PD-1 Blockade Prevents the Development and Progression of Carcinogen-Induced Oral Premalignant Lesions.

Oral squamous cell carcinoma (OSCC) is preceded by progressive oral premalignant lesions (OPL). Therefore, therapeutic strategies that prevent malignant progression of OPLs are expected to reduce the incidence of OSCC development. Immune checkpoint inhibitors that target the interaction of programmed death receptor 1 (PD-1) on T cells with the PD-1 ligand PD-L1 on cancer cells have been shown to extend the survival of patients with advanced OSCC. Here, we used the 4-nitroquinoline-1-oxide (4-NQO) mouse model of oral carcinogenesis to test the hypothesis that PD-1 blockade may control the progression of OPLs. Mice were exposed to 4-NQO in their drinking water and then randomly assigned to two treatment groups that received either a blocking antibody for PD-1 or a control IgG. We found that anti-PD-1 treatment significantly reduced the number of oral lesions that developed in these mice and prevented malignant progression. Low-grade dysplastic lesions responded to PD-1 blockade with a significant increase in the recruitment of CD8+ and CD4+ T cells and the accumulation of CTLA-4+ T cells in their microenvironment. Notably, PD-1 inhibition was accompanied by induction of IFNγ, STAT1 activation and the production of the T-cell effector granzyme B in infiltrating cells, and by the induction of apoptosis in the epithelial cells of the oral lesions, suggesting that T-cell activation mediates the immunopreventive effects of anti-PD-1. These results support the potential clinical benefit of PD-1 immune checkpoint blockade to prevent OSCC development and progression and suggest that CTLA-4 inhibitors may enhance the preventive effects of anti-PD-1. Cancer Prev Res; 10(12); 684-93. ©2017 AACRSee related editorial by Gutkind et al., p. 681.

Alleviation of 4-nitroquinoline 1-oxide induced oxidative stress by Oroxylum indicum (L.) leaf extract in albino Wistar rats.

BACKGROUND: 4-nitroquinoline 1-oxide (4-NQO) is a mutagen known to be responsible for causing cancer by generating oxidative stress in humans. Oroxylum indicum (L.) possesses various bioactive compounds with antioxidant properties. In this connection, the present study aims to analyze the alleviation of 4-NQO induced oxidative stress in albino Wistar rats using O. indicum (L.) leaf extract. METHODS: O. indicum (L.) belonging to the family Bignoniaceae, has anticancer and anti-inflammatory properties. In this study, we observed severe oxidative stress in 4-NQO induced albino Wistar rats when compared to untreated control. Alleviation of this condition was seen after the oral administration of O. indicum (L.) leaf extract at 50, 100, and 200 mg/kg body weight. RESULTS: 4-NQO (50 ppm) administration in drinking water resulted in the generation of reactive oxygen species (ROS) leading to cellular damage, lipid peroxidation and imbalance in antioxidant status. Administration of O. indicum (L.) leaf extract has alleviated the level of 4-NQO induced oxidative stress by increasing the antioxidant status and decreasing the elevation of liver markers in serum. CONCLUSIONS: Results clearly suggest that O. indicum (L.) leaf extract when administered orally in a dose dependent manner has the ability to overcome the oxidative stress induced by 4-NQO with hepatoprotective and lipid protective properties.

Gene expression profiling signatures for the diagnosis and prevention of oral cavity carcinogenesis-genome-wide analysis using RNA-seq technology.

We compared the changes in global gene expression between an early stage (the termination of the carcinogen treatment and prior to the appearance of frank tumors) and a late stage (frank squamous cell carcinoma (SCC)) of tongue carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model of human oral cavity and esophageal squamous cell carcinoma. Gene ontology and pathway analyses show that increases in "cell cycle progression" and "degradation of basement membrane and ECM pathways" are early events during SCC carcinogenesis and that changes in these pathways are even greater in the actual tumors. Myc, NFκB complex (NFKB1/RELA), and FOS transcription networks are the major transcriptional networks induced in early stage tongue carcinogenesis. Decreases in metabolism pathways, such as in "tricarboxylic acid cycle" and "oxidative phosphorylation", occurred only in the squamous cell carcinomas and not in the early stages of carcinogenesis. We detected increases in ALDH1A3, PTGS2, and KRT1 transcripts in both the early and late stages of carcinogenesis. The identification of the transcripts and pathways that change at an early stage of carcinogenesis provides potentially useful information for early diagnosis and for prevention strategies for human tongue squamous cell carcinomas.

