ABSTRACT
The 4-biphenylnitrenium ion (BPN), a reactive metabolic intermediate of the tobacco smoke carcinogen 4-aminobiphenyl (4-ABP), can react with nucleophilic sulfanyl groups in glutathione (GSH) as well as in proteins. The main site of attack of these S-nucleophiles was predicted using simple orientational rules of aromatic nucleophilic substitution. Thereafter, a series of presumptive 4-ABP metabolites and adducts with cysteine were synthesized, namely, S-(4-amino-3-biphenyl)cysteine (ABPC), N-acetyl-S-(4-amino-3-biphenyl)cysteine (4-amino-3-biphenylmercapturic acid, ABPMA), S-(4-acetamido-3-biphenyl)cysteine (AcABPC), and N-acetyl-S-(4-acetamido-3-biphenyl)cysteine (4-acetamido-3-biphenylmercapturic acid, AcABPMA). Then, globin and urine of rats dosed with a single ip dose of 4-ABP (27 mg/kg b.w.) was analyzed by HPLC-ESI-MS2. ABPC was identified in acid-hydrolyzed globin at levels of 3.52 ± 0.50, 2.74 ± 0.51, and 1.25 ± 0.12 nmol/g globin (mean ± S.D.; n = 6) on days 1, 3, and 8 after dosing, respectively. In the urine collected on day 1 (0-24 h) after dosing, excretion of ABPMA, AcABPMA, and AcABPC amounted to 1.97 ± 0.88, 3.09 ± 0.75, and 3.69 ± 1.49 nmol/kg b.w. (mean ± S.D.; n = 6), respectively. On day 2, excretion of the metabolites decreased by one order of magnitude followed by a slower decrease on day 8. Regarding the possible formation of AcABPC from ABPC, N-acetylation of the amino group at the biphenyl moiety prior to that at cysteine appears to be very unlikely. Thus, the structure of AcABPC indicates the involvement of N-acetyl-4-biphenylnitrenium ion (AcBPN) and/or its reactive ester precursors in in vivo reactions with GSH and protein-bound cysteine. ABPC in globin might become an alternative biomarker of the dose of toxicologically relevant metabolic intermediates of 4-ABP.
Subject(s)
Carcinogens , Tobacco Smoke Pollution , Rats , Animals , Carcinogens/chemistry , Globins/chemistry , Cysteine/chemistry , Aminobiphenyl Compounds/chemistry , Nicotiana/chemistry , SmokeABSTRACT
Humans are exposed to carcinogenic chemicals via occupational and environmental exposures. Common chemicals of concern that can occur in exposures together are aromatic amines (e.g., 4-aminobiphenyl [4-ABP] and ß-naphthylamine [BNA]) and hexavalent chromium (Cr[VI]). Arylamine N-acetyltransferases 1 and 2 (NAT1 and NAT2) are key to the metabolism of aromatic amines and their genotoxicity. The effects of Cr(VI) on the metabolism of aromatic amines remains unknown as well as how it may affect their ensuing toxicity. The objective of the research presented here is to investigate the effects of Cr(VI) on the metabolism and genotoxicity of 4-ABP and BNA in immortalized human lung epithelial cells (BEP2D) expressing NAT1 and NAT2. Exposure to Cr(VI) for 48 h increased NAT1 activity (linear regression analysis: P < 0.0001) as measured by N-acetylation of para-aminobenzoic acid (PABA) in BEP2D cells but not NAT2 N-acetylation of sulfamethazine, which are prototypic NAT1 and NAT2 substrates respectively. Cr(VI) also increased the N-acetylation of 4-ABP and BNA. In BEP2D cells the N-acetylation of 4-ABP (1-3 µM) exhibited a dose-dependent increase (linear regression analysis: P < 0.05) following co-incubation with 0-3 µM Cr(VI). In BEP2D cells, incubation with Cr(VI) caused dose-dependent increases (linear regression analysis: P < 0.01) in expression of CYP1A1 protein and catalytic activity. For genotoxicity, BEP2D cells were exposed to 4-ABP or BNA with/without Cr(VI) for 48 h. We observed dose-dependent increases (linear regression analysis: P < 0.01) in phospho-γH2AX protein expression for combined treatment of 4-ABP or BNA with Cr(VI). Further using a CYP1A1 inhibitor (α-naphthoflavone) and NAT1 siRNA, we found that CYP1A1 inhibition did not reduce the increased N-acetylation or genotoxicity of BNA by Cr(VI), while NAT1 inhibition did reduce increases in BNA N-acetylation and genotoxicity by Cr(VI). We conclude that during co-exposure of aromatic amines and Cr(VI) in human lung cells, Cr(VI) increased NAT1 activity contributing to increased 4-ABP and BNA genotoxicity.
