs) in DMSO, making them promising fluorescence probes for PA detection and removal, simultaneously, for real-time application. The quenching constant (KPA) of nanosensor with PA was obtained to be 4.3×104 M-1 in DMSO with a limit of detection (LOD) up to 2.2 µM as a consequence of turn off sensing.[6] The sensing mechanism is probed via UV–Vis spectroscopy, steady-state and time-resolved fluorescence spectroscopy. In addition to solution-phase sensing, this nanosensor also showed an excellent ability for the removal of detected PA molecules with the use of an external magnet, staging as a possibility for the potential application of low-cost and stand-off sensor.

The Adverse Effect of Air Pollution with Polycyclic Aromatic hydrocarbon (PAH) on 8-OXO-DG and gene expression (HOGG1) in Midland Refineries Company-Daura Refinery Workers

Introduction: Polycyclic aromatic hydrocarbons (PAHs) are a group of different kind of hazardous organic chemicals which are considered to be the top of pollutants that released by petroleum industries exploration activities to the environment. PAH Metabolites that affect initiation of cancer by reaction with DNA are practically modified chemically by enzymes. PAH Mutagenic metabolites include radical PAH cations, diol epoxides, and quinones. Reactive oxygen species (ROS) mostly produce by routine oxidation processes in mitochondria is responsible for many types of DNA damage. The aim of study identify the DNA alteration due to air pollution by modulation of gene expression and regulatory gene Human 8-oxoguanine DNA Glycosylase (HOOG1) and association with 7,8-dihydro-8-oxoguanine (8-oxo-dg). Methods: there were 168 participants included in this study divided in three groups (control, office workers and in field workers) .PAH, 8-OXO-DG and gene expression (HOGG1) detected for each participant by GC/MS, competitive ELISA and quantitative-competitive reverse transcription-PCR respectively. Results: PAH were not detected in the blood of control group, and there was significant difference in the concentration of PAH between office and field workers. The concentration of (8-oxo DG) and (hOGG1) was significantly higher in field worker than in office worker and control group, significant difference was found between office workers and control. Discussion: It is observed that increased levels of PAH and its metabolites when exposed to polluted air. PAHs metabolism is associated with production of ROS as well as oxidative damaged. Among this damage, the most common lesion is (8-OHdG) .Increase in (8-OHdG) levels associated with increase oxidative stress and (HOGG1) gene. Conclusion: (a) increased PAH and its metabolites levels in serum causing high 8-OHdG and (HOGG1) expression levels in refineries workers (b) there is a correlation between 8-OHdG and hOGG1 expression with PAH.

Nordihydroguaiaretic Acid as a Novel Substrate and Inhibitor of Catechol O-Methyltransferase Modulates 4-Hydroxyestradiol-Induced Cyto- and Genotoxicity in MCF-7 Cells.

Nordihydroguaiaretic acid (NDGA) is a major lignan metabolite found in Larrea spp., which are widely used in South America to treat various diseases. In breast tissue, estradiol is metabolized to the catechol estrogens such as 4-hydroxyestradiol (4-OHE2), which have been proposed to be cancer initiators potentially involved in mammary carcinogenesis. Catechol-O-methyltransferase (COMT) catalyzes the O-methylation of catechol estrogens to their less toxic methoxy derivatives, such as 4-O-methylestradiol (4-MeOE2). The present study investigated the novel biological activities of NDGA in relation to COMT and the effects of COMT inhibition by NDGA on 4-OHE2-induced cyto- and genotoxicity in MCF-7 human breast cancer cells. Two methoxylated metabolites of NDGA, 3-O-methylNDGA (3-MNDGA) and 4-O-methyl NDGA (4-MNDGA), were identified in the reaction mixture containing human recombinant COMT, NDGA, and cofactors. Km values for the COMT-catalyzed metabolism of NDGA were 2.6 µM and 2.2 µM for 3-MNDGA and 4-MNDGA, respectively. The COMT-catalyzed methylation of 4-OHE2 was inhibited by NDGA at an IC50 of 22.4 µM in a mixed-type mode of inhibition by double reciprocal plot analysis. Molecular docking studies predicted that NDGA would adopt a stable conformation at the COMT active site, mainly owing to the hydrogen bond network. NDGA is likely both a substrate for and an inhibitor of COMT. Comet and apurinic/apyrimidinic site quantitation assays, cell death, and apoptosis in MCF-7 cells showed that NDGA decreased COMT-mediated formation of 4-MeOE2 and increased 4-OHE2-induced DNA damage and cytotoxicity. Thus, NDGA has the potential to reduce COMT activity in mammary tissues and prevent the inactivation of mutagenic estradiol metabolites, thereby increasing catechol estrogen-induced genotoxicities.