Characterization of the mutagenic spectrum of 4-nitroquinoline 1-oxide (4-NQO) in Aspergillus nidulans by whole genome sequencing.

4-Nitroquinoline 1-oxide (4-NQO) is a highly carcinogenic chemical that induces mutations in bacteria, fungi, and animals through the formation of bulky purine adducts. 4-NQO has been used as a mutagen for genetic screens and in both the study of DNA damage and DNA repair. In the model eukaryote Aspergillus nidulans, 4-NQO-based genetic screens have been used to study diverse processes, including gene regulation, mitosis, metabolism, organelle transport, and septation. Early work during the 1970s using bacterial and yeast mutation tester strains concluded that 4-NQO was a guanine-specific mutagen. However, these strains were limited in their ability to determine full mutagenic potential, as they could not identify mutations at multiple sites, unlinked suppressor mutations, or G:C to C:G transversions. We have now used a whole genome resequencing approach with mutant strains generated from two independent genetic screens to determine the full mutagenic spectrum of 4-NQO in A. nidulans. Analysis of 3994 mutations from 38 mutant strains reveals that 4-NQO induces substitutions in both guanine and adenine residues, although with a 19-fold preference for guanine. We found no association between mutation load and mutagen dose and observed no sequence bias in the residues flanking the mutated purine base. The mutations were distributed randomly throughout most of the genome. Our data provide new evidence that 4-NQO can potentially target all base pairs. Furthermore, we predict that current practices for 4-NQO-induced mutagenesis are sufficient to reach gene saturation for genetic screens with feasible identification of causative mutations via whole genome resequencing.

Combination of bexarotene and the retinoid CD1530 reduces murine oral-cavity carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide.

We investigated the effects of bexarotene (a retinoid X receptor agonist), CD1530 (a retinoic acid receptor γ selective agonist), and the combination of these two drugs for the prevention of oral carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model of human oral-cavity and esophageal squamous-cell carcinoma previously generated in our laboratory. We observed decreased numbers of neoplastic tongue lesions and reduced lesion severity in the 4-NQO plus CD1530 (4N+C) and 4-NQO plus bexarotene plus CD1530 (4N+B+C) groups compared with the 4-NQO group. RNA-Seq analyses showed increases in transcripts in cell proliferation/cell cycle progression pathways in the 4-NQO vs. the untreated group. In addition, ß-catenin and matrix metallopeptidase 9 (MMP9) protein levels and reactive oxygen species (ROS), as assessed by 4-hydroxynonenal (4-HNE) staining, were elevated in tongue tissues 17 wk after the termination of the 4-NQO treatment. The 4N+B, 4N+C, and 4N+B+C groups showed dramatically lower levels of ß-catenin, MMP9, and 4-HNE staining compared with the 4-NQO group. The major reduction in 4-HNE staining in the retinoid treatment groups suggests a novel mechanism of action, reduction of ROS, by which bexarotene and CD1530 inhibit carcinogenesis.

Confirmation of Frm2 as a novel nitroreductase in Saccharomyces cerevisiae.

Nitroreductases comprise a group of FMN- or FAD-dependent enzymes that reduce nitrosubstituted compounds by using NAD(P)H, and are found in bacterial species and yeast. Although there is little information on the biological functions of nitroreductases, some studies suggest their possible involvement in oxidative stress responses. In the yeast Saccharomyces cerevisiae, a putative nitroreductase protein, Frm2, has been identified based on its sequence similarity with known bacterial nitroreductases. Frm2 has been reported to function in the lipid signaling pathway. To study the functions of Frm2, we measured the nitroreductase activity of purified Frm2 on 4-nitroquinoline-N-oxide (4-NQO) using NADH. LC-MS analysis of the reaction products revealed that Frm2 reduced NQO into 4-aminoquinoline-N-oxide (4-AQO) via 4-hydroxyaminoquinoline (4-HAQO). An Frm2 deletion mutant exhibited growth inhibition in the presence of 4-NQO. Thus, in this study, we demonstrate a novel nitroreductase activity of Frm2 and its involvement in the oxidative stress defense system.

Simultaneous occurrence of energy transfer and photoinduced electron transfer in interactions of hen egg white lysozyme with 4-nitroquinoline-1-oxide.