Subject(s)
Arylamine N-Acetyltransferase , Carcinogens , 2-Naphthylamine , Acetylation , Acetyltransferases/metabolism , Amines/toxicity , Aminobiphenyl Compounds , Arylamine N-Acetyltransferase/genetics , Arylamine N-Acetyltransferase/metabolism , Carcinogens/metabolism , Carcinogens/toxicity , Chromium , Cytochrome P-450 CYP1A1/metabolism , Epithelial Cells/metabolism , Humans , Isoenzymes/genetics , Lung/metabolismABSTRACT
Electrocatalytic oxidation (EO) of carcinogenic 4-aminobiphenyl (4-ABP) aromatic amine was performed using Ti-RuO2 anodes. Current (I), pH, electrolysis time (t), and 4-ABP initial concentration (Co ) were selected as EO parameters, and their effects on %4-ABP removal (R1 ) and energy consumed (R2 ) were studied. Experimental design, parameters optimization, and their interaction with responses R1 and R2 were performed using response surface methodology. At optimized parameters, %TOC removal and 4-BP mineralization current efficiency (%MCE) were assessed to evaluate the potential of Ti/RuO2 anodes towards 4-ABP mineralization. Simultaneous TOC and 4-ABP degradation kinetics were also studied to evaluate the competition in 4-ABP mineralization and degradation. Further, UPLC-Q-TOF-MS analysis was performed to identify the 4-ABP transformation products during the EO, and a mechanism describing the EO transformation was proposed. At optimum parameters (I = 1.2 A; pH = 4.0; t = 30 min; Co = 30 ppm), responses were found to be R1 = 60.25%; R2 = 2.49 kWh/g of 4-ABP removed. %TOC removal and %MCE were 52.4% and 34.2%, respectively. PRACTITIONER POINTS: 4-Aminobiphenyl electro-oxidation (EO) was explored using Ti/RuO2 anode. Achieved 34.2% mineralization current efficiency, 52.4% TOC and 61.3% TKN removal. Three electro-oxidation transformation products of 4-ABP were detected. 4-Aminobiphenyl was found degrading at ≈1.6 times higher rate than TOC A plausible EO transformation pathway and mechanism was proposed.
Subject(s)
Wastewater , Water Pollutants, Chemical , Amines , Aminobiphenyl Compounds , Electrodes , Kinetics , Oxidation-Reduction , Titanium , Wastewater/analysis , Water Pollutants, Chemical/analysisABSTRACT
Exposure of humans to carcinogenic aromatic amines (AAs) occurs daily. AAs are bioactivated in cells into products that attack DNA, primarily leading to N-linked C8-dG adducts. Previous work on DNA containing a single AA-derived adduct (monoadducted DNA) has shown a structure-function relationship between the damaged DNA conformation and cellular outcomes. However, relatively little is known about the conformation and biological outcomes of DNA containing two bulky adducts (diadducted DNA) in close proximity. To fill this current void in the literature, the present work uses quintuplet 0.5 µs MD simulations to understand the structural impact of DNA exposure to the potent bladder carcinogen 4-aminobiphenyl (ABP), which is found in cigarette smoke and select dyes, and results in the widely studied N-linked ABPdG adduct. Specifically, 18 unique DNA duplexes were investigated that contain one or two ABPdG adducts in the anti and/or syn glycosidic orientation(s) in all combinations of three G positions in the NarI mutation hotspot for AAs (5'-G1G2CG3CC). Monoadducted DNA displays sequence-dependent conformational heterogeneity, with the G1 site having the greatest anti preference, and highlights the range of helical structures associated with the syn lesion orientation [i.e., stacked (S), intercalated (I), and wedge (W) conformations]. Diadducted DNA results in interesting lesion separation effects on the conformational heterogeneity, including a greater anti preference for neighboring adducts (G1G2) and a greater syn preference for next-nearest neighbor damaged sites (G2G3) compared to monoadducted DNA. As a result, an increase in the number of ABPdG adducts changes the conformational heterogeneity of ABP-exposed DNA depending on the relative positions of the lesions and thereby could result in increased or decreased toxicity upon human exposure to elevated levels of ABP.
Subject(s)
Carcinogens , Molecular Dynamics Simulation , Aminobiphenyl Compounds , Carcinogens/chemistry , Carcinogens/toxicity , DNA/chemistry , DNA Adducts , DNA Damage , Humans , Nucleic Acid ConformationABSTRACT
A new method was developed for selectively and rapidly detecting carcinogen 4-aminobiphenyl, with lower limit of detection and wider linear range. Up-conversion nanoparticles ß-NaGdF4:Yb3+, Er3+ was the first time to choose as light-emitting signal component. Molecularly imprinted polymers (MIPs) with specific recognition ability were successfully coated on the surface of ß-NaGdF4:Yb3+, Er3+ to obtain a nano fluorescent probe for detecting 4-aminobiphenyl. The effect of addition amount of ß-NaGdF4:Yb3+, Er3+ on the detection ability of ß-NaGdF4:Yb3+, Er3+@MIPs was studied, and composite fluorescence nanoprobe with the best performance was obtained. ß-NaGdF4:Yb3+, Er3+@MIPs were characterized by transmission electron microscopy, X-ray powder diffractometer, Fourier transform infrared spectroscopy and thermogravimetric analysis. The fluorescence intensity of ß-NaGdF4:Yb3+, Er3+@MIPs decreased significantly compared with molecularly non-imprinted polymers ß-NaGdF4:Yb3+, Er3+@NIPs (the maximum emission peak is at 541 nm) in the presence of 4-aminobiphenyl. Adsorption isotherm and adsorption kinetics between UCNP@MIPs and 4-ABP have been investigated and a satisfactory imprinting factor is 2.5. The detection mechanism is proved to be based on Langmuir adsorption and internal filtration effect. Under optimal experimental conditions, the limit of detection and quantification are 0.16 µM and 0.53 µM, respectively. The linear range of response is 1-50 µM, and RSD is less than 6.7%. This method was applied to determining river water samples in order to evaluate the practicability, and the good recovery rate is between 98.89% and 109.7%. These evidences demonstrate that ß-NaGdF4:Yb3+, Er3+@MIPs is successfully used for the detection of 4-aminobiphenyl.
