High Salt-Resistant Urethanase Degrades Ethyl Carbamate in Soy Sauce.

Urethanase is a promising biocatalyst for degrading carcinogen ethyl carbamate (EC) in fermented foods. However, their vulnerability to high ethanol and/or salt and acidic conditions severely limits their applications. In this study, a novel urethanase from Alicyclobacillus pomorum (ApUH) was successfully discovered using a database search. ApUH shares 49.4% sequence identity with the reported amino acid sequences. It belongs to the Amidase Signature family and has a conserved "K-S-S" catalytic triad and the characteristic "GGSS" motif. The purified enzyme overexpressed in Escherichia coli exhibits a high EC affinity (Km, 0.306 mM) and broad pH tolerance (pH 4.0-9.0), with an optimum pH 7.0. Enzyme activity remained at 58% in 12% (w/v) NaCl, and 80% in 10% (v/v) ethanol or after 1 h treatment with the same ethanol solution at 37 °C. ApUH has no hydrolytic activity toward urea. Under 30 °C, the purified enzyme (200 U/L) degraded about 15.4 and 43.1% of the EC in soy sauce samples (pH 5.0, 6.0), respectively, in 5 h. Furthermore, the enzyme also showed high activity toward the class 2A carcinogen acrylamide in foods. These attractive properties indicate their potential applications in the food industry.

CYP2E1 in 1,4-dioxane metabolism and liver toxicity: insights from CYP2E1 knockout mice study.

1,4-Dioxane (DX), an emerging water contaminant, is classified as a Group 2B liver carcinogen based on animal studies. Understanding of the mechanisms of action of DX liver carcinogenicity is important for the risk assessment and control of this environmental pollution. Previous studies demonstrate that high-dose DX exposure in mice through drinking water for up to 3 months caused liver mild cytotoxicity and oxidative DNA damage, a process correlating with hepatic CYP2E1 induction and elevated oxidative stress. To access the role of CYP2E1 in DX metabolism and liver toxicity, in the current study, male and female Cyp2e1-null mice were exposed to DX in drinking water (5000 ppm) for 1 week or 3 months. DX metabolism, redox and molecular investigations were subsequently performed on male Cyp2e1-null mice for cross-study comparisons to similarly treated male wildtype (WT) and glutathione (GSH)-deficient Gclm-null mice. Our results show that Cyp2e1-null mice of both genders were resistant to DX-induced hepatocellular cytotoxicity. In male Cyp2e1-null mice exposed to DX for 3 months, firstly, DX metabolism to ß-hydroxyethoxyacetic acid was reduced to ~ 36% of WT levels; secondly, DX-induced hepatic redox dysregulation (lipid peroxidation, GSH oxidation, and activation of NRF2 antioxidant response) was substantially attenuated; thirdly, liver oxidative DNA damage was at a comparable level to DX-exposed WT mice, accompanied by suppression of DNA damage repair response; lastly, no aberrant proliferative or preneoplastic lesions were noted in DX-exposed livers. Overall, this study reveals, for the first time, that CYP2E1 is the main enzyme for DX metabolism at high dose and a primary contributor to DX-induced liver oxidative stress and associated cytotoxicity. High dose DX-induced genotoxicity may occur via CYP2E1-independent pathway(s), potentially involving impaired DNA damage repair.

One-Week Kava Dietary Supplementation Increases Both Urinary N- and O-Glucuronides of NNAL, a Lung Carcinogen Major Metabolite, among Smokers.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (commonly known as NNK) is one of the most prevalent and potent pulmonary carcinogens in tobacco products that increases the human lung cancer risk. Kava has the potential to reduce NNK and tobacco smoke-induced lung cancer risk by enhancing urinary excretion of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the major metabolite of NNK) and thus reducing NNK-induced DNA damage. In this study, we quantified N-glucuronidated NNAL (NNAL-N-gluc), O-glucuronidated NNAL (NNAL-O-gluc), and free NNAL in the urine samples collected before and after 1-week kava dietary supplementation. The results showed that kava increased both NNAL-N-glucuronidation and O-glucuronidation. Since NNAL-N-glucuronidation is dominantly catalyzed by UGT2B10, its representative single-nucleotide polymorphisms (SNPs) were analyzed among the clinical trial participants. Individuals with any of the four analyzed SNPs appear to have a reduced basal capacity in NNAL-N-glucuronidation. Among these individuals, kava also resulted in a smaller extent of increases in NNAL-N-glucuronidation, suggesting that participants with those UGT2B10 SNPs may not benefit as much from kava with respect to enhancing NNAL-N-glucuronidation. In summary, our results provide further evidence that kava enhances NNAL urinary detoxification via an increase in both N-glucuronidation and O-glucuronidation. UGT2B10 genetic status has not only the potential to predict the basal capacity of the participants in NNAL-N-glucuronidation but also potentially the extent of kava benefits.