The carcinogenic drug 4-nitroquinoline-1-oxide (4NQO) has been found to bind with the protein hen egg white lysozyme as evident from fluorescence quenching experiments. The binding constant and stoichiometry have been determined. The values of the thermodynamic parameters indicate that the interaction is an enthalpy-driven spontaneous phenomenon. The experimental value of change in free energy is similar to that obtained from the docking study. The far UV circular dichroism spectra show some changes in the secondary structure of protein. The high value of bimolecular quenching constant leads to the possibility of Förster resonance energy transfer (FRET). Along with FRET, the photoinduced electron transfer (PET) from tryptophan residue of protein to 4NQO has also been evident from the transient absorption spectra obtained in laser flash photolysis experiments. The simultaneous occurrence of FRET and PET is the key factor for quenching of intrinsic fluorescence of the protein as it binds with the drug.

A mouse model for oral squamous cell carcinoma.

Despite recent advances, the prognosis of oral squamous cell carcinoma is still poor. Therapeutic options such as radiotherapy, chemotherapy, surgery and the novel treatment option gene therapy are being investigated in animal models. Diverse models have been studied to induce oral squamous cell carcinomas. The carcinogenic 4-nitroquinoline-1-oxide (4NQO) model has proven to be successful although until now it is unknown at what time point the established tumor is a representative squamous cell carcinoma and has a suitable volume for scientific treatment. For this end we applied 4NQO 3 times a week during 16 weeks and measured the volume of tumor tissue each week until the end of the experiment at 40 weeks. Concurrent histopathology at different time points up to the end of the experiment revealed that all mice bearing oral tumors were diagnosed with squamous cell carcinoma. Immunohistochemistry with markers cyclin D1 and E-cadherin revealed that the generated mouse oral tumors showed strong similarities with the described immunopathology in human oral tumors. The 4NQO model is a suitable alternative for preclinical gene therapy experiments with primary oral tumors. Future survey of therapeutic options in the carcinogenic 4NQO model should be conducted around 40 weeks after the start of the treatment.

In vitro inhibitory activity of probiotic spore-forming bacilli against genotoxins.

AIMS: To investigate the ability of bacilli of various species (Bacillus clausii, Bacillus subtilis, Bacillus lentus, Bacillus pumilus. Bacillus megaterium, Bacillus firmus, Bacillus sp.) and origins (probiotic and collection strains) to counteract the activity of some representative DNA-reactive agents. METHODS AND RESULTS: The inhibitory effect of 21 bacilli strains, previously characterized by tDNA-PCR, on four genotoxins, was examined in vitro using the short-term assay SOS-Chromotest. All strains had a high inhibitory activity against 4-nitroquinoline-1-oxide and N-methyl-N'-nitro-nitrosoguanidine (direct agents), whereas the inhibitory activity was high or moderate against 2-amino-3,4-dimethylimidazo[4,5-f]quinoline and aflatoxin B1 (indirect agents). Antigenotoxicity was observed in vegetative cells, but not heat-treated cells or spore suspensions. The spectroscopic properties of compounds were modified after cell co-incubation and all the strains maintained high viability after exposure to the genotoxins. CONCLUSIONS: No relevant differences in antigenotoxicity were evidenced among strains of the examined species or between probiotic and collection strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Although derived from an in vitro model, the results suggest that Bacillus-based probiotics could be useful for reducing the gastrointestinal risk originating from genotoxic agents.

Interaction of 4-nitroquinoline-1-oxide with indole derivatives and some related biomolecules: a study with magnetic field.

Laser flash photolysis and an external magnetic field have been used for the study of the interaction of 4-nitroquinoline-1-oxide (4NQO) with some indole derivatives, amino acids, tyrosine and tryptophan, and model proteins, lysozyme and bovine serum albumin. In an aprotic medium, photoinduced electron transfer (PET) from indoles to 4NQO is accompanied by proton transfer from the indole moieties irrespective of the substitution at the N-1 position. For 1,2-dimethylindole, however, proton abstraction is hindered possibly due to steric effects. In a protic medium, obviously proton transfer is possible from the medium and is the dominating reaction following PET. The effect of an external magnetic field is very small for all the systems studied. This is attributed to a competition between geminate proton abstraction by the 4NQO radical anion from the partner radical cation and escape of the 4NQO radical anion to the medium followed by proton transfer. The latter process is more predominant, and the former one, which produces a small population of geminate spin-correlated radical pairs, leads to a minor field effect. Another interesting observation is the affinity of 4NQO toward the tryptophan residues in a protein environment. It is seen that PET takes place preferably from the tryptophan residues rather than from the tyrosine residues.

Genotoxicity of dental resin polymerization initiators in vitro.

