Development of a QuEChERS-Based Method for Determination of Carcinogenic 2‑Nitrofluorene and 1‑Nitropyrene in Rice Grains and Vegetables: A Comparative Study with Benzo[<i>a</i>]pyrene

Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) are ubiquitous environmental pollutants attracting increasing attention because of their potent mutagenicity to humans. Previous studies of nitro-PAHs focused on investigating their formation mechanisms and detecting them in atmospheric environment; however, few studies have reported their occurrence in food samples, and regulations on nitro-PAHs are still lacking. We report in this study the development and application of a quick, easy, cheap, effective, rugged, and safe (QuEChERS) method for determination of nitro-PAHs in rice and vegetable samples. Analysis of the collected samples by the validated method revealed 1-nitropyrene and 2-nitrofluorene were widespread food contaminants. A comparative study with benzo­[<i>a</i>]­pyrene, the commonly used marker for PAH exposure, showed that carcinogenic nitro-PAHs existed in rice and vegetables at similar concentrations. Dietary exposure risk, which was estimated based on the surveillance data, suggested 3.28–5.03 ng/kg/day of nitro-PAHs exposure for Hong Kong citizens from rice grains and vegetables

Automated In-Injector Derivatization Combined with High-Performance Liquid Chromatography–Fluorescence Detection for the Determination of Semicarbazide in Fish and Bread Samples

Semicarbazide (<b>1</b>) is a widespread genotoxic food contaminant originating as a metabolic byproduct of the antibiotic nitrofurazone used in fish farming or as a thermal degradation product of the common flour additive azodicarbonamide. The goal of this study is to develop a simple and sensitive high-performance liquid chromatography coupled with fluorescence detection (HPLC–FLD) method for the detection of compound <b>1</b> in food products. In comparison to existing methods for the determination of compound <b>1</b>, the reported method combining online precolumn derivatization and HPLC–FLD is less labor-intensive, produces higher sample throughput, and does not require the use of expensive analytical instruments. After validation of accuracy and precision, this method was applied to determine the amount of compound <b>1</b> in fish and bread samples. Comparative studies using an established liquid chromatography coupled with tandem mass spectrometry method did not yield systematically different results, indicating that the developed HPLC–FLD method is accurate and suitable for the determination of compound <b>1</b> in fish and bread samples

Comparison of DNA and RNA Adduct Formation: Significantly Higher Levels of RNA than DNA Modifications in the Internal Organs of Aristolochic Acid-Dosed Rats

Prolonged exposure to aristolochic acid (AA) contaminated slimming drugs and food is believed to be associated with the development of endemic nephropathy in Belgian women and in farmers living alongside the Danube River. Decades of research has revealed the pathophysiology of carcinogenesis of AA, and the molecular mechanisms underlying renal interstitial fibrosis remain unclear. We hypothesized that RNA modification may have contributed to the observed toxicity of AA. Thus, a highly sensitive and selective ultra-high performance liquid chromatography-coupled tandem mass spectrometric method was developed to quantify RNA-AA adducts in target and nontarget organs of AA-dosed rats. The results revealed, for the first time, that AA forms RNA adducts <i>in vitro</i> and <i>in vivo</i>. Comparative studies on DNA revealed that RNA is modified by AA at frequencies approximately 6-fold higher than that of DNA in both kidney and liver tissue in AA-dosed rats. Results also demonstrated that guanosine is modified by AA at frequencies significantly higher than that of adenosine, 2-deoxyadenosine, and 2-deoxyguanosine in both organs of the AA-dosed. This finding suggests that guanosine is a major target for AA and that guanosine adducts of AA might be critical lesions in the pathophysiology of AA-induced toxicity. It is anticipated that the results of our study may open up a new area of investigating the nephrotoxicity and/or carcinogenicity by quantifying RNA adducts using the UPLC-MS/MS technique of high sensitivity and selectivity

Quantification of Aristolochic Acid-RNA Adducts in the Urine of Aristolochic Acid-Treated Rats by Liquid Chromatography–Tandem Mass Spectrometry

Balkan endemic nephropathy (BEN) is a peculiar renal disease affecting thousands of farmers living in the suburban areas of the Balkan countries. Emerging evidence suggested that BEN is an environmental disease caused by chronic food poisoning with aristolochic acid (AA). We have developed a sensitive liquid chromatography–tandem mass spectrometry method to detect urinary RNA-AA adducts. Results revealed high levels of RNA-AA adducts in the urine samples collected from AA-treated rats. To the best of our knowledge, this study is the first to report on the detection of urinary RNA-AA adducts. Compared with previous studies that quantified DNA-AA adducts, this method is more sensitive and user-friendly

Facile Formation of a DNA Adduct of Semicarbazide on Reaction with Apurinic/Apyrimidinic Sites in DNA

Mutagenic semicarbazide (SEM) is a hydrazine-containing food contaminant found in a wide variety of foods. Despite decades of research, the toxicity of SEM remains incompletely understood. In this study, we demonstrate for the first time that SEM reacts rapidly with apurinic/apyrimidinic sites in an endogenous DNA lesion to form covalently bonded DNA adducts <i>in vitro</i> and in bacteria. Specifically, we performed high-performance liquid chromatography with high accuracy and tandem mass spectrometry to characterize the DNA adduct formed by reacting SEM with 2′-deoxyribose and single- and double-stranded oligonucleotides containing abasic sites under physiologically relevant conditions. By analyzing the reaction mixture at different time points, the reaction kinetics of SEM with DNA was also elucidated. Moreover, by using a highly sensitive and selective liquid chromatography–tandem mass spectrometry method, we show that SEM induces the dose-dependent formation of DNA adducts in Escherichia coli. The results from our studies provide the first direct evidence suggesting that SEM may exert genotoxicity by forming covalently bonded DNA adducts

