Proteomics Study of DNA-Protein Crosslinks in Methylmethanesulfonate and Fe2+-EDTA-Exposed Human Cells.

The formation of covalently bound DNA-protein crosslinks (DPCs) is linked to the pathophysiology of cancers and many other degenerative diseases. Knowledge of the proteins that were frequently involved in forming DPCs will improve our understanding of the etiological mechanism of diseases and facilitate the establishment of preventive measures and treatment methods. By using SDS-PAGE and nano-LC coupled Orbitrap LC-MS/MS analyses, we identified, for the first time, that the major DNA-cross-linked proteins in HeLa cells exposed to a methylating agent (methylmethanesulfonate) or hydroxyl free radicals are transcription-associated proteins. In particular, histone H2B3B and poly(rC) binding protein 2 were identified as the most frequent DPC-forming proteins.

Liquid chromatography-tandem mass spectrometry analysis of aristolochic acids in soil samples collected from Serbia: Link to Balkan endemic nephropathy.

RATIONALE: Over the past six decades, residents of farming villages in multiple countries of the Balkan peninsula have been suffering from a unique type of chronic renal disease, Balkan endemic nephropathy (BEN). It was speculated that environmental pollution by aristolochic acids (AAs) produced naturally by Aristolochia clematitis L., a weed that grows in the area, was causing the disease. However, the human exposure pathway to this class of phytotoxin remains obscure. Knowledge of the sink and stability of AAs in the environment would assist in the formulation of policy reducing exposure risk. METHODS: Using our newly developed liquid chromatography/tandem mass spectrometry method of high sensitivity and selectivity, we analysed over 130 soil samples collected from cultivation fields in southern Serbia for the presence of AAs. The environmental stability of AAs was also investigated by incubating soil samples spiked with AAs at various temperatures. RESULTS: The analysis detected AA-I in over two-fifths of the tested samples at sub-µg/kg to µg/kg levels, with higher concentrations observed in more acidic farmland soil. Furthermore, analysis of soil samples incubated at various temperatures revealed half-lives of over 2 months, indicating that AAs are relatively resistant to degradation. CONCLUSIONS: Cultivation soil in southern Serbia is being extensively contaminated with AAs released from the decomposition of A. clematitis weeds. Since AAs are resistant to degradation, it is possible that AAs could have been taken up by root absorption and transported to the edible part of food crops. Prolonged exposure to AA-contaminated food grown from polluted soil could be one of the main aetiological mechanisms of BEN observed in the area.

Quantitation of N6-Formyl-lysine Adduct Following Aristolochic Acid Exposure in Cells and Rat Tissues by Liquid Chromatography-Tandem Mass Spectrometry Coupled with Stable Isotope-Dilution Method.

N6-Formyl-lysine (FLys) is an abundant and lasting protein adduct formed when formaldehyde generated by nitrosative/oxidative stress and inflammation reacts with lysine residues. It is believed that the post-translational N6-formylation of lysine is associated with a variety of pathological processes and human diseases. Thus, FLys may serve well as a dosimetric biomarker for exposure to formaldehyde and other oxidative stress-inducing toxicants. However, since current methods for FLys determination are tedious and time-consuming, we developed and validated an aqueous normal phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with isotope-dilution method for the rigorous quantification of FLys with enhanced sensitivity and selectivity. After validating the accuracy and precision of the method with a synthetic peptide containing FLys, the method was applied to quantitate the concentration-dependent formation of FLys in cells exposed to formaldehyde and Fe2+-EDTA, an OH radical-mediated oxidant. The study reveals formaldehyde and Fe2+-EDTA produced FLys at a frequency of 20.2 and 4.1 per 104 lysine per mM, respectively, after correcting for losses during protein digestion steps. The study was further extended to quantitate the concentration-dependent formation of FLys in aristolochic acid I (AA-I) exposed Escherichia coli cells and rat tissues. This study demonstrates for the first time that AA-I exposure induces time- and dose-dependent formation of FLys in cellular proteins. Furthermore, results show AA-I exposure leads to organotropic N6-formylation of lysine, with elevated levels of FLys detectable in the kidney, which is the one of the tumor targeting organs of AAs. Previous studies have also revealed AA exposure induced renal interstitial fibrosis in both laboratory rodents and humans, by a yet to be determined molecular mechanism. These data shed light on the potential caustative role of N6-formylation in the pathophysiology of AA nephrotoxicity and carcinogenicity.

Cooking methods employing natural anti-oxidant food additives effectively reduced concentration of nephrotoxic and carcinogenic aristolochic acids in contaminated food grains.