Dietary Nitrosamines from Processed Meat Intake as Drivers of the Fecal Excretion of Nitrosocompounds.

Diet is one of the main exogenous sources of potentially carcinogenic nitrosamines (NAs) along with tobacco and cosmetics. Several factors can affect endogenous N-nitroso compounds (NOCs) formation and therefore the potential damage of the intestinal mucosa at initial colorectal cancer stages. To address this issue, 49 volunteers were recruited and classified according to histopathological analyses. Lifestyle and dietary information were registered after colonoscopy. The mutagenicity of fecal supernatants was assayed by a modified Ames test. Fecal heme-derived NOCs and total NOC concentrations were determined by selective denitrosation and chemiluminescence-based detection. Results revealed processed meats as the main source of dietary nitrites and NAs, identifying some of them as predictors of the fecal concentration of heme-derived and total NOCs. Furthermore, increased fecal NOC concentrations were found as the severity of colonic mucosal damage increased from the control to the adenocarcinoma group, these concentrations being strongly correlated with the intake of the NAs N-nitrosodimethylamine, N-nitrosopiperidine, and N-nitrosopyrrolidine. Higher fecal NOC concentrations were also noted in higher fecal mutagenicity samples. These results could contribute to a better understanding of the importance of modulating dietary derived xenobiotics as related with their impact on the intestinal environment and colonic mucosa damage.

Gut microbiota carcinogen metabolism causes distal tissue tumours.

Exposure to environmental pollutants and human microbiome composition are important predisposition factors for tumour development1,2. Similar to drug molecules, pollutants are typically metabolized in the body, which can change their carcinogenic potential and affect tissue distribution through altered toxicokinetics3. Although recent studies demonstrated that human-associated microorganisms can chemically convert a wide range of xenobiotics and influence the profile and tissue exposure of resulting metabolites4,5, the effect of microbial biotransformation on chemical-induced tumour development remains unclear. Here we show that the depletion of the gut microbiota affects the toxicokinetics of nitrosamines, which markedly reduces the development and severity of nitrosamine-induced urinary bladder cancer in mice6,7. We causally linked this carcinogen biotransformation to specific gut bacterial isolates in vitro and in vivo using individualized bacterial culture collections and gnotobiotic mouse models, respectively. We tested gut communities from different human donors to demonstrate that microbial carcinogen metabolism varies between individuals and we showed that this metabolic activity applies to structurally related nitrosamine carcinogens. Altogether, these results indicate that gut microbiota carcinogen metabolism may be a contributing factor for chemical-induced carcinogenesis, which could open avenues to target the microbiome for improved predisposition risk assessment and prevention of cancer.

AKR1C2 genetic variants mediate tobacco carcinogens metabolism involving bladder cancer susceptibility.