Quantitation of Thioprolines in Grape Wine by Isotope Dilution–Liquid Chromatography–Tandem Mass Spectrometry

Cysteine reacts with reactive carbonyls to form thioprolines, which have been demonstrated to possess various pharmaceutical properties. Therefore, thioproline formation is considered as a major detoxification pathway for carcinogenic reactive carbonyls. In this study, we report the initial identification of thiazolidine-4-carboxylic acid (<b>1</b>) and 2-methylthiazolidine-4-carboxylic acid (<b>2</b>), two very common thioprolines, formed by reacting formaldehyde and acetaldehyde with cysteine in grape wine samples. We have developed an isotope dilution–liquid chromatography–tandem mass spectrometry method featuring high sensitivity (limit of detection of ≤1.5 ng/mL) and selectivity to quantitate compounds <b>1</b> and <b>2</b>. The method after validated to be highly accurate (recovery of ≥92%) and precise [intraday relative standard deviation (RSD) of ≤4.1% and interday RSD of ≤9.7%] was applied to determine the varying compound <b>1</b> and <b>2</b> contents in grape wine samples. Results revealed the grape type and storage duration-dependent formation of thioprolines in grape wines. Overall, the results are expected to facilitate compound-dependent investigations of the health benefits of grape wine, and our findings could be adopted to predict the age of grape wine

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 <i>Escherichia coli</i> (<i>E. coli</i>) protein. The study was then extended to quantitate thioproline in protein obtained from formaldehyde- and oxidant-exposed <i>E. coli</i>. Furthermore, <i>N</i>⁶-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

Uptake and Accumulation of Nephrotoxic and Carcinogenic Aristolochic Acids in Food Crops Grown in Aristolochia clematitis-Contaminated Soil and Water

Emerging evidence has suggested aristolochic acids (AAs) are linked to the development of Balkan endemic nephropathy (BEN), a chronic renal disease affecting numerous farmers living in the Balkan peninsula. However, the pathway by which AAs enter the human food chain and cause kidney disease remains poorly understood. Using our previously developed analytical method with high sensitivity and selectivity (Chan, W.; Lee, K. C.; Liu, N.; Cai, Z. <i>J. Chromatogr. A</i> <b>2007</b>, <i>1164</i>, 113–119), we quantified AAs in lettuce, tomato, and spring onion grown in AA-contaminated soil and culture medium. Our study revealed that AAs were being taken up from the soil and bioaccumulated in food crops in a time- and dose-dependent manner. To the best of our knowledge, this study is the first to identify one of the possible pathways by which AAs enter our food chain to cause chronic food poisoning. Results also demonstrated that AAs were resistant to the microbial activity of the soil/water

Quantitation of the DNA Adduct of Semicarbazide in Organs of Semicarbazide-Treated Rats by Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry: A Comparative Study with the RNA Adduct

Semicarbazide is a widespread food contaminant that is produced by multiple pathways. However, the toxicity of semicarbazide to human health remains unclear. Using a highly accurate and sensitive isotope-dilution liquid chromatography–tandem mass spectrometry method, we identified and quantitated in this study for the first time the DNA and RNA adduct of semicarbazide in DNA/RNA isolated from the internal organs of semicarbazide-exposed rats. The analysis revealed a dose-dependent formation of the adducts in the internal organs of the semicarbazide-dosed rats and with the highest adduct levels identified in the stomach and small intestine. Furthermore, results showed significantly higher levels of the RNA adduct (4.1–7.0 times) than that of the DNA adducts. By analyzing DNA/RNA samples isolated from rat organs in semicarbazide-dosed rats at different time points postdosing, the adduct stability <i>in vivo</i> was also investigated. These findings suggest that semicarbazide could have exerted its toxicity by affecting both the transcription and translation processes of the cell

Quantification of DNA and Protein Adducts of 1‑Nitropyrene: Significantly Higher Levels of Protein than DNA Adducts in the Internal Organs of 1‑Nitropyrene Exposed Rats

1-Nitropyrene (1NP) level is closely associated with the mutagenicity of diesel exhaust and is being used as the marker molecule for diesel exhaust. Thus, quantitation of the exposure to 1NP may provide an efficient method for biomonitoring human exposure to diesel exhaust and risk assessment. Using ultra-performance liquid chromatography coupled with fluorescence or tandem mass spectrometric detection methods, we quantitated and compared in this study the DNA and protein adducts of 1NP in internal organs of 1NP-exposed rats. While previous studies using radioactivity-based detection methods were descriptive in nature and focused on the mutation-associated genetic materials, the results of our quantitative analysis showed, for the first time, a significantly higher concentration of the protein adduct than the DNA adduct in the tissue samples. The data also revealed higher in vivo stability of the protein adduct than that of the DNA adduct. Our results provide solid evidence that demonstrates that the protein adduct might be a more-sensitive dosimeter for 1-NP and, thus, diesel-exhaust exposure