MITF regulates IDH1, NNT, and a transcriptional program protecting melanoma from reactive oxygen species.

Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte function, development and plays a significant role in melanoma pathogenesis. MITF genomic amplification promotes melanoma development, and it can facilitate resistance to multiple therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo. Some of the MITF target genes involved, such as IDH1 and NNT, are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state.

Long-term impact of Hurricane Maria on point-of-use drinking water quality in Puerto Rico and associated potential adverse health effects.

Drinking water security in Puerto Rico (PR) is increasingly challenged by both regulated and emerging anthropogenic contaminants, which was exacerbated by the Hurricane Maria (HM) due to impaired regional water cycle and damaged water infrastructure. Leveraging the NIEHS PROTECT (Puerto Rico Testsite for Exploring Contamination Threats) cohort, this study assessed the long-term tap water (TW) quality changes from March 2018 to November 2018 after HM in PR, by innovatively integrating two different effect-based quantitative toxicity assays with a targeted analysis of 200 organic and 22 inorganic pollutants. Post-hurricane PR TW quality showed recovery after >6-month period as indicated by the decreased number of contaminants showing elevated average concentrations relative to pre-hurricane samples, with significant difference of both chemical and toxicity levels between northern and southern PR. Molecular toxicity profiling and correlation revealed that the HM-accelerated releases of certain pesticides and PPCPs could exert increased cellular oxidative and/or AhR (aryl hydrocarbon receptor)-mediated activities that may persist for more than six months after HM. Maximum cumulative ratio and adverse outcome pathway (AOP) assessment identified the top ranked detected TW contaminants (Cu, Sr, V, perfluorooctanoic acid) that potentially associated with different adverse health effects such as inflammation, impaired reproductive systems, cancers/tumors, and/or organ toxicity. These insights can be incorporated into the regulatory framework for post-disaster risk assessment, guiding water quality control and management for public health protection.

MITF regulates IDH1 and NNT and drives a transcriptional program protecting cutaneous melanoma from reactive oxygen species.

Microphthalmia-associated transcription factor (MITF) plays pivotal roles in melanocyte development, function, and melanoma pathogenesis. MITF amplification occurs in melanoma and has been associated with resistance to targeted therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo . Some of the MITF target genes involved, such as IDH1 and NNT , are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state. One Sentence Summary: MITF promote melanoma survival via increasing ROS tolerance.

A high-specificity aniline-based mass tag for aldehyde detection.

RATIONALE: We studied an aldehyde-labeling reagent, N-{2-[(4-aminophenoxy)methyl]benzyl}-N,N-diethylethanaminium bromide (CAX-A), containing an aniline functional group for the detection of aldehydes with high specificity. METHODS: Six standard aldehydes were labeled by CAX-A and analyzed using LC-ESI-Orbitrap-MS. The aldehydes (each 40 nmol) were derivatized with CAX-A in the presence of sodium cyanoborohydride at room temperature overnight. The labeling reaction was applied to two urine samples for the detection of putative aldehydes. RESULTS: All six standard CAX-aldehyde derivatives were detected as precursor ions by dilution to 830 fmol/injection (signal-to-noise [S/N] ratio 587-1573). A total of 2184 MS1 features were detected overall in urine and blanks, of which 14 were putative aldehydes found only in urine. CONCLUSIONS: CAX-A can provide three levels of specificity for aldehyde detection. First is the known labeling specificity of the aniline functional group for aldehydes, which we confirmed here by observing a significant peak only from the aldehyde (S/N = 3388) when a mixture of an aldehyde, a ketone (no peak), and a quinone (S/N = 2.3) was tested. Second is the ease of formation of an analyte-characteristic first product ion (via anchimeric-assisted loss of triethylamine as a neutral) in MS2 from a CAX-labeled analyte. Third is the formation of a characteristic second product ion via loss of CO in MS3. CAX-A enables the specific, convenient detection of putative aldehydes in urine.

DNA Adductomics by mass tag prelabeling.