Tobacco carcinogens metabolism-related genes (TCMGs) could generate reactive metabolites of tobacco carcinogens, which subsequently contributed to multiple diseases. However, the association between genetic variants in TCMGs and bladder cancer susceptibility remains unclear. In this study, we derived TCMGs from metabolic pathways of polycyclic aromatic hydrocarbons and tobacco-specific nitrosamines, and then explored genetic associations between TCMGs and bladder cancer risk in two populations: a Chinese population of 580 cases and 1101 controls, and a European population of 5930 cases and 5468 controls, along with interaction and joint analyses. Expression patterns of TCMGs were sourced from Nanjing Bladder Cancer (NJBC) study and publicly available datasets. Among 43 TCMGs, we observed that rs7087341 T > A in AKR1C2 was associated with a reduced risk of bladder cancer in the Chinese population [odds ratio (OR) = 0.84, 95% confidence interval (CI) = 0.72-0.97, P = 1.86 × 10-2]. Notably, AKR1C2 rs7087341 showed an interaction effect with cigarette smoking on bladder cancer risk (Pinteraction = 5.04 × 10-3), with smokers carrying the T allele increasing the risk up to an OR of 3.96 (Ptrend < 0.001). Genetically, rs7087341 showed an allele-specific transcriptional regulation as located at DNA-sensitive regions of AKR1C2 highlighted by histone markers. Mechanistically, rs7087341 A allele decreased AKR1C2 expression, which was highly expressed in bladder tumors that enhanced metabolism of tobacco carcinogens, and thereby increased DNA adducts and reactive oxygen species formation during bladder tumorigenesis. These findings provided new insights into the genetic mechanisms underlying bladder cancer.

Mass Spectrometry-Based Method to Measure Aflatoxin B1 DNA Adducts in Formalin-Fixed Paraffin-Embedded Tissues.

Aflatoxin B1 (AFB1) is a potent human liver carcinogen produced by certain molds, particularly Aspergillus flavus and Aspergillus parasiticus, which contaminate peanuts, corn, rice, cottonseed, and ground and tree nuts, principally in warm and humid climates. AFB1 undergoes bioactivation in the liver to produce AFB1-exo-8,9-epoxide, which forms the covalently bound cationic AFB1-N7-guanine (AFB1-N7-Gua) DNA adduct. This adduct is unstable and undergoes base-catalyzed opening of the guanine imidazolium ring to form two ring-opened diastereomeric 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy-aflatoxin B1 (AFB1-FapyGua) adducts. The AFB1 formamidopyrimidine (Fapy) adducts induce G → T transversion mutations and are likely responsible for the carcinogenic effects of AFB1. Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB1-N7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB1-FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB1 DNA adducts in humans, impeding research advances in this potent liver carcinogen. In contrast, formalin-fixed paraffin-embedded (FFPE) specimens used for histopathological analysis are often accessible for molecular studies. However, ensuring nucleic acid quality presents a challenge due to incomplete reversal of formalin-mediated DNA cross-links, which can preclude accurate quantitative measurements of DNA adducts. In this study, employing ion trap or high-resolution accurate Orbitrap mass spectrometry, we demonstrate that ring-opened AFB1-FapyGua adducts formed in AFB1-exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB1-FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS2 measurements can detect AFB1-FapyGua at a quantification limit of 4.0 adducts per 108 bases when only 0.8 µg of DNA is assayed on the column. Thus, our breakthrough DNA retrieval technology can be adapted to screen for AFB1 DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.

Tissue Organoid Cultures Metabolize Dietary Carcinogens Proficiently and Are Effective Models for DNA Adduct Formation.

Human tissue three-dimensional (3D) organoid cultures have the potential to reproduce in vitro the physiological properties and cellular architecture of the organs from which they are derived. The ability of organoid cultures derived from human stomach, liver, kidney, and colon to metabolically activate three dietary carcinogens, aflatoxin B1 (AFB1), aristolochic acid I (AAI), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was investigated. In each case, the response of a target tissue (liver for AFB1; kidney for AAI; colon for PhIP) was compared with that of a nontarget tissue (gastric). After treatment cell viabilities were measured, DNA damage response (DDR) was determined by Western blotting for p-p53, p21, p-CHK2, and γ-H2AX, and DNA adduct formation was quantified by mass spectrometry. Induction of the key xenobiotic-metabolizing enzymes (XMEs) CYP1A1, CYP1A2, CYP3A4, and NQO1 was assessed by qRT-PCR. We found that organoids from different tissues can activate AAI, AFB1, and PhIP. In some cases, this metabolic potential varied between tissues and between different cultures of the same tissue. Similarly, variations in the levels of expression of XMEs were observed. At comparable levels of cytotoxicity, organoids derived from tissues that are considered targets for these carcinogens had higher levels of adduct formation than a nontarget tissue.