Subject(s)
Molecular Imprinting , Nanoparticles , Adsorption , Aminobiphenyl CompoundsABSTRACT
Aristolochic acid (AA-I) induces upper urothelial tract cancer (UUTC) and bladder cancer (BC) in humans. AA-I forms the 7-(2'-deoxyadenosin-N6-yl)aristolactam I (dA-AL-I) adduct, which induces multiple A:T-to-T:A transversion mutations in TP53 of AA-I exposed UTUC patients. This mutation is rarely reported in TP53 of other transitional cell carcinomas and thus recognized as an AA-I mutational signature. A:T-to-T:A transversion mutations were recently detected in bladder tumors of patients in Asia with known AA-I-exposure, implying that AA-I contributes to BC. Mechanistic studies on AA-I genotoxicity have not been reported in human bladder. In this study, we examined AA-I DNA adduct formation and mechanisms of toxicity in the human RT4 bladder cell line. The biological potencies of AA-I were compared to 4-aminobiphenyl, a recognized human bladder carcinogen, and several structurally related carcinogenic heterocyclic aromatic amines (HAA), which are present in urine of smokers and omnivores. AA-I (0.05-10 µM) induced a concentration- and time-dependent cytotoxicity. AA-I (100 nM) DNA adduct formation occurred at over a thousand higher levels than the principal DNA adducts formed with 4-ABP or HAAs (1 µM). dA-AL-I adduct formation was detected down to a 1 nM concentration. Studies with selective chemical inhibitors provided evidence that NQO1 is the major enzyme involved in AA-I bio-activation in RT4 cells, whereas CYP1A1, another enzyme implicated in AA-I toxicity, had a lesser role in bio-activation or detoxification of AA-I. AA-I DNA damage also induced genotoxic stress leading to p53-dependent apoptosis. These biochemical data support the human mutation data and a role for AA-I in BC.
Subject(s)
Aristolochic Acids/toxicity , Carcinogens/toxicity , DNA Damage/drug effects , Urinary Bladder/drug effects , Aminobiphenyl Compounds/toxicity , Aristolochic Acids/administration & dosage , Carcinogens/administration & dosage , Cell Line, Tumor , Cytochrome P-450 CYP1A1/metabolism , DNA Adducts/metabolism , Dose-Response Relationship, Drug , Humans , Mutation , NAD(P)H Dehydrogenase (Quinone)/metabolism , Tumor Suppressor Protein p53/genetics , Urinary Bladder/pathology , Urinary Bladder Neoplasms/pathologyABSTRACT
4-Aminobiphenyl (4-ABP) may cause DNA damage in human liver cells (HepG2 and L-02). Propolis exhibits antioxidant properties through reactive oxygen species (ROS) scavenging. We determined the effects of propolis in alleviating 4-ABP -induced DNA damage using the comet assay. Results revealed that propolis could significantly alleviated oxidative damaged DNA by 4-ABP. Furthermore, we proved that inhibition of cytochrome P450 2E1 (CYP2E1) expression by propolis could contribute to the decreased oxidative DNA damage in the treated cells, as the conversion of 4-ABP into its metabolite, N-hydroxy-ABP (HOABP), was blocked; after all, HOABP showed more genotoxic than its parent chemical, 4-ABP. With the homologous recombination assay, propolis failed to induce DNA repair enzymes. Furthermore, the expression of RAD51, Ku70/Ku80, and OGG1 in treated cells were determined with the western blot, revealing that the expression of these protein were unchanged in comparison with those in nontreated cells. However, propolis could protect the treated cells from DNA damage. In conclusion, propolis could antagonize 4-ABP-induced oxidative DNA damage though the removal of ROS and inhibition of CYP2E1 expression in the treated cells.