The polymerization initiators for resins cured using visible light usually consist of a photosensitizer, primarily camphorquinone (CQ), and a reducing agent, which is often a tertiary amine (DMPT, DMAEMA), while the initiator used for self-curing resins consists of benzoyl peroxide (BPO) and a tertiary amine (DMPT). The genotoxicities of camphorquinone (CQ), benzoyl peroxide (BPO), dimethyl-para-toluidine (DMPT), 2-dimethylamino-ethyl-methacrylate (DMAEMA), and 1-allyl-2-thiourea (ATU) were examined using the bioluminescent bacterial genotoxicity test. 4-Nitroquinoline-N-oxide (4NQO) was prepared for comparison with these chemicals. Acetone solutions of the five polymerization initiators and 4NQO were prepared. Benzoyl peroxide (BPO), dimethyl-para-toluidine (DMPT), and 1-allyl-2-thiourea (ATU) showed significant genotoxic activity at 24 h in the bioluminescent bacterial genotoxicity test, at concentrations of approximately 5 microM, 4 mM, and 1 mM, respectively. 2-Dimethyloamino-ethyl-methacrylate (DMAEMA) did not have genotoxic activity and CQ had questionable genotoxic activity. In comparison, 4NQO had strong genotoxicity, at 4 microM, roughly the same as that of BPO. Therefore, BPO should be used carefully in clinical dentistry.

Inhibition of 4-nitroquinoline-1-oxide genotoxicity by Bacillus strains.

The effect of 16 Bacillus strains from pharmaceutical probiotic preparations (Bacillus spp.) and collection (B. subtilis, B. firmus, B. megaterium, B. pumilus) on genotoxicity induced by the known mutagen 4-nitroquinoline-1-oxide (4-NQO) was studied using the short-term bacterial assay SOS-chromotest. with Escherichia coli PQ37 as the tester organism. It was found that the activity of 0.1 mM 4-NQO was reduced (P < 0.01) after coincubation with Bacillus suspensions (10(8) CFU/ml for 150 min at 37 degrees C). All isolates showed potential for deactivating 4-NQO, and genotoxicity inhibition ranged from 92.9 to 100%. There were no appreciable differences in behaviour observed among probiotic and collection strains or in relation to species. The observed antigenotoxicity was associated with a clear-cut modification of 4-NQO molecular characteristics.

Lactic acid bacteria isolated from dairy products inhibit genotoxic effect of 4-nitroquinoline-1-oxide in SOS-chromotest.

Antigenotoxic activity against 4-nitroquinoline-1-oxide (4-NQO) of lactic acid bacteria isolated from commercial dairy products was studied using SOS-Chromotest. The supernatants from bacteria-genotoxin co-incubations in general exhibited a strong suppression on SOS-induction produced by 4-NQO on the tester organism Escherichia coli PQ37 (sfiA::lacZ). High genotoxicity inhibition (>75%) was found for 31/67 of the examined bacteria and the maximum values of some strains within the species were as follows: Lactobacillus casei, 99.1%; L. plantarum, 93.3%; L. rhamnosus, 93.4%; L. acidophilus, 90.9%; L. delbrueckii subsp. bulgaricus, 85.7% and Bifidobacterium bifidum, 89.6%; Strains with low antigenotoxicity (5-60%) were evidenced in both L. acidophilus and L. delbrueckii subsp. bulgaricus, whereas some inactive strains were found only in L. casei and L. delbrueckii subsp. bulgaricus. Cell exposure to 100 degrees C for 15 min prevented antigenotoxicity and no effect was evidenced for cell-free spent media. The active strains survived at 0.1 mM 4-NQO exposure and generally presented some relevant functional properties, such as tolerance to bile (0.5%) or acid environment (pH 2.0) and adherence to Caco-2 enterocytes. Antigenotoxicity was always associated with modification of the 4-NQO absorbance profile.

Enzymatic and nonenzymatic production of free radicals from the carcinogens 4-nitroquinoline N-oxide and 4-hydroxylaminoquinoline N-oxide.

The anion radicals of 4-nitroquinoline N-oxide (4-NQO) and 4-nitrosoquinoline N-oxide (4-NOQO) carcinogens were detected and characterized by electron spin resonance (ESR) spectroscopy. The structures of the radical intermediates were examined by density functional theory (DFT) at the level of hybrid unrestricted uBecke3LYP. The formation of superoxide anion radical catalyzed by flavin-containing enzymes such as cytochrome P450 reductase or xanthine oxidase in the presence of 4-NQO or 4-nitroquinoline N-oxide was studied by spin-trapping experiments. In this case, the ESR signal of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-superoxide radical adduct was observed, and its formation was inhibited by superoxide dismutase (SOD). No ESR signal was detected when the two-electron-transferring flavoenzyme DT-diaphorase (NADPH-quinone oxidoreductase) was used. The above is consistent with a one-electron reduction in the metabolism of these nitro compounds to anion free radicals by various flavoenzyme reductases.