RATIONALE: As a new approach to DNA adductomics, we directly reacted intact, double-stranded (ds)-DNA under warm conditions with an alkylating mass tag followed by analysis by liquid chromatography/mass spectrometry. This method is based on the tendency of adducted nucleobases to locally disrupt the DNA structure (forming a "DNA bubble") potentially increasing exposure of their nucleophilic (including active hydrogen) sites for preferential alkylation. Also encouraging this strategy is that the scope of nucleotide excision repair is very broad, and this system primarily recognizes DNA bubbles. METHODS: A cationic xylyl (CAX) mass tag with limited nonpolarity was selected to increase the retention of polar adducts in reversed-phase high-performance liquid chromatography (HPLC) for more detectability while maintaining resolution. We thereby detected a diversity of DNA adducts (mostly polar) by the following sequence of steps: (1) react DNA at 45°C for 2 h under aqueous conditions with CAX-B (has a benzyl bromide functional group to label active hydrogen sites) in the presence of triethylamine; (2) remove residual reagents by precipitating and washing the DNA (a convenient step); (3) digest the DNA enzymatically to nucleotides and remove unlabeled nucleotides by nonpolar solid-phase extraction (also a convenient step); and (4) detect CAX-labeled, adducted nucleotides by LC/MS2 or a matrix-assisted laser desorption/ionization (MALDI)-MS technique. RESULTS: Examples of the 42 DNA or RNA adducts detected, or tentatively so based on accurate mass and fragmentation data, are as follows: 8-oxo-dGMP, ethyl-dGMP, hydroxyethyl-dGMP (four isomers, all HPLC-resolved), uracil-glycol, apurinic/apyrimidinic sites, benzo[a]pyrene-dGMP, and, for the first time, benzoquinone-hydroxymethyl-dCMP. Importantly, these adducts are detected in a single procedure under a single set of conditions. Sensitivity, however, is only defined in a preliminary way, namely the latter adduct seems to be detected at a level of about 4 adducts in 109 nucleotides (S/N ~30). CONCLUSIONS: CAX-Prelabeling is an emerging new technique for DNA adductomics, providing polar DNA adductomics in a practical way for the first time. Further study of the method is encouraged to better characterize and extend its performance, especially in scope and sensitivity.

Ligation-Mediated Polymerase Chain Reaction Detection of 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine and 5-Hydroxycytosine at the Codon 176 of the p53 Gene of Hepatitis C-Associated Hepatocellular Carcinoma Patients.

Molecular mechanisms underlying Hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) pathogenesis are still unclear. Therefore, we analyzed the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and other oxidative lesions at codon 176 of the p53 gene, as well as the generation of 3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG), in a cohort of HCV-related HCC patients from Italy. Detection of 8-oxodG and 5-hydroxycytosine (5-OHC) was performed by ligation mediated-polymerase chain reaction assay, whereas the levels of M1dG were measured by chromatography and mass-spectrometry. Results indicated a significant 130% excess of 8-oxodG at -TGC- position of p53 codon 176 in HCV-HCC cases as compared to controls, after correction for age and gender, whereas a not significant increment of 5-OHC at -TGC- position was found. Then, regression models showed an 87% significant excess of M1dG in HCV-HCC cases relative to controls. Our study provides evidence that increased adduct binding does not occur randomly on the sequence of the p53 gene but at specific sequence context in HCV-HCC patients. By-products of lipid peroxidation could also yield a role in HCV-HCC development. Results emphasize the importance of active oxygen species in inducing nucleotide lesions at a p53 mutational hotspot in HCV-HCC patients living in geographical areas without dietary exposure to aflatoxin B1.

Impact of Hurricane Maria on Drinking Water Quality in Puerto Rico.

This study performed a comprehensive assessment of the impact of Hurricane Maria (HM) on drinking water quality in Puerto Rico (PR) by integrating targeted chemical analysis of both inorganic (18 trace elements) and organic trace pollutants (200 micropollutants) with high-throughput quantitative toxicogenomics and in vitro biomarkers-based toxicity assays. Average concentrations of 14 detected trace elements and 20 organic micropollutants showed elevation after HM. Arsenic, sucralose, perfluorooctanoic acid (PFOA), atrazine-2-hydroxy, benzotriazole, acesulfame, and prometon were at significantly (p < 0.05) higher levels in the post-HM than in the pre-HM samples. Thirteen micropollutants, including four pesticides, were only detected in posthurricane samples. Spatial comparison showed higher pollutant and toxicity levels in the samples from northern PR (where eight Superfund sites are located) than in those from southern PR. Distinctive pathway-specific molecular toxicity fingerprints for water extracts before and after HM and at different locations revealed changes in toxicity nature that likely resulted from the impact of HM on drinking water composition. Correlation analysis and Maximum Cumulative Ratio assessment suggested that metals (i.e., arsenic) and PFOA were the top ranked pollutants that have the potential to cause increased risk after HM, providing a possible direction for future water resource management and epidemiological studies.

Jettison-MS of Nucleic Acid Species.