Subject(s)
Cytochrome P-450 CYP2E1 , Propolis , Aminobiphenyl Compounds/pharmacology , Carcinogens , Cytochrome P-450 CYP2E1/metabolism , DNA Damage , Humans , Liver , Oxidative Stress , Propolis/pharmacologyABSTRACT
TP53 harbors somatic mutations in more than half of human tumors with some showing characteristic mutation spectra that have been linked to environmental exposures. In bladder cancer, a unique distribution of mutations amongst several codons of TP53 has been hypothesized to be caused by environmental carcinogens including 4-aminobiphenyl (4-ABP). 4-ABP undergoes metabolic activation to N-hydroxy-4-aminobiphenyl (N-OH-4-ABP) and forms pre-mutagenic adducts in DNA, of which N-(deoxyguanosin-8-yl)-4-ABP (dG-C8-4-ABP) is the major one. Human TP53 knock-in mouse embryo fibroblasts (HUFs) are a useful model to study the influence of environmental carcinogens on TP53-mutagenesis. By performing the HUF immortalization assay (HIMA) TP53-mutant HUFs are generated and mutations can be identified by sequencing. Here we studied the induction of mutations in human TP53 after treatment of primary HUFs with N-OH-4-ABP. In addition, mutagenicity in the bacterial lacZ reporter gene and the formation of dG-C8-4-ABP, measured by 32 P-postlabelling analysis, were determined in N-OH-4-ABP-treated primary HUFs. A total of 6% TP53-mutants were identified after treatment with 40 µM N-OH-4-ABP for 24 hr (n = 150) with G>C/C>G transversion being the main mutation type. The mutation spectrum found in the TP53 gene of immortalized N-OH-4-ABP-treated HUFs was unlike the one found in human bladder cancer. DNA adduct formation (~40 adducts/108 nucleotides) was detected after 24 hr treatment with 40 µM N-OH-4-ABP, but lacZ mutagenicity was not observed. Adduct levels decreased substantially (sixfold) after a 24 hr recovery period indicating that primary HUFs can efficiently repair the dG-C8-4-ABP adduct possibly before mutations are fixed. In conclusion, the observed difference in the N-OH-4-ABP-induced TP53 mutation spectrum to that observed in human bladder tumors do not support a role of 4-ABP in human bladder cancer development.
Subject(s)
Aminobiphenyl Compounds/toxicity , DNA Adducts , DNA Damage , Mutagenesis , Mutagens/toxicity , Mutation , Tumor Suppressor Protein p53/genetics , Animals , Humans , Mice , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
We report a new application of the single-entity electrochemistry (SEE) to in situ measure a partition coefficient at intact nanoemulsions (NEs). The partition coefficient at intact NEs is the most crucial physicochemical property to determine the uptake of delivery molecules inside NEs. It, however, has not been unequivocally elucidated by currently existing techniques based on ex situ measurements. Herein, we apply the single-entity electrochemistry (SEE) to directly and quantitatively measure the partition coefficient at NEs in situ. In this work, we use NEs featured with amphiphilic triblock copolymer (Pluronic F-127) as a model system to extract/preconcentrate 2-aminobiphenyl (2-ABP) dissolved in the water and demonstrate a new application of SEE to in situ quantitatively estimate the amounts of 2-ABP distributed into each intact NE. Our SEE measurements reveal that the partitioning is governed by extraction of 2-ABP inside NEs rather than its adsorption on the NE surface, and this extraction is remarkably efficient with up to â¼8 orders of magnitude of the preconcentration factor, thus leading to the unprecedentedly large partition coefficient of 1.9 (±1.4) × 1010. This result implies that not only the thermodynamic distribution but also the intermolecular interaction of extracted compounds inside NEs could play a significant role in the apparent partition coefficient (P = 1.9 (±1.4) × 1010). The experimentally determined partition coefficient was validated by molecular dynamics (MD) simulations with showing a stabilizing role of intermolecular interaction in the partitioned system. We further verified our methodology with other compounds exhibiting aromatic properties, e.g., ferrocenemethanol. Significantly, our new approach can be readily applicable to investigate practical NEs commercially marketed for drug, food, and cosmetics.
Subject(s)
Aminobiphenyl Compounds/chemistry , Electrochemical Techniques , Nanoparticles/chemistry , Emulsions/chemistry , Particle Size , Surface PropertiesABSTRACT
4-Aminobiphenyl (4-ABP), a well-known human carcinogen, can cause oxidative DNA damage and induce miR-513a-5p. However, the interplay between miR-513a-5p and DNA damage remains unclear. In our result of ChIP assay, we speculated that p53 as transcription factor could regulate miR-513a-5p expression. In addition, we found that miR-513a-5p-induced by 4-ABP could suppress p53 expression and HR repair activity. On the other hand, the levels of p53, miR-513a-5p, and γH2AX were attenuated by 5 mM N-acetyl-l-cysteine (NAC) pretreatment, indicating that the reactive oxygen species (ROS)-dependent p53-miR-513a-5p was involved in DSB repair in 4-ABP-treated cells. These findings indicated that the ROS/p53/miR-513a-5p/p53 loop axis plays a relevant role in regulating HR repair which may facilitate our understanding of molecular mechanisms regarding how miR-513a-5p impacts DSB repair in 4-ABP-treated cells.
