Voltammetric studies of the interaction of genotoxic 2-nitrofluorene with DNA.

The interaction of genotoxic environmental pollutant 2-nitrofluorene (2-NF) with double-stranded DNA has been studied using a hanging mercury drop electrode (HMDE) as an electrochemical sensor. Two types of DNA damage were investigated and electrochemically detected using cyclic voltammetry and differential pulse voltammetry: (i) DNA damage caused by the direct interaction with 2-NF and (ii) DNA damage caused by short-lived radicals generated by the electrochemical reduction of 2-NF. For the study of the direct interaction, the HMDE was modified by DNA and the interaction of DNA with 2-NF was studied after their mutual interaction right at the HMDE surface, or DNA was preincubated with 2-NF in solution and, subsequently, the interaction was studied voltammetrically. Using both detection techniques, the formation of DNA-2-NF complex was observed and the mutual interaction was interpreted as an intercalation between DNA base pairs. On the basis of obtained results, we suppose that expected formation of 8-oxoguanosine leads to guanosine-cytidine base pair interruption and DNA double-strand break formation. The binding constants (K) of the DNA-2-NF complex formed in solution and on the HMDE surface (DNA/HMDE) were determined from the changes in the voltammetric peaks of the studied analyte.

Endocrine-disrupting metabolic activation of 2-nitrofluorene catalyzed by human cytochrome P450 1A1: A QM/MM approach.

Nitropolycyclic aromatic hydrocarbons (NPAHs) present one of the most important airborne pollutants. Recent studies have shown that one of the most abundant NPAHs, 2-Nitrofluorene (NF), was supposed to be converted to endocrine-disrupting metabolites by cytochrome P450 1A1 (CYP1A1) in human cells. However, the mechanism is still largely unexplored. Here the metabolic activation and transformation mechanism of NF catalyzed by CYP1A1 were systematically studied with the aid of Molecular Dynamics, Density Functional Theory and Quantum Mechanics/Molecular Mechanics techniques. We evidence that CYP1A1 can activate NF through two elementary processes: (i) electrophilic addition (12.4 kcal·mol-1) or hydrogen abstraction (38.2 kcal·mol-1) and (ii) epoxidation (5.9 and 8.7 kcal·mol-1) or NIH shift (12.5 and 14.9 kcal·mol-1) or proton shuttle (12.1 kcal·mol-1). Electrophilic addition was found to be the rate-determining step while epoxidation rather than NIH shift or proton shuttle is the more feasible pathway after electrophilic addition. Metabolites 6,7-epoxide-2-nitrofluorene and 7,8-epoxide-2-nitrofluorene were identified as the major epoxidation products. Epoxides are unstable and easy to react with hydrated hydrogen ions and hydroxyls to produce endocrine disrupter 7-hydroxy-2-nitrofluorene. Toxic analysis shows that some of the metabolites are more toxic to model aquatic organisms (e.g. Green algea) than NF. Binding affinity analysis to human sex hormone binding globulin reveals that NF metabolites all have endocrine-disrupting potential. This study provides a comprehensive understanding on the biotransformation process of NF and may aid future studies on various NPAHs activation catalyzed by human P450 enzyme.

Combination of precolumn nitro-reduction and ultraperformance liquid chromatography with fluorescence detection for the sensitive quantification of 1-nitronaphthalene, 2-nitrofluorene, and 1-nitropyrene in meat products.

Carcinogenic nitropolycyclic aromatic hydrocarbons (nitro-PAHs) are ubiquitous in the ambient environment. They are emitted predominantly from internal combustion engines and by reacting polycyclic aromatic hydrocarbons with nitrogen oxide. The emerging evidence that nitro-PAHs are taken up by plants and bioaccumulatd in the food chain has aroused worldwide concerns for the potential of chronic poisoning through dietary intake. Therefore, analytical methods of high sensitivity are extremely important for assessing the risk of human exposure to nitro-PAHs. This paper describes the development of a simple and robust ultraperformance liquid chromatography coupled fluorescence detector (UPLC-FLD) method for the sensitive determination of nitro-PAHs in meat products. The method entails precolumn reduction of the otherwise nonfluorescent nitro-PAHs to amino-PAHs which strongly fluoresce for their determination by UPLC-FLD analysis. The developed method was validated for extraction efficiency, accuracy, precision, and detection limit and has been successfully applied in quantifying 1-nitronaphthalene (1-NN), 2-nitrofluorene (2-NF), and 1-nitropyrene (1-NP) in fresh and cured meat products. The results showed that the combination of Fe/H(+)-induced nitro-reduction and UPLC-FLD analysis allows sensitive quantification of 1-NN, 2-NF, and 1-NP at detection limits of 0.59, 0.51, and 0.31 µg/kg, respectively, which is at least 10 times lower than those of the existing analytical methods.

Way forward in case of a false positive in vitro genotoxicity result for a cosmetic substance?

The currently used regulatory in vitro mutagenicity/genotoxicity test battery has a high sensitivity for detecting genotoxicants, but it suffers from a large number of irrelevant positive results (i.e. low specificity) thereby imposing the need for additional follow-up by in vitro and/or in vivo genotoxicity tests. This could have a major impact on the cosmetic industry in Europe, seen the imposed animal testing and marketing bans on cosmetics and their ingredients. Afflicted, but safe substances could therefore be lost. Using the example of triclosan, a cosmetic preservative, we describe here the potential applicability of a human toxicogenomics-based in vitro assay as a potential mechanistically based follow-up test for positive in vitro genotoxicity results. Triclosan shows a positive in vitro chromosomal aberration test, but is negative during in vivo follow-up tests. Toxicogenomics analysis unequivocally shows that triclosan is identified as a compound acting through non-DNA reactive mechanisms. This proof-of-principle study illustrates the potential of genome-wide transcriptomics data in combination with in vitro experimentation as a possible weight-of-evidence follow-up approach for de-risking a positive outcome in a standard mutagenicity/genotoxicity battery. As such a substantial number of cosmetic compounds wrongly identified as genotoxicants could be saved for the future.

Dietary safety of cycloastragenol from Astragalus spp.: subchronic toxicity and genotoxicity studies.

Extracts, teas, and other preparations of Astragalus roots (e.g., Radix Astragali) are historically recognized traditional medicines and foods. Cycloastragenol (CAG), a bioactive triterpene aglycone from Astragalus root extracts, is being developed as a modern dietary ingredient. To this end, studies assessing subchronic toxicity and genotoxic potential were conducted. In the subchronic study with recovery component, rats ingested 0, 40, 80, or 150 mg/kg/d CAG by oral gavage for ⩾91 consecutive days. No treatment-related mortalities occurred and no cardiac effects were identified. Although several endpoints among those monitored (i.e., clinical observations, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, gross pathology, organ weights, or histopathology) exhibited statistically significant effects, none was adverse. The oral no-observed-adverse-effect level (NOAEL) for CAG was >150 mg/kg/d in male and female rats. CAG (⩽5000 µg/plate) did not induce mutagenicity in Salmonella typhimurium or Escherichia coli tester strains. Although the in vitro chromosome aberration assay gave a moderately positive response (likely due to poor solubility) for one intermediate concentration (1.50mM) with metabolic activation, responses were negative in all other test groups. Finally, in the in vivo micronucleus assay no clastogenicity was observed in peripheral erythrocytes from mice administered 2000 mg/kg CAG by intraperitoneal injection.