Emerging evidence suggests that aristolochic acids (AA) produced naturally by a common weed Aristolochia clematitis in the cultivation fields is contaminating the food products in Balkan Peninsula and acting as the etiological agent in the development of Balkan endemic nephropathy. In this study, we investigated the combined use of natural anti-oxidative "food additives" and different cooking methods to find a solution for the widespread contamination of AA in food products. The results indicated that the addition of healthy dietary supplements (such as cysteine, glutathione, ascorbic acid, citric acid and magnesium) during cooking, is a highly efficient method in lowering the concentration of AA in the final food products. Because previous observation indicated one of the toxicological mechanisms by which AA exert its toxicity is to induce oxidative stress in internal organs, it is anticipated that these added anti-oxidants will also help to attenuate the nephrotoxicity of AA.

Plant Uptake and Metabolism of Nitrofuran Antibiotics in Spring Onion Grown in Nitrofuran-Contaminated Soil.

Environmental pollution caused by the discharge of mutagenic and carcinogenic nitrofurans to the aquatic and soil environment is an emerging public health concern because of the potential in producing drug-resistant microbes and being uptaken by food crops. Using liquid chromatography-tandem mass spectrometry analysis and with spring onion (Allium wakegi Araki) as the plant model, we investigated in this study the plant uptake and accumulation of nitrofuran from a contaminated environment. Our study revealed for the first time high uptake and accumulation rates of nitrofuran in the edible parts of the food crop. Furthermore, results indicated highly efficient plant metabolism of the absorbed nitrofuran within the plant, leading to the formation of genotoxic hydrazine-containing metabolites. The results from this study may disclose a previously unidentified human exposure pathway through contaminated food crops.

Identification of Protein Thiazolidination as a Novel Molecular Signature for Oxidative Stress and Formaldehyde Exposure.

Chemical modifications of proteins have been well-documented to play important roles in normal cell physiology such as cell signaling and protein functions. They have also been demonstrated to be one of the milestones in the pathophysiology of many human diseases such as cancer, age-related pathology, and neurodegenerative disorders. Here, we report the initial identification of a novel protein modification, cysteine thiazolidination, through reaction with endogenous and exogenous formaldehyde with cysteine residues in proteins. Using an isotope-dilution liquid chromatography-tandem mass spectrometric (LC-MS3) method, we initiated the study by quantitating thioproline in formaldehyde-treated Escherichia coli (E. coli) protein. The study was then extended to quantitate thioproline in protein obtained from formaldehyde- and oxidant-exposed E. coli. Furthermore, N6-formyllysine, a well-defined formylation product between formaldehyde and lysine, was exploited in a comparative study to evaluate the relative reactivity and amount of cysteine thiazolidination in the reaction of formaldehyde with proteins. It is anticipated that cysteine thiazolidination may serve as a novel biomarker for oxidative stress and formaldehyde exposure.

The mechanisms of radical SAM/cobalamin methylations: an evolving working hypothesis.

ALL ABOUT ME: Pierre and co-workers have revealed mechanistic details of a tryptophan methyltransferase (TsrM) involved in the biosynthesis of the thiopeptide antibiotic, thiostrepton. Utilising cobalamin and a [4Fe-4S] cluster to generate 2-methyltryptophan from tryptophan, a key difference between this enzyme and other radical SAM methyltransferases is that the reaction is not initiated by a single-electron reduction of SAM to generate 5'-dA⋅.

The rare fluorinated natural products and biotechnological prospects for fluorine enzymology.

Nature has hardly evolved a biochemistry of fluorine although there is a low-level occurrence of fluoroacetate found in selected tropical and subtropical plants. This compound, which is generally produced in low concentrations, has been identified in the plants due to its high toxicity, although to date the biosynthesis of fluoroacetate in plants remains unknown. After that, fluorinated entities in nature are extremely rare, and despite increasingly sophisticated screening and analytical methods applied to natural product extraction, it has been 25 years since the last bona fide fluorinated natural product was identified from an organism. This was the reported isolation of the antibiotic 4-fluorothreonine and the toxin fluoroacetate in 1986 from Streptomyces cattleya. This bacterium has proven amenable to biochemical investigation, the fluorination enzyme (fluorinase) has been isolated and characterized, and the biosynthetic pathway to these bacterial metabolites has been elucidated. Also the fluorinase gene has been cloned into a host bacterium (Salinispora tropica), and this has enabled the de novo production of a bioactive fluorinated metabolite from fluoride ion, by genetic engineering. Biotechnological manipulation of the fluorinase offers the prospects for the assembly of novel fluorinated metabolites by fermentation technology. This is particularly attractive, given the backdrop that about 15-20% of pharmaceuticals licensed each year (new chemical entities) contain a fluorine atom.