Subject(s)
Aminobiphenyl Compounds/toxicity , Carcinogens/toxicity , MicroRNAs/genetics , Recombinational DNA Repair/drug effects , Tumor Suppressor Protein p53/metabolism , Cell Line , DNA Damage , Humans , Reactive Oxygen Species/metabolismABSTRACT
Liver cancer is the third most common cause of cancer-related death but is almost 4-fold more prevalent in men than in women. Increased risk in men may be due in part to elevated chronic inflammation, which is a crucial driving force for many cancers. Male mice also have a greater incidence of liver cancer than females after postnatal exposure to procarcinogens such as 4-aminobiphenyl (ABP) or diethylnitrosamine (DEN), or in mice that transgenically express hepatitis B virus (HBV) proteins. Liver damage, inflammation and proliferation are central to liver cancer development, and previous studies have shown that hepatocellular damage, inflammation and proliferation are acutely elevated to a greater extent in adult male mice than in females after high-dose exposure to DEN. In contrast, postnatal exposure of mice to tumor-inducing doses of either DEN or ABP produces no such acute responses. However, it is not known whether sex differences in responses to postnatal carcinogen exposure or to HBV protein expression may develop over time following sexual maturation. We conducted an extended time course study to compare markers of liver damage, inflammation and proliferation between male and female mice exposed postnatally to 600 nmol ABP or 10 mg/kg DEN, and also in HBV transgenic (HBVTg) mice, over the duration of time that mice are normally maintained for standard liver tumor development protocols. Postnatal exposure to either ABP or DEN produced no evidence of either acute or chronic hepatocyte damage, liver inflammation or proliferation in either male or female mice. In contrast, HBVTg mice showed increased liver damage, inflammation and proliferation with age, but with no observed sex difference. These findings suggest that although chronic liver damage, inflammation and proliferation may be drivers for liver cancer development, they are unlikely to contribute directly to observed sex differences in liver tumor risk.
Subject(s)
Carcinogenesis/chemically induced , Cell Proliferation/drug effects , Chemical and Drug Induced Liver Injury/pathology , Liver Neoplasms, Experimental/pathology , Aging/pathology , Aminobiphenyl Compounds/toxicity , Animals , Carcinogenesis/pathology , Diethylnitrosamine/toxicity , Female , Hepatitis B virus/metabolism , Liver Function Tests , Liver Neoplasms, Experimental/chemically induced , Male , Mice , Mice, Inbred C57BL , Sex Characteristics , Viral Proteins/biosynthesisABSTRACT
Rationale: Cartilage stem/progenitor cells (CSPC) are a promising cellular source to promote endogenous cartilage regeneration in osteoarthritis (OA). Our previous work indicates that ribosomal s6 kinase 3 (RSK-3) is a target of 4-aminobiphenyl, a chemical enhancing CSPC-mediated cartilage repair in OA. However, the primary function and mechanism of RSK-3 in CSPC-mediated cartilage pathobiology remain undefined. Methods: We systematically assessed the association of RSK-3 with OA in three mouse strains with varying susceptibility to OA (MRL/MpJ>CBA>STR/Ort), and also RSK-3-/- mice. Bioinformatic analysis was used to identify the possible mechanism of RSK-3 affecting CSPC, which was further verified in OA mice and CSPC with varying RSK-3 expression induced by chemicals or gene modification. Results: We demonstrated that the level of RSK-3 in cartilage was positively correlated with cartilage repair capacities in three mouse strains (MRL/MpJ>CBA>STR/Ort). Enhanced RSK-3 expression by 4-aminobiphenyl markedly attenuated cartilage injury in OA mice and inhibition or deficiency of RSK-3 expression, on the other hand, significantly aggravated cartilage damage. Transcriptional profiling of CSPC from mice suggested the potential role of RSK-3 in modulating cell proliferation. It was further shown that the in vivo and in vitro manipulation of the RSK-3 expression indeed affected the CSPC proliferation. Mechanistically, ribosomal protein S6 (rpS6) was activated by RSK-3 to accelerate CSPC growth. Conclusion: RSK-3 is identified as a key regulator to enhance cartilage repair, at least partly by regulating the functionality of the cartilage-resident stem/progenitor cells.
Subject(s)
Cartilage/cytology , Chondrocytes/cytology , Osteoarthritis/therapy , Regeneration , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Ribosomal Protein S6/metabolism , Stem Cells/cytology , Aminobiphenyl Compounds/pharmacology , Animals , Carcinogens/pharmacology , Cartilage/drug effects , Cartilage/metabolism , Cells, Cultured , Chondrocytes/drug effects , Chondrocytes/metabolism , Disease Models, Animal , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Inbred MRL lpr , Mice, Knockout , Osteoarthritis/metabolism , Osteoarthritis/pathology , Stem Cells/drug effects , Stem Cells/metabolismABSTRACT
4-Aminobiphenyl (4-ABP), a well-known human carcinogen, has been shown to cause oxidative DNA damage and induce miR-630 expression in HepG2 cells treated with 18.75 µM-300 µM for 24 h. However, the underlying mechanism regarding the epigenetic regulation of miR-630 on DNA damage repair in liver cells is still not understood and needs to be investigated. In present study, our results showed that miR-630 was upregulated, resulting in mediating a decrease of DNA homologous recombination (HR) repair in L-02, HepG2 or Hep3B cells. Results from a luciferase reporting experiment showed that RAD18 and MCM8 were the potential targets of miR-630 during DNA damage induction. The downregulation of RAD18 or MCM8 by miR-630 was accompanied by inhibition of HR repair. Conversely, inhibiting miR-630 enhanced the expression of RAD18 and MCM8, and rescued HR repair. Additionally, we proved that the transcription factor CREB was related to miR-630 biogenesis in liver cells. Moreover, the levels of CREB, miR-630 expression, and double-strand breaks (DSBs) were attenuated by 5 mM N-acetyl-L-cysteine (NAC) pretreatment, indicating that reactive oxygen species (ROS)-dependent CREB-miR-630 was involved in DSB repair. These findings indicated that the ROS/CREB/-miR-630 axis plays a relevant role in the regulation of RAD18 and MCM8 in HR repair, which may facilitate our understanding of molecular mechanisms regarding the role of miR-630 downregulating DNA damage repair in liver cells.