While MALDI-MS of intact genomic DNA is unheard of, actually many DNA adducts can be detected in this way under certain MALDI conditions: relatively high molar ratio of DNA nucleobases to matrix (0.01 to 0.3), hot matrix (CCA), and high laser fluence. This is because many DNA adducts create "bubbles" on dsDNA (disruption of base pairing), making it easier for these adducts as modified nucleobases to be jettisoned by the laser-derived energy of MALDI (jettison mass spectrometry or JeMS). The method also works for other nucleic acid species, namely nucleobases, nucleosides, nucleotides, and RNA. Examples of what we have detected in this way are as follows: methyladenine in E. coli DNA, 5-hydroxymethylcytosine in human brain DNA, melphalan-adenine in leukocyte DNA from patients on corresponding chemotherapy, wybutosine in tRNA, benzyl DNA adducts in E. coli cell culture treated with benzyl bromide, and various DNA adducts formed in test tube exposure experiments with calf thymus DNA. Noteworthy, in the chemotherapy study, principle component analysis of the data encourages the hypothesis that patient DNA undergoes much more damage than just melphalan adducts. Overall, our work leads to the preliminary generalization that about 5 fmol of a nucleobase deficient in base pairing, and present in a MALDI spot, will be detected by JeMS (on the equipment that we used), irrespective of the type of nucleic acid species which houses it, as long as the nucleobase is relatively basic such as A, C, or G.

A Cross-Sectional Study on 3-(2-Deoxy-ß-D-Erythro-Pentafuranosyl)Pyrimido[1,2-α]Purin-10(3H)-One Deoxyguanosine Adducts among Woodworkers in Tuscany, Italy.

Occupational exposure to wood dust has been estimated to affect 3.6 million workers within the European Union (EU). The most serious health effect caused by wood dust is the nasal and sinonasal cancer (SNC), which has been observed predominantly among woodworkers. Free radicals produced by inflammatory reactions as a consequence of wood dust could play a major role in SNC development. Therefore, we investigated the association between wood dust and oxidative DNA damage in the cells of nasal epithelia, the target site of SNC. We have analyzed oxidative DNA damage by determining the levels of 3-(2-deoxy-ß-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG), a major-peroxidation-derived DNA adduct and a biomarker of cancer risk in 136 woodworkers compared to 87 controls in Tuscany, Italy. We then examined the association of M1dG with co-exposure to volatile organic compounds (VOCs), exposure length, and urinary 15-F2t isoprostane (15-F2t-IsoP), a biomarker of oxidant status. Wood dust at the workplace was estimated by the Information System for Recording Occupational Exposures to Carcinogens. M1dG was measured using 32P-postlabeling and mass spectrometry. 15-F2t-IsoP was analyzed using ELISA. Results show a significant excess of M1dG in the woodworkers exposed to average levels of 1.48 mg/m3 relative to the controls. The overall mean ratio (MR) between the woodworkers and the controls was 1.28 (95% C.I. 1.03-1.58). After stratification for smoking habits and occupational status (exposure to wood dust alone and co-exposure to VOCs), the association of M1dG with wood dust (alone) was even greater in non-smokers workers, MR of 1.43 (95% C.I. 1.09-1.87). Conversely, not consistent results were found in ex-smokers and current smokers. M1dG was significantly associated with co-exposure to VOCs, MR of 1.95 (95% C.I. 1.46-2.61), and occupational history, MR of 2.47 (95% C.I. 1.67-3.62). Next, the frequency of M1dG was significantly correlated to the urinary excretion of 15-F2t-IsoP, regression coefficient (ß) = 0.442 ± 0.172 (SE). Consistent with the hypothesis of a genotoxic mechanism, we observed an enhanced frequency of M1dG adducts in woodworkers, even at the external levels below the regulatory limit. Our data implement the understanding of SNC and could be useful for the management of the adverse effects caused by this carcinogen.

The response of Escherichia coli to the alkylating agents chloroacetaldehyde and styrene oxide.

DNA damage is ubiquitous and can arise from endogenous or exogenous sources. DNA-damaging alkylating agents are present in environmental toxicants as well as in cancer chemotherapy drugs and are a constant threat, which can lead to mutations or cell death. All organisms have multiple DNA repair and DNA damage tolerance pathways to resist the potentially negative effects of exposure to alkylating agents. In bacteria, many of the genes in these pathways are regulated as part of the SOS reponse or the adaptive response. In this work, we probed the cellular responses to the alkylating agents chloroacetaldehyde (CAA), which is a metabolite of 1,2-dichloroethane used to produce polyvinyl chloride, and styrene oxide (SO), a major metabolite of styrene used in the production of polystyrene and other polymers. Vinyl chloride and styrene are produced on an industrial scale of billions of kilograms annually and thus have a high potential for environmental exposure. To identify stress response genes in E. coli that are responsible for tolerance to the reactive metabolites CAA and SO, we used libraries of transcriptional reporters and gene deletion strains. In response to both alkylating agents, genes associated with several different stress pathways were upregulated, including protein, membrane, and oxidative stress, as well as DNA damage. E. coli strains lacking genes involved in base excision repair and nucleotide excision repair were sensitive to SO, whereas strains lacking recA and the SOS gene ybfE were sensitive to both alkylating agents tested. This work indicates the varied systems involved in cellular responses to alkylating agents, and highlights the specific DNA repair genes involved in the responses.

