Evaluation of the cytotoxicity of transition metal complexes: DNA cleavage of copper complexes in cells

Dissertação de mestrado, Inovação Química e Regulamentação, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015Transition metal complexes, particularly copper(II) compounds have received an increasing attention for cytotoxic effect. Copper compounds coordinated to various types of ligands exhibit considerable nuclease activity that is mostly measured by using the Agarose Gel Electrophoresis (AGE) of pDNA digested by the compounds of interest. In attempt to gain more insight into the cytotoxicity of transition metal complexes, namely their binding and cleavage capacity towards DNA, copper(II) compound [Cu(L)Cl](CH3OH) was prepared using a Schiff base ligand, 2-(pyridin-2-yl)hydrazono)methyl)phenol. The latter has been synthesized by condensation reaction between 2-hydrazinopuridine and salicylaldehyde and characterized by x-ray crystallography, mass spectrometry (MS), NMR and IR spectroscopy. [Cu(L)Cl](CH3OH) DNA binding properties were examined by spectrophotometric DNA titration (UV-visible spectroscopy) and EtBr displacement assay (fluorescence spectroscopy) using ct-DNA. The nuclease activity was evaluated by conversion of the Sc DNA into Nck and Lin forms using the AGE of pBR322 and pA1 DNA. A new method for evaluating the extent of DNA cleavage within living cells has been developed. The method was studied on Mach1 E. coli bacteria cells using vanadyl acetylacetonate, VIVO(acac)2, and copper(II) complex, [Cu(L)Cl](CH3OH). This new technique consists of bacterial cells culture, exposure of bacterial cells to concerned compounds, pDNA purification, and the AGE of pDNA extracted from exposed cells. DNA binding results show that the complex interacts with ct-DNA. The calculated values of intrinsic binding (Kb) and Stern-Volmer quenching (Ksv) constants were of 5.98 x 104 M-1 and 399.9 M-1, respectively. In contrary to pRB322 DNA, [Cu(L)Cl](CH3OH cleaves pA1 pDNA cleavage in presence of mercaptopropionic acid (MPA). Furthermore, VIVO(acac)2 DNA cleavage inside living cells was observed by using the new developed procedure

Determining Toxic Potencies of Water-Soluble Contaminants in Wastewater Influents and Effluent Using Gene Expression Profiling in C. elegans as a Bioanalytical Tool

With chemical analysis, it is impossible to qualify and quantify the toxic potency of especially hydrophilic bioactive contaminants. In this study, we applied the nematode C. elegans as a model organism for detecting the toxic potency of whole influent wastewater samples. Gene expression in the nematode was used as bioanalytical tool to reveal the presence, type and potency of molecular pathways induced by 24-h exposure to wastewater from a hospital (H), nursing home (N), community (C), and influent (I) and treated effluent (E) from a local wastewater treatment plant. Exposure to influent water significantly altered expression of 464 genes, while only two genes were differentially expressed in nematodes treated with effluent. This indicates a significant decrease in bioactive pollutant-load after wastewater treatment. Surface water receiving the effluent did not induce any genes in exposed nematodes. A subset of 209 genes was differentially expressed in all untreated wastewaters, including cytochromes P450 and C-type lectins related to the nematode's xenobiotic metabolism and immune response, respectively. Different subsets of genes responded to particular waste streams making them candidates to fingerprint-specific wastewater sources. This study shows that gene expression profiling in C. elegans can be used for mechanism-based identification of hydrophilic bioactive compounds and fingerprinting of specific wastewaters. More comprehensive than with chemical analysis, it can demonstrate the effective overall removal of bioactive compounds through wastewater treatment. This bioanalytical tool can also be applied in the process of identification of the bioactive compounds via a process of toxicity identification evaluation. [GRAPHICS]

Microarray analysis of Caenorhabditis elegans exposed to four indirect-acting toxicants

In this study, we exposed Caenorhabditis elegans wild types N2 to the model indirect-acting toxicants aflatoxin B1 (AFB1), benzo(a)pyrene (B(a)P), the PCB mixture Aroclor 1254 (PCB1254), and 2,3,7,8-tetrachlorodibenzodioxin (TCDD). In microarray experiments, we studied one concentration of AFB1 (30 μM), one concentration of B(a)P (30 μM), two concentrations of PCB1254 (1 μM and 30 μM), and two concentrations of TCDD (1 μM and 10 μM). As a control M9 medium with 0.5% DMSO was used. Age synchronized worms at developmental L4 larval stage were exposed to treatment for 24 hours. After flash freezing the samples, RNA was isolated, labeled and hybridized on oligo microarray (Agilent) slides

Microarray analysis of Caenorhabditis elegans exposed to four indirect-acting toxicants