Subject(s)
Aminobiphenyl Compounds/pharmacology , DNA-Binding Proteins/antagonists & inhibitors , Liver/metabolism , MicroRNAs/metabolism , Minichromosome Maintenance Proteins/antagonists & inhibitors , Recombinational DNA Repair/drug effects , Ubiquitin-Protein Ligases/antagonists & inhibitors , Acetylcysteine/pharmacology , Cell Line , Cyclic AMP Response Element-Binding Protein/biosynthesis , DNA Breaks, Double-Stranded/drug effects , Free Radical Scavengers/pharmacology , Homologous Recombination , Humans , Liver/drug effects , Reactive Oxygen Species/metabolismABSTRACT
In this work, a rapid and sensitive thin-layer chromatography combined with surface-enhanced Raman spectroscopy method was established for rapid detection of benzidine and 4-aminobiphenyl in migration from food contact materials based on Au nanoparticle doped metal-organic framework. Benzidine and 4-aminobiphenyl were firstly separated by thin-layer chromatography to solve the limitation of their overlapping Raman peaks. Then the target molecules were monitored by adding AuNPs/MIL-101(Cr) on the sample spots. Under the optimum conditions, the concentration of benzidine and 4-aminobiphenyl can be quantitatively measured in the range of 2.0-20.0 and1.0-15.0 µg/L, respectively with good linear relationship, and the limits of detection were 0.21 and 0.23 µg/L, respectively. Furthermore, the developed method was applied to analyze benzidine and 4-aminobiphenyl in migration of different food contact materials. The recoveries of benzidine and 4-aminobiphenyl for migration of food contact materials, including paper cups, polypropylene food containers, and polyethylene glycol terephthalate bottles, were 80.6-116.0 and 80.7-118% with relative standard deviations of 1.1-9.1 and 3.1-9.9%, respectively. Surface-enhanced Raman scattering detection was performed conveniently in the on-plate mode without additional elution process. The method shows great potential in rapid monitoring of hazardous substances with overlapping characteristic Raman peaks in food contact materials.
Subject(s)
Aminobiphenyl Compounds/analysis , Benzidines/analysis , Food Contamination/analysis , Food Packaging , Metal-Organic Frameworks/chemistry , Chromatography, Thin Layer , Gold/chemistry , Metal Nanoparticles/chemistry , Spectrum Analysis, Raman , Surface PropertiesABSTRACT
Muscarinic M3 receptor antagonists and inverse agonists displaying high affinity and subtype selectivity over the antitarget M2 are valuable pharmacological tools and may enable improved treatment of chronic obstructive pulmonary disease (COPD), asthma, or urinary incontinence. On the basis of known M3 antagonists comprising a piperidine or quinuclidine unit attached to a biphenyl carbamate, 5-fluoro substitution was responsible for M3 subtype selectivity over M2, while 3'-chloro substitution substantially increased affinity through a σ-hole interaction. Resultantly, two piperidinyl- and two quinuclidinium-substituted biphenyl carbamates OFH243 (13n), OFH244 (13m), OFH3911 (14n), and OFH3912 (14m) were discovered, which display two-digit picomolar affinities with Ki values from 0.069 to 0.084 nM, as well as high selectivity over the M2 subtype (46- to 68-fold). While weak inverse agonistic properties were determined for the biphenyl carbamates 13m and 13n, neutral antagonism was observed for 14m and 14n and tiotropium under identical assay conditions.