Circadian dysfunction induces NAFLD-related human liver cancer in a mouse model.

BACKGROUND & AIMS: Chronic circadian dysfunction increases the risk of non-alcoholic fatty liver disease (NAFLD)-related hepatocellular carcinoma (HCC), but the underlying mechanisms and direct relevance to human HCC have not been established. In this study, we aimed to determine whether chronic circadian dysregulation can drive NAFLD-related carcinogenesis from human hepatocytes and human HCC progression. METHODS: Chronic jet lag of mice with humanized livers induces spontaneous NAFLD-related HCCs from human hepatocytes. The clinical relevance of this model was analysed by biomarker, pathological/histological, genetic, RNA sequencing, metabolomic, and integrated bioinformatic analyses. RESULTS: Circadian dysfunction induces glucose intolerance, NAFLD-associated human HCCs, and human HCC metastasis independent of diet in a humanized mouse model. The deregulated transcriptomes in necrotic-inflammatory humanized livers and HCCs bear a striking resemblance to those of human non-alcoholic steatohepatitis (NASH), cirrhosis, and HCC. Stable circadian entrainment of hosts rhythmically paces NASH and HCC transcriptomes to decrease HCC incidence and prevent HCC metastasis. Circadian disruption directly reprogrammes NASH and HCC transcriptomes to drive a rapid progression from hepatocarcinogenesis to HCC metastasis. Human hepatocyte and tumour transcripts are clearly distinguishable from mouse transcripts in non-parenchymal cells and tumour stroma, and display dynamic changes in metabolism, inflammation, angiogenesis, and oncogenic signalling in NASH, progressing to hepatocyte malignant transformation and immunosuppressive tumour stroma in HCCs. Metabolomic analysis defines specific bile acids as prognostic biomarkers that change dynamically during hepatocarcinogenesis and in response to circadian disruption at all disease stages. CONCLUSION: Chronic circadian dysfunction is independently carcinogenic to human hepatocytes. Mice with humanized livers provide a powerful preclinical model for studying the impact of the necrotic-inflammatory liver environment and neuroendocrine circadian dysfunction on hepatocarcinogenesis and anti-HCC therapy. IMPACT AND IMPLICATIONS: Human epidemiological studies have linked chronic circadian dysfunction to increased hepatocellular carcinoma (HCC) risk, but direct evidence that circadian dysfunction is a human carcinogen has not been established. Here we show that circadian dysfunction induces non-alcoholic steatohepatitis (NASH)-related carcinogenesis from human hepatocytes in a murine humanized liver model, following the same molecular and pathologic pathways observed in human patients. The gene expression signatures of humanized HCC transcriptomes from circadian-disrupted mice closely match those of human HCC with the poorest prognostic outcomes, while those from stably circadian entrained mice match those from human HCC with the best prognostic outcomes. Our studies establish a new model for defining the mechanism of NASH-related HCC and highlight the importance of circadian biology in HCC prevention and treatment.

The adsorption of 2-amino-1-methyl-6-phenyl-imidazolium [4, 5-B] pyridine (PhIP) by lactic acid bacteria 37X-15 and its peptidoglycan.

The heterocyclic amine 2-amino-1-methyl-6-phenyl-imidazolium [4, 5-B] pyridine (PhIP), commonly found in roasted meat products, is considered a potential carcinogen. This study is to explore the underlying mechanisms involved in the adsorption of PhIP by lactic acid bacteria 37X-15 and its peptidoglycan. The scanning electron microscope results suggested that the strain's adsorption on PhIP occurs on the cell wall, primarily composed of peptidoglycan. The fourier-transformed infrared spectroscopy results indicated that PhIP adsorption by both lactic acid bacteria 37X-15 and its peptidoglycan primarily involved OH and NH binding groups. Different adsorption conditions affected the adsorption rate of PhIP by peptidoglycan. The optimal values for each adsorption condition were 2 h, 37 °C, and pH 6 when the maximum adsorption rate reached. This study provides a new direction for the application of lactic acid bacteria and its peptidoglycan in food safety.

Assessment of no-observed-effect-levels for DNA adducts formation by genotoxic carcinogens in fetal turkey livers.