In this study, we exposed Caenorhabditis elegans wild types N2 to the model indirect-acting toxicants aflatoxin B1 (AFB1), benzo(a)pyrene (B(a)P), the PCB mixture Aroclor 1254 (PCB1254), and 2,3,7,8-tetrachlorodibenzodioxin (TCDD). In microarray experiments, we studied one concentration of AFB1 (30 μM), one concentration of B(a)P (30 μM), two concentrations of PCB1254 (1 μM and 30 μM), and two concentrations of TCDD (1 μM and 10 μM). As a control M9 medium with 0.5% DMSO was used. Age synchronized worms at developmental L4 larval stage were exposed to treatment for 24 hours. After flash freezing the samples, RNA was isolated, labeled and hybridized on oligo microarray (Agilent) slides

Microarray analysis of Caenorhabditis elegans exposed to three hydrophilic genotoxicants in water in duplicate

In this study, we exposed Caenorhabditis elegans wild types N2 to two concentrations of the model genotoxicants formaldehyde (HCHO), N-ethyl-N-nitrosourea (ENU), and methyl methanesulfonate (MMS).We performed a concentration-response test to determine the non-toxic concentration range for studying the transcriptional effects of compounds. In microarray experiments, we studied two concentrations (1 mM and 5 mM) for each compound and a control of M9 medium. Age synchronized worms at developmental L4 larval stage were exposed to treatment for two hours. After flash freezing the samples, RNA was isolated, labeled and hybridized on oligo microarray (Agilent) slides

Microarray analysis of Caenorhabditis elegans exposed to three hydrophilic genotoxicants in water

In this study, we exposed Caenorhabditis elegans wild types N2 to two concentrations of the model genotoxicants formaldehyde (HCHO), N-ethyl-N-nitrosourea (ENU), and methyl methanesulfonate (MMS).We performed a concentration-response test to determine the non-toxic concentration range for studying the transcriptional effects of compounds. In microarray experiments, we studied two concentrations (1 mM and 5 mM) for each compound and a control of M9 medium. Age synchronized worms at developmental L4 larval stage were exposed to treatment for four hours. After flash freezing the samples, RNA was isolated, labeled and hybridized on oligo microarray (Agilent) slides

Microarray analysis of Caenorhabditis elegans exposed to three hydrophilic genotoxicants in water in duplicate

In this study, we exposed Caenorhabditis elegans wild types N2 to two concentrations of the model genotoxicants formaldehyde (HCHO), N-ethyl-N-nitrosourea (ENU), and methyl methanesulfonate (MMS).We performed a concentration-response test to determine the non-toxic concentration range for studying the transcriptional effects of compounds. In microarray experiments, we studied two concentrations (1 mM and 5 mM) for each compound and a control of M9 medium. Age synchronized worms at developmental L4 larval stage were exposed to treatment for two hours. After flash freezing the samples, RNA was isolated, labeled and hybridized on oligo microarray (Agilent) slides

Microarray analysis of Caenorhabditis elegans exposed to three hydrophilic genotoxicants in water

In this study, we exposed Caenorhabditis elegans wild types N2 to two concentrations of the model genotoxicants formaldehyde (HCHO), N-ethyl-N-nitrosourea (ENU), and methyl methanesulfonate (MMS).We performed a concentration-response test to determine the non-toxic concentration range for studying the transcriptional effects of compounds. In microarray experiments, we studied two concentrations (1 mM and 5 mM) for each compound and a control of M9 medium. Age synchronized worms at developmental L4 larval stage were exposed to treatment for four hours. After flash freezing the samples, RNA was isolated, labeled and hybridized on oligo microarray (Agilent) slides

Development of a transcription-based bioanalytical tool to quantify the toxic potencies of hydrophilic compounds in water using the nematode Caenorhabditis elegans

Low concentrations of environmental contaminants can be difficult to detect with current analytical tools, yet they may pose a risk to human and environmental health. The development of bioanalytical tools can help to quantify toxic potencies of biologically active compounds even of hydrophilic contaminants that are hard to extract from water samples. In this study, we exposed the model organism Caenorhabditis elegans synchronized in larval stage L4 to hydrophilic compounds via the water phase and analyzed the effect on gene transcription abundance. The nematodes were exposed to three direct-acting genotoxicants (1 mM and 5 mM): N-ethyl-N-nitrosourea (ENU), formaldehyde (HCHO), and methyl methanesulfonate (MMS). Genome-wide gene expression analysis using microarrays revealed significantly altered transcription levels of 495 genes for HCHO, 285 genes for ENU, and 569 genes for MMS in a concentration-dependent manner. A relatively high number of differentially expressed genes was downregulated, suggesting a general stress in nematodes treated with toxicants. Gene ontology and Kyoto encyclopedia of genes and genomes analysis demonstrated that the upregulated genes were primarily associated with metabolism, xenobiotic detoxification, proteotoxic stress, and innate immune response. Interestingly, genes downregulated by MMS were linked to the inhibition of neurotransmission, and this is in accordance with the observed decreased locomotion in MMS-exposed nematodes. Unexpectedly, the expression level of DNA damage response genes such as cell-cycle checkpoints or DNA-repair proteins were not altered. Overall, the current study shows that gene expression profiling of nematodes can be used to identify the potential mechanisms underlying the toxicity of chemical compounds. C. elegans is a promising test organism to further develop into a bioanalytical tool for quantification of the toxic potency of a wide array of hydrophilic contaminants