Subject(s)
Aminobiphenyl Compounds/chemistry , Drug Inverse Agonism , Halogens/chemistry , Muscarinic Agonists/chemistry , Muscarinic Antagonists/chemistry , Receptor, Muscarinic M3/agonists , Receptor, Muscarinic M3/antagonists & inhibitors , Aminobiphenyl Compounds/pharmacology , Animals , Caco-2 Cells , HEK293 Cells , Halogens/pharmacology , Humans , Male , Molecular Docking Simulation/methods , Muscarinic Agonists/metabolism , Muscarinic Agonists/pharmacology , Muscarinic Antagonists/pharmacology , Protein Binding/physiology , Protein Structure, Secondary , Rats , Rats, Sprague-Dawley , Receptor, Muscarinic M3/metabolismABSTRACT
Arylamine N-acetyltransferases are xenobiotic-metabolizing enzymes responsible for detoxification of many drugs and carcinogens. Two N-acetyltransferase proteins (NAT1 and NAT2) are expressed in humans and they both N-acetylate aromatic amine carcinogens such as 4-aminobiphenyl. Arylamines such as 4-aminobiphenyl represent a large class of chemical carcinogens. Exposure to 4-aminobiphenyl occurs in the chemical, dye and rubber industries as well as in hair dyes, paints, and cigarette smoke. NAT2 is subject to a genetic polymorphism resulting in rapid, intermediate and slow acetylator phenotypes. We investigated the role of the NAT2 genetic polymorphisms on the N-acetylation of 4-aminobiphenyl in cryopreserved human hepatocytes in which NAT2 genotype and deduced phenotype were determined. Differences in sulfamethazine (selectively N-acetylated via NAT2) and 4-aminobiphenyl (N-acetylated by both NAT1 and NAT2) N-acetylation rates among rapid, intermediate, and slow NAT2 acetylator genotypes were tested for significance by one-way analysis of variance. In vitro 4-aminobiphenyl N-acetyltransferase activities differed significantly between rapid, intermediate and slow acetylators at 10 µM (P = 0.0102) or 100 µM (P = 0.0028). N-acetylation of 4-aminobiphenyl in situ also differed significantly between human hepatocytes from rapid, intermediate, and slow acetylators at 10 µM (P = 0.0015) and 100 µM (P = 0.0216). A gene dose-response relationship was exhibited as intermediate acetylators catalyzed 4-aminobiphenyl N-acetylation both in vitro and in situ at rates arithmetically between rapid and slow acetylators. In conclusion, N-acetylation of 4-aminobiphenyl is NAT2 genotype-dependent in human hepatocytes. These results suggest refinement of the exposure limit and safety for arylamine carcinogens according to NAT2 genotype.
Subject(s)
Aminobiphenyl Compounds/metabolism , Arylamine N-Acetyltransferase/genetics , Arylamine N-Acetyltransferase/metabolism , Hepatocytes/enzymology , Acetylation , Carcinogens/metabolism , Cryopreservation , Genetic Association Studies , Genotype , Hepatocytes/metabolism , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Phenotype , Polymorphism, Genetic , Sulfamethazine/metabolismABSTRACT
Purpose: The present study evaluated biochemical as well as biophysical mechanisms behind the synergistic effects of curcumin and resveratrol during prostate carcinogenesis.Methods: The rats were segregated into five groups that included normal control, 3,2'-dimethyl-4-aminobiphenyl (DMAB)treated, DMAB + curcumin treated, DMAB + resveratrol-treated and DMAB + curcumin + resveratrol-treated.Results: The DMAB treatment resulted in a significant increase in the levels of lipid peroxidation (LPO) in DMAB treated rats. Also, significant changes were recorded in the enzyme activities of both drug metabolising enzyme and antioxidant enzymes after DMAB treatment. Further, radiorespirometric studies showed a significant increase in the 14C-glucose turnover as well as 14C-glucose uptake in the prostate slices of DMAB treated rats. Moreover, a significant rise in cell proliferation was confirmed indirectly by enhanced uptake of 3H-thymidine in the prostate slices of DMAB treated rats. Interestingly, combined treatment of curcumin and resveratrol to DMAB treated animals resulted in a significant decrease in lipid peroxidation, 14C glucose uptakes/turnover and 3H-thymidine uptake in the DMAB treated rats. Besides this, curcumin and resveratrol in combination significantly modulated biochemical indices including drug-metabolising enzymes; antioxidant enzymes in DMBA treated rats.Conclusion: The study, therefore, concludes that the combination of curcumin and resveratrol holds strong modulatory potential against prostate carcinogenesis.
Subject(s)
Antineoplastic Agents/pharmacology , Curcumin/pharmacology , Prostatic Neoplasms/drug therapy , Resveratrol/pharmacology , Aminobiphenyl Compounds/pharmacology , Animals , Antioxidants/administration & dosage , Antioxidants/pharmacology , Cell Proliferation/drug effects , Curcumin/administration & dosage , Curcumin/pharmacokinetics , Disease Models, Animal , Drug Therapy, Combination , Glucose/metabolism , Lipid Peroxidation/drug effects , Male , Random Allocation , Rats , Rats, Wistar , Resveratrol/administration & dosage , Resveratrol/pharmacokinetics , Thymidine/metabolismABSTRACT
Carcinogenic aromatic amines such as 4-aminobiphenyl (ABP) and 2-aminofluorene (AF) require metabolic activation to form electrophilic intermediates that mutate DNA leading to carcinogenesis. Bioactivation of these carcinogens includes N-hydroxylation catalyzed by CYP1A2 followed by O-acetylation catalyzed by arylamine N-acetyltransferase 2 (NAT2). To better understand the role of NAT2 genetic polymorphism in ABP- and AF-induced mutagenesis and DNA damage, nucleotide excision repair-deficient (UV5) Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. ABP and AF both caused significantly (P < 0.001) greater mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in the UV5/CYP1A2/NAT2*4 acetylator cell line compared to the UV5, UV5/CYP1A2, and UV5/CYP1A2/NAT2*5B cell lines. ABP- and AF-induced hprt mutant cDNAs were sequenced and over 80% of the single-base substitutions were at G:C base pairs. DNA damage also was quantified by γH2AX in-cell western assays and by identification and quantification of the two predominant DNA adducts, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) by liquid chromatography-mass spectrometry. DNA damage and adduct levels were dose-dependent, correlated highly with levels of hprt mutants, and were significantly (P < 0.0001) greater in the UV5/CYP1A2/NAT2*4 rapid acetylator cell line following treatment with ABP or AF as compared to all other cell lines. Our findings provide further clarity on the importance of O-acetylation in CHO mutagenesis assays for aromatic amines. They provide evidence that NAT2 genetic polymorphism modifies aromatic amine-induced DNA damage and mutagenesis that should be considered in human risk assessments following aromatic amine exposures. Environ. Mol. Mutagen. 61:235-245, 2020. © 2019 Wiley Periodicals, Inc.