For genotoxic carcinogens, covalent binding to DNA is a critical initiating event in tumorigenesis. The present research investigated dose-effect relationships of three genotoxic carcinogens representing different structural classes, 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P) and quinoline (QUI), to assess the existence of no-observed-effect-levels (NOELs) for the formation of DNA adducts. Carcinogens were administered into the air sac of fertilized turkey eggs over wide dose ranges in three daily injections on days 22 to 24 of incubation. DNA adducts were measured in the fetal turkey livers by the 32P-nucleotide postlabeling (NPL) assay. B[a]P and QUI produced DNA adducts in a dosage-related manner and exhibited NOELs at 0.65 and 0.35 mg/kg bw/day, respectively. In contrast, 2-AAF formed DNA adducts at all tested dosages down to 0.005 mg/kg bw/day. Benchmark dose (BMD) analysis identified the potencies of 2-AAF and QUI to be similar, while B[a]P was the least potent compound. Overall, findings in fetal turkey livers demonstrated that exposure levels to genotoxic compounds that do not result in DNA adducts can exist but are not evident with all carcinogens of this type. The use of mechanistic dose-effect studies for genotoxic endpoints can provide critical information for prioritization of concerns for risk assessment.

27-Hydroxylation of oncosterone by CYP27A1 switches its activity from pro-tumor to anti-tumor.

Oncosterone (6-oxo-cholestane-3ß,5α-diol; OCDO) is an oncometabolite and a tumor promoter on estrogen receptor alpha-positive breast cancer (ER(+) BC) and triple-negative breast cancers (TN BC). OCDO is an oxysterol formed in three steps from cholesterol: 1) oxygen addition at the double bond to give α- or ß- isomers of 5,6-epoxycholestanols (5,6-EC), 2) hydrolyses of the epoxide ring of 5,6-ECs to give cholestane-3ß,5α,6ß-triol (CT), and 3) oxidation of the C6 hydroxyl of CT to give OCDO. On the other hand, cholesterol can be hydroxylated by CYP27A1 at the ultimate methyl carbon of its side chain to give 27-hydroxycholesterol ((25R)-Cholest-5-ene-3beta,26-diol, 27HC), which is a tumor promoter for ER(+) BC. It is currently unknown whether OCDO and its precursors can be hydroxylated at position C27 by CYP27A1, as is the impact of such modification on the proliferation of ER(+) and TN BC cells. We investigated, herein, whether 27H-5,6-ECs ((25R)-5,6-epoxycholestan-3ß,26-diol), 27H-CT ((25R)-cholestane-3ß,5α,6ß,26-tetrol) and 27H-OCDO ((25R)-cholestane-6-oxo-3ß,5α,26-triol) exist as metabolites and can be produced by cells expressing CYP27A1. We report, for the first time, that these compounds exist as metabolites in humans. We give pharmacological and genetic evidence that CYP27A1 is responsible for their production. Importantly, we found that 27-hydroxy-OCDO (27H-OCDO) inhibits BC cell proliferation and blocks OCDO and 27-HC-induced proliferation in BC cells, showing that this metabolic conversion commutes the proliferative properties of OCDO into antiproliferative ones. These data suggest an unprecedented role of CYP27A1 in the control of breast carcinogenesis by inhibiting the tumor promoter activities of oncosterone and 27-HC.

Design, Synthesis, and Biological Studies of Flavone-Based Esters and Acids as Potential P450 2A6 Inhibitors.

As a potential means for smoking cessation and consequently prevention of smoking-related diseases and mortality, in this study, our goal was to investigate the inhibition of nicotine metabolism by P450 2A6. Smoking is the main cause of many diseases and disabilities and harms nearly every organ of the body. As reported by the Centers for Disease Control and Prevention (CDC), more than 16 million Americans are living with diseases caused by smoking. On average, the life expectancy of a smoker is about 10 years less than a nonsmoker. Smoking cessation can substantially reduce the incidence of smoking-related diseases, including cancer. At least, 70 of the more than 7000 cigarette smoke components, including polycyclic aromatic hydrocarbons, N-nitrosamines, and aromatic amines, are known carcinogens. Nicotine is the compound responsible for the addictive and psychopharmacological effects of tobacco. Cytochrome P450 enzymes are responsible for the phase I metabolism of many tobacco components, including nicotine. Nicotine is mainly metabolized by cytochrome P450s 2A6 and 2A13 to cotinine. This metabolism decreases the amount of available nicotine in the bloodstream, leading to increased smoking behavior and thus exposure to tobacco toxicants and carcinogens. Here, we report the syntheses and P450 2A6 inhibitory activities of a number of new flavone-based esters and acids. Three of the flavone derivatives studied were found to be potent competitive inhibitors of the enzyme. Docking studies were used to determine the possible mechanisms of the activity of these inhibitors.