Integrating exhaled breath diagnostics by disease-sniffing dogs with instrumental laboratory analysis.

Dogs have been studied for many years as a medical diagnostic tool to detect a pre-clinical disease state by sniffing emissions directly from a human or an in vitro biological sample. Some of the studies report high sensitivity and specificity in blinded case-control studies. However, in these studies it is completely unknown as to which suites of chemicals the dogs detect and how they ultimately interpret this information amidst confounding background odors. Herein, we consider the advantages and challenges of canine olfaction for early (meaningful) detection of cancer, and propose an experimental concept to narrow the molecular signals used by the dog for sample classification to laboratory-based instrumental analysis. This serves two purposes; first, in contrast to dogs, analytical methods could be quickly up-scaled for high throughput sampling. Second, the knowledge gained from identifying probative chemicals could be helpful in learning more about biochemical pathways and disease progression. We focus on exhaled breath aerosol, arguing that the semi-volatile fraction should be given more attention. Ultimately, we conclude that the interaction between dog-based and instrument-based research will be mutually beneficial and accelerate progress towards early detection of cancer by breath analysis.

Cold-induced aqueous acetonitrile phase separation: A salt-free way to begin quick, easy, cheap, effective, rugged, safe.

Cooling a 1:1 (v/v) solution of acetonitrile and water at -16° C is known to result in two clear phases. We will refer to this event as "cold-induced aqueous acetonitrile phase separation (CIPS)". On a molar basis, acetonitrile is 71.7% and 13.6% in the upper and lower phases, respectively, in our study. The phase separation proceeds as a descending cloud of microdroplets. At the convenient temperature (typical freezer) employed here the lower phase is rather resistant to solidification, although it emerges from the freezer as a solid if various insoluble matter is present at the outset. In a preliminary way, we replaced the initial (salting-out) step of a representative QuEChERS procedure with CIPS, applying this modified procedure ("CIPS-QuEChERS") to a homogenate of salmon (and partly to beef). Three phases resulted, where only the upper, acetonitrile-rich phase is a liquid (that is completely clear). The middle phase comprises ice and precipitated lipids, while the lower phase is the residual matrix of undissolved salmon or meat. Treating the upper phase from salmon, after isolation, with anhydrous MgSO4 and C18-Si (typical QuEChERS dispersive solid phase extraction sorbents), and injecting into a GC-MS in a nontargeted mode, gives two-fold more preliminary hits for chemicals, and also number of spiked pesticides recovered, relative to that from a comparable QuEChERS method. In part, this is because of much higher background signals in the latter case. Further study of CIPS-QuEChERS is encouraged, including taking advantage of other QuERChERS conditions.

Bulky DNA Adducts, Tobacco Smoking, Genetic Susceptibility, and Lung Cancer Risk.

The generation of bulky DNA adducts consists of conjugates formed between large reactive electrophiles and DNA-binding sites. The term "bulky DNA adducts" comes from early experiments that employed a 32P-DNA postlabeling approach. This technique has long been used to elucidate the association between adducts and carcinogen exposure in tobacco smoke studies and assess the predictive value of adducts in cancer risk. Molecular data showed increased DNA adducts in respiratory tracts of smokers vs nonsmokers. Experimental studies and meta-analysis demonstrated that the relationship between adducts and carcinogens was linear at low doses, but reached steady state at high exposure, possibly due to metabolic and DNA repair pathway saturation and increased apoptosis. Polymorphisms of metabolic and DNA repair genes can increase the effects of environmental factors and confer greater likelihood of adduct formation. Nevertheless, the central question remains as to whether bulky adducts cause human cancer. If so, lowering them would reduce cancer incidence. Pooled and meta-analysis has shown that smokers with increased adducts have increased risk of lung cancer. Adduct excess in smokers, especially in prospective longitudinal studies, supports their use as biomarkers predictive of lung cancer.

Magnetic Hyperthermia and Oxidative Damage to DNA of Human Hepatocarcinoma Cells.