Novel visible and ultraviolet light photogeneration of hydroxyl radicals by 2-methyl-4-nitro-quinoline-N-oxide (MNO) and 4,4'-dinitro-(2,2')bipyridinyl-N,N'-dioxide (DBD).

Chemicals that upon absorption of light generate hydroxyl radicals (.OH), free of other damaging species under physiological conditions, are useful tools for the study of the biological effects of .OH radical and for its utilization for analytical purposes. We report the novel property of 2-methyl-4-nitro-quinoline-N-oxide (MNO) and 4,4'-dinitro-(2,2')bipyridinyl-N,N'-dioxide (DBD) to act as photogenerators of .OH with UV and visible light. Upon irradiation with 360-400 nm light MNO and DBD generate free radicals that convert coumarin carboxylic acid (CCA) to fluorescent 7-OH-CCA; the .OH radical scavengers dimethylsulfoxide (DMSO) and ethanol eliminate the induction of 7-OH-CCA fluorescence. Upon 400 nm illumination in the presence of MNO, supercoiled plasmid DNA is converted to circular and strand breakage is significantly reduced in the presence of DMSO and completely absent in the absence of MNO. The conversion of CCA to 7-OH-CCA and of supercoiled plasmid to circular DNA are also observed in the absence of oxygen. Taken together, these data indicate that MNO and DBD constitute novel .OH-generating compounds. Because currently known .OH-photogenerating compounds require UV illumination (< 360 nm) that also damages DNA and cells directly, the property of MNO to generate .OH upon 400 nm illumination is advantageous when studies on cells, DNA and other biomolecules are conducted.

A modified SOS-Chromotest procedure to test for genotoxicity and cytotoxicity in sediments directly without extraction.

A modified SOS-Chromotest bioassay using a chromogenic pad (pad procedure) was developed to test for genotoxicity in sediments directly without extraction. This test is based on the de novo synthesis of beta-galactosidase enzyme by a genetically-engineered E. coli strain PQ37. In the bioassay, an exponential growth phase antibiotic-containing culture of the test bacterium is introduced into a series of tubes with the first tube containing 0.1 gram of sediment. Serial dilutions are then made and the tubes of sediment plus bacterial culture are incubated at 37 degrees C for four hours, followed by placing a drop of each mixture on a chromogenic pad and additional incubation for 20 hours at 37 degrees C. The solid particulates are then washed off with tap water and positive (genotoxic) activity is noted by the presence of a distinctive blue colour on the pad. The SOS-Chromotest pad procedure may be best used as a relative measure of genotoxicity by comparing results to a reference sample. In addition it can also determine sediment cytotoxicity by comparing samples spiked with a genotoxic standard (i.e., 4-nitroquinoline-N-oxide). Preliminary results suggest that this new bioassay is highly sensitive, consistent and discriminating.

The ultimate carcinogen of 4-nitroquinoline 1-oxide does not react with Z-DNA and hyperreacts with B-Z junctions.

DNA secondary and tertiary structures are known to affect the reaction between the double helix and several damaging agents. We have previously shown that the tertiary structure of DNA influences the reactivity of 4-acetoxyaminoquinoline 1-oxide (Ac-4-HAQO), the ultimate carcinogen of 4-nitroquinoline 1-oxide (4-NQO), being more reactive with naturally supercoiled DNA than with relaxed DNA. The relative proportion of the three main stable adducts and of an unstable adduct, that resulted in strand scission and/or AP sites, was also affected by the degree of supercoiling of plasmid DNA. In this study we examined the influence of Z-DNA structure on the reactivity of Ac-4-HAQO by mapping the distribution of the two main Ac-4-HAQO adducts, C8-guanine and N2-guanine, along a (dC-dG)16 sequence inserted at the BamHI site of pBR322 plasmid DNA. This insert adopted the left-handed Z and right-handed B structure depending on the superhelical density of the plasmid. Sites of C8-guanine adduct formation were determined by hot piperidine cleavage of Ac-4-HAQO modified DNA, while N2-guanine adducts were mapped by the arrest of the 3'-5' exonuclease activity of T4 DNA polymerase. The results showed that Ac-4-HAQO did not react with guanine residues when the (dC-dG)16 sequence was in Z conformation, while hyperreactivity at the B-Z junction was observed. These results indicate that Ac-4-HAQO can probe the polymorphism of DNA at the nucleotide level.