miRNA-214-3p stimulates carcinogen-induced mammary epithelial cell apoptosis in mammary cancer-resistant species.

Mammary cancer incidence varies greatly across species and underlying mechanisms remain elusive. We previously showed that mammosphere-derived epithelial cells from species with low mammary cancer incidence, such as horses, respond to carcinogen 7, 12-Dimethylbenz(a)anthracene-induced DNA damage by undergoing apoptosis, a postulated anti-cancer mechanism. Additionally, we found that miR-214-3p expression in mammosphere-derived epithelial cells is lower in mammary cancer-resistant as compared to mammary cancer-susceptible species. Here we show that increasing miR-214 expression and decreasing expression of its target gene nuclear factor kappa B subunit 1 in mammosphere-derived epithelial cells from horses abolishes 7,12-Dimethylbenz(a)anthracene-induced apoptosis. A direct interaction of miR-214-3p with another target gene, unc-5 netrin receptor A, is also demonstrated. We propose that relatively low levels of miR-214 in mammosphere-derived epithelial cells from mammals with low mammary cancer incidence, allow for constitutive gene nuclear factor kappa B subunit 1 expression and apoptosis in response to 7, 12-Dimethylbenz(a)anthracene. Better understanding of the mechanisms regulating cellular responses to carcinogens improves our overall understanding of mammary cancer resistance mechanisms.

Hypermethylation of the ADIRF promoter regulates its expression level and is involved in NNK-induced malignant transformation of lung bronchial epithelial cells.

The carcinogenic mechanism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a well-known tobacco carcinogen, has not been fully elucidated in epigenetic studies. 5-Methylcytosine (5mC) modification plays a major role in epigenetic regulation. In this study, the 5mC level increased in both BEAS-2B human bronchial epithelium cells treated with 100 mg/L NNK for 24 h and NNK-induced malignant-transformed BEAS-2B cells (2B-NNK cells), suggesting that 5mC modification is associated with the malignant transformation mechanism of NNK. Using a combination of Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq), RNA sequencing (RNA-seq), and bioinformatics analysis of data from the Genomic Data Commons database, we found that the Adipogenesis regulatory factor (ADIRF) promoter region was abnormally hypermethylated, yielding low ADIRF mRNA expression, and that ADIRF overexpression could inhibit the proliferation, migration, and invasion of 2B-NNK cells. This finding suggests that ADIRF plays a tumor suppressor role in the NNK-induced malignant transformation of cells. Subsequently, using 5-Aza-2'-deoxycytidine (5-Aza-2'-dC) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Catalytically Dead Cas9 (dCas9 system), we verified that the demethylation of the ADIRF promoter region in 2B-NNK cells inhibited the proliferation, migration, and invasion ability of the cells and increased their apoptosis ability. These results suggest that abnormal 5mC modification of the ADIRF promoter plays a positive regulatory role in the pathogenesis of NNK-induced lung cancer. This study offers a new experimental basis for the epigenetic mechanism of NNK-induced lung cancer.

Differentiated and non-differentiated HepaRG™ cells: A possible in-vitro model system for early hepatocarcinogenesis and non-genotoxic carcinogens.