Subject(s)
Aminobiphenyl Compounds/metabolism , Arylamine N-Acetyltransferase/genetics , Carcinogens/metabolism , Fluorenes/metabolism , Polymorphism, Genetic , Acetylation , Aminobiphenyl Compounds/toxicity , Animals , Arylamine N-Acetyltransferase/metabolism , CHO Cells , Carcinogens/toxicity , Cricetinae , Cricetulus , DNA Damage/drug effects , Fluorenes/toxicity , Humans , Mutagenesis/drug effects , Mutagenicity TestsABSTRACT
Rationale The small molecule Kartogenin (KGN) promotes cartilage regeneration in osteoarthritis (OA) by activating stem cells differentiation, but its pharmacological mode-of-action remains unclear. KGN can be cleaved into 4-aminobiphenyl (4-ABP) and phthalic acid (PA) following enzymolysis of an amide bond. Therefore, this study investigated whether 4-ABP or PA exerted the same action as KGN. Methods KGN, 4-ABP and PA were analyzed in cartilage of mice after oral, intravenous or intra-articular administration of KGN by liquid chromatography-mass spectrometry method. Their effect on proliferation and chondrogenic differentiation of mesenchymal stem cells (MSC) was evaluated in vitro. Furthermore, their effect on cartilage preservation was tested in mice OA model induced by destabilization of medial meniscus. OA severity was quantified using OARSI histological scoring. Transcriptional analysis was used to find the possible targets of the chemicals, which were further validated. Results We demonstrated that while oral or intra-articular KGN delivery effectively ameliorated OA phenotypes in mice, only 4-ABP was detectable in cartilage. 4-ABP could induce chondrogenic differentiation and proliferation of MSC in vitro and promote cartilage repair in OA mouse models mainly by increasing the number of CD44+/CD105+ stem-cell and prevention of matrix loss. These effect of 4-ABP was stronger than that of KGN. Transcriptional profiling of 4-ABP-stimulated MSC suggested that RPS6KA2 and the PI3K-Akt pathway were 4-ABP targets; 4-ABP could activate the PI3K-Akt pathway to promote MSC proliferation and repair OA injury, which was blocked in RPS6KA2-knockdown MSC or RPS6KA2-deficient mice.Conclusion 4-ABP bio-distribution in cartilage promotes proliferation and chondrogenic differentiation of MSC, and repairs osteoarthritic lesions via PI3K-Akt pathway activation.
Subject(s)
Aminobiphenyl Compounds/metabolism , Anilides/metabolism , Cartilage/metabolism , Phthalic Acids/metabolism , Regeneration , Administration, Oral , Anilides/administration & dosage , Anilides/pharmacology , Animals , Antigens, CD/metabolism , Cartilage/drug effects , Cartilage/injuries , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Chondrogenesis/drug effects , Humans , Hydrolysis , Male , Meniscus/drug effects , Meniscus/pathology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Mice , Phosphatidylinositol 3-Kinases/metabolism , Phthalic Acids/administration & dosage , Phthalic Acids/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Regeneration/drug effects , Signal Transduction/drug effects , Tissue Distribution/drug effectsABSTRACT
Cathepsin B plays key roles in tumor progression with its overexpression being associated with invasive and metastatic phenotypes and is a primary target of protease activated antibody-directed prodrug therapy. It therefore represents a potential therapeutic and diagnostic target and effort has been made to develop fluorescent probes to report on Cathepsin B activity in cells and animal models of cancer. We have designed, synthesized, and thoroughly evaluated four novel "turn on" probes that employ a lysosomotropic dansylcadaverine dye to report on Cathepsin B activity. Enzyme activity assays using a recombinant human enzyme and cancer cell lysates coupled with confocal microscopy experiments demonstrated that one of the probes, derivatized with the self-immolative prodrug linker p-aminobenzyl alcohol, can selectively report on Cathepsin B in biological samples including live cells.