Nanotechnology is addressing major urgent needs for cancer treatment. We conducted a study to compare the frequency of 3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) adducts, biomarkers of oxidative stress and/or lipid peroxidation, on human hepatocarcinoma HepG2 cells exposed to increasing levels of Fe3O4-nanoparticles (NPs) versus untreated cells at different lengths of incubations, and in the presence of increasing exposures to an alternating magnetic field (AMF) of 186 kHz using 32P-postlabeling. The levels of oxidative damage tended to increase significantly after ≥24 h of incubations compared to controls. The oxidative DNA damage tended to reach a steady-state after treatment with 60 µg/mL of Fe3O4-NPs. Significant dose-response relationships were observed. A greater adduct production was observed after magnetic hyperthermia, with the highest amounts of oxidative lesions after 40 min exposure to AMF. The effects of magnetic hyperthermia were significantly increased with exposure and incubation times. Most important, the levels of oxidative lesions in AMF exposed NP treated cells were up to 20-fold greater relative to those observed in nonexposed NP treated cells. Generation of oxidative lesions may be a mechanism by which magnetic hyperthermia induces cancer cell death.

3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine adducts of workers exposed to asbestos fibers.

Asbestos is the commercial name for a group of silicate minerals naturally occurring in the environment and widely used in the industry. Asbestos exposure has been associated with pulmonary fibrosis, mesothelioma, and malignancies, which may appear after a period of latency of 20-40 years. Mechanisms involved in the carcinogenic effects of asbestos are still not fully elucidated, although the oxidative stress theory suggests that phagocytic cells produce large amounts of reactive oxygen species, due to their inability to digest asbestos fiber. We have conducted a mechanistic study to evaluate the association between 3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) adducts, a biomarker of oxidative stress and lipid peroxidation, and asbestos exposure in the peripheral blood of 327 subjects living in Tuscany and Liguria, Italy, stratified by occupational exposure to asbestos. Adduct frequency was significantly greater into exposed subjects with respect to the controls. M1dG per 108 normal nucleotides were 4.0±0.5 (SE) in 156 asbestos workers, employed in mechanic, naval, petrochemical, building industries, and in pottery and ceramic plants, versus a value of 2.3±0.1 (SE) in 171 controls (p<0.001). After stratification for occupational history, the effects persisted in 54 current asbestos workers, mainly employed in building renovation industry (2.9±0.3 (SE)), and in 102 former asbestos workers (4.5±0.7 (SE)), with p-values of 0.033, and <0.001, respectively. A significant effect of smoking on heavy smokers was found (p=0.005). Our study gives additional support to the oxidative stress theory, where M1dG may reflect an additional potential mechanism of asbestos-induced toxicity.

Recommendations for quantitative analysis of small molecules by matrix-assisted laser desorption ionization mass spectrometry.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been used for quantitative analysis of small molecules for many years. It is usually preceded by an LC separation step when complex samples are tested. With the development several years ago of "modern MALDI" (automation, high repetition laser, high resolution peaks), the ease of use and performance of MALDI as a quantitative technique greatly increased. This review focuses on practical aspects of modern MALDI for quantitation of small molecules conducted in an ordinary way (no special reagents, devices or techniques for the spotting step of MALDI), and includes our ordinary, preferred methods The review is organized as 18 recommendations with accompanying explanations, criticisms and exceptions.

Porous extraction paddle: a solid-phase extraction technique for studying the urine metabolome.

RATIONALE: A method was needed to accomplish solid-phase extraction of a large urine volume in a convenient way where resources are limited, towards a goal of metabolome and xenobiotic exposome analysis at another, distant location. METHODS: A porous extraction paddle (PEP) was set up, comprising a porous nylon bag containing extraction particles that is flattened and immobilized between two stainless steel meshes. Stirring the PEP after attachment to a shaft of a motor mounted on the lid of the jar containing the urine accomplishes extraction. The bag contained a mixture of nonpolar and partly nonpolar particles to extract a diversity of corresponding compounds. RESULTS: Elution of a urine-exposed, water-washed PEP with aqueous methanol containing triethylammonium acetate (conditions intended to give a complete elution), followed by MALDI-TOF/TOF-MS, demonstrated that a diversity of compounds had been extracted ranging from uric acid to peptides. CONCLUSIONS: The PEP allows the user to extract a large liquid sample in a jar simply by turning on a motor. The technique will be helpful in conducting metabolomics and xenobiotic exposome studies of urine, encouraging the extraction of large volumes to set up a convenient repository sample (e.g. 2 g of exposed adsorbent in a cryovial) for shipment and re-analysis in various ways in the future, including scaled-up isolation of unknown chemicals for identification. Copyright © 2016 John Wiley & Sons, Ltd.