Many xenobiotics are non-genotoxic carcinogens (NGC) in rodent liver. Their mode of action (MoA) and health risks for humans are unclear and no in-vitro tests are available to predict NGC. Human HepaRG™ cells in the differentiated (d-HepaRG) and non-differentiated state (nd-HepaRG) were studied as new approach methodology (NAM) for NGC. Cell-biological assays were performed with d-/nd-HepaRG and human hepatoma/hepatocarcinoma cell lines to characterize the benign/malignant phenotype. Reaction of d-/nd-HepaRG to several liver growth factors and NGC (phenobarbital, PB; cyproterone acetate, CPA; WY-14643) was compared to unaltered and premalignant rat hepatocytes in ex-vivo culture. Enzyme induction by NGC was checked by RT-qPCR/oligo-arrays. Growth, anchorage-independency, migration, clonogenicity, and in-vivo tumorigenicity of nd-HepaRG ranged between benign d-HepaRG and malignant hepatoma/hepatocarcinoma cells. All growth factors elevated DNA replication of d-/nd-HepaRG cells, similarly to unaltered/premalignant rat hepatocytes. NGC induced their prototypical enzymes in the rat and human cells, but elicited a growth response only in the unaltered/premalignant rat hepatocytes and not in human d-/nd-HepaRG cells. To conclude, a benign/premalignant phenotype of d-/nd-HepaRG cells and a reactivity towards several hepatic growth factors and NGC, as known from human hepatocytes, are essential components for an in-vitro model for early stage human hepatocarcinogenesis.The potential value as new approach methodology (NAM) for NGC is discussed.

Nuclear DNA and Mitochondrial Damage of the Cooked Meat Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Human Neuroblastoma Cells.

Animal fat and iron-rich diets are risk factors for Parkinson's disease (PD). The heterocyclic aromatic amines (HAAs) harman and norharman are neurotoxicants formed in many foods and beverages, including cooked meats, suggesting a role for red meat in PD. The structurally related carcinogenic HAAs 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (AαC) also form in cooked meats. We investigated the cytotoxicity, DNA-damaging potential, and mitochondrial damage of HAAs and their genotoxic HONH-HAA metabolites in galactose-dependent SH-SY5Y cells, a human neuroblastoma cell line relevant for PD-related neurotoxicity. All HAAs and HONH-HAAs induced weak toxicity except HONH-PhIP, which was 1000-fold more potent than the other chemicals. HONH-PhIP DNA adduct formation occurred at 300-fold higher levels than adducts formed with HONH-MeIQx and HONH-AαC, assuming similar cellular uptake rates. PhIP-DNA adduct levels occurred at concentrations as low as 1 nM and were threefold or higher and more persistent in mitochondrial DNA than nuclear DNA. N-Acetyltransferases (NATs), sulfotransferases, and kinases catalyzed PhIP-DNA binding and converted HONH-PhIP to highly reactive ester intermediates. DNA binding assays with cytosolic, mitochondrial, and nuclear fractions of SH-SY5Y fortified with cofactors revealed that cytosolic AcCoA-dependent enzymes, including NAT1, mainly carried out HONH-PhIP bioactivation to form N-acetoxy-PhIP, which binds to DNA. Furthermore, HONH-PHIP and N-acetoxy-PhIP inhibited mitochondrial complex-I, -II, and -III activities in isolated SH-SY5Y mitochondria. Mitochondrial respiratory chain complex dysfunction and DNA damage are major mechanisms in PD pathogenesis. Our data support the possible role of PhIP in PD etiology.

Effect of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and benzo[a]pyrene exposure on the development of metabolic syndrome in mice.

AIM: The prevalence of metabolic syndrome (MetS), a cluster of serious medical conditions that raise the risk of lung cancer, has increased worldwide. Tobacco smoking (TS) potentially increases the risk of developing MetS. Despite the potential association of MetS with lung cancer, preclinical models that mimic human diseases, including TS-induced MetS, are limited. Here we evaluated the impact of exposure to tobacco smoke condensate (TSC) and two representative tobacco carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNK) and benzo[a]pyrene (BaP), on MetS development in mice. MATERIALS AND METHODS: FVB/N or C57BL/6 mice were exposed to vehicle, TSC, or NNK and BaP (NB) twice weekly for 5 months. The serum levels of total cholesterol (TCHO), triglycerides, high-density lipoprotein (HDL), blood glucose, and metabolites, along with glucose tolerance and body weight, were measured. KEY FINDINGS: Compared with those of vehicle-treated mice, mice with TSC or NB exposure displayed major phenotypes associated with MetS, including increased serum levels of TCHO, triglycerides, and fasting and basal blood glucose and decreased glucose tolerance, and serum levels of HDL. These MetS-associated changes were found in both FVB/N and C57BL/6 mice that were susceptible or resistant to carcinogen-induced tumorigenesis, respectively, indicating that tumor formation is not involved in the TSC- or NB-mediated MetS. Moreover, oleic acid and palmitoleic acid, which are known to be associated with MetS, were significantly upregulated in the serum of TSC- or NB-treated mice compared with those in vehicle-treated mice. SIGNIFICANCE: Both TSC and NB caused detrimental health problems, leading to the development of MetS in experimental mice.

Computational Prediction of Metabolic α-Carbon Hydroxylation Potential of N-Nitrosamines: Overcoming Data Limitations for Carcinogenicity Assessment.

Recent withdrawal of several drugs from the market due to elevated levels of N-nitrosamine impurities underscores the need for computational approaches to assess the carcinogenicity risk of nitrosamines. However, current approaches are limited because robust animal carcinogenicity data are only available for a few simple nitrosamines, which do not represent the structural diversity of the many possible nitrosamine drug substance related impurities (NDSRIs). In this paper, we present a novel method that uses data on CYP-mediated metabolic hydroxylation of CH2 groups in non-nitrosamine xenobiotics to identify structural features that may also help in predicting the likelihood of metabolic α-carbon hydroxylation in N-nitrosamines. Our approach offers a new avenue for tapping into potentially large experimental data sets on xenobiotic metabolism to improve risk assessment of nitrosamines. As α-carbon hydroxylation is the crucial rate-limiting step in nitrosamine metabolic activation, identifying and quantifying the influence of various structural features on this step can provide valuable insights into their carcinogenic potential. This is especially important considering the scarce information available on factors that affect NDSRI metabolic activation. We have identified hundreds of structural features and calculated their impact on hydroxylation, a significant advancement compared to the limited findings from the small nitrosamine carcinogenicity data set. While relying solely on α-carbon hydroxylation prediction is insufficient for forecasting carcinogenic potency, the identified features can help in the selection of relevant structural analogues in read across studies and assist experts who, after considering other factors such as the reactivity of the resulting electrophilic diazonium species, can establish the acceptable intake (AI) limits for nitrosamine impurities.

Systematic Review of Roles of Arecoline and Arecoline N-Oxide in Oral Cancer and Strategies to Block Carcinogenesis.

Betel quid and areca nut are complex mixture carcinogens, but little is known about whether their derived single-agent arecoline or arecoline N-oxide (ANO) is carcinogenic, and the underlying mechanisms remain unclear. In this systematic review, we analyzed recent studies on the roles of arecoline and ANO in cancer and strategies to block carcinogenesis. In the oral cavity, flavin-containing monooxygenase 3 oxidizes arecoline to ANO, and both alkaloids conjugate with N-acetylcysteine to form mercapturic acid compounds, which are excreted in urine, reducing arecoline and ANO toxicity. However, detoxification may not be complete. Arecoline and ANO upregulated protein expression in oral cancer tissue from areca nut users compared to expression levels in adjacent normal tissue, suggesting a causal relationship between these compounds and oral cancer. Sublingual fibrosis, hyperplasia, and oral leukoplakia were diagnosed in mice subjected to oral mucosal smearing of ANO. ANO is more cytotoxic and genotoxic than arecoline. During carcinogenesis and metastasis, these compounds increase the expression of epithelial-mesenchymal transition (EMT) inducers such as reactive oxygen species, transforming growth factor-ß1, Notch receptor-1, and inflammatory cytokines, and they activate EMT-related proteins. Arecoline-induced epigenetic markers such as sirtuin-1 hypermethylation, low protein expression of miR-22, and miR-886-3-p accelerate oral cancer progression. Antioxidants and targeted inhibitors of the EMT inducers used reduce the risk of oral cancer development and progression. Our review findings substantiate the association of arecoline and ANO with oral cancer. Both of these single compounds are likely carcinogenic to humans, and their mechanisms and pathways of carcinogenesis are useful indicators for cancer therapy and prognosis.