M. Matilde Marques

Studies on the Use of Ionic Liquids as Potential Extractants of Phenolic Compounds and Metal Ions

Abstract The present work was aimed at investigating the usefulness of 1‐(n‐alkyl)‐3‐methylimidazolium‐derived ionic liquids (ILs) in liquid–liquid separation processes. For this purpose, a series of 1‐(n‐alkyl)‐3‐methylimidazolium tetrafluoroborates and hexafluorophosphates were prepared by standard synthetic methods and characterized by 1H NMR. Experiments were performed to assess the influence of the alkyl group and the anion on the physical properties of the IL. Equilibrium studies on the extraction of heavy metal ions (copper, zinc, and chromium) and some common pollutant aromatic compounds (phenol, tyrosol, and p‐hydroxybenzoic acid) from aqueous media then were performed with some selected ILs. The effects of temperature and the pH on the distribution ratio of solutes between the IL/aqueous phases also were analyzed. The results suggest that some of the ionic liquids tested are adequate to extract metal ions and organic compounds from aqueous solutions, with a high efficiency in some instances.

Protein Adducts As Prospective Biomarkers of Nevirapine Toxicity

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against human immunodeficiency virus type-1 (HIV-1), mostly to prevent mother-to-child HIV-1 transmission in developing countries. Despite its clinical efficacy, NVP administration is associated with a variety of toxic responses that include hepatotoxicity and skin rash. Although the reasons for the adverse effects of NVP administration are still unclear, increasing evidence supports the involvement of metabolic activation to reactive electrophiles. In particular, Phase II activation of the NVP metabolite 12-hydroxy-NVP is thought to mediate NVP binding to bionucleophiles, which may be at the onset of toxicity. In the present study, we investigated the nature and specific locations of the covalent adducts produced in human serum albumin and human hemoglobin by reaction in vitro with the synthetic model electrophile 12-mesyloxy-NVP, used as a surrogate for the Phase II metabolite 12-sulfoxy-NVP. Multiple sites of modification were identified by two different mass spectrometry-based methodologies, liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-TOF-TOF-MS). These two distinct methodologies, which in some instances afforded complementary information, allowed the identification of multiple adducts involving cysteine, lysine, tryptophan, histidine, serine, and the N-terminal valine of hemoglobin. Tryptophan, which is not a common site of covalent protein modification, was the NVP-modified amino acid residue detected in the two proteins and consistently identified by both LC-ESI-MS/MS and MALDI-TOF-TOF-MS. The propensity of tryptophan to react with the NVP-derived electrophile is further emphasized by the fact that human serum albumin possesses a single tryptophan residue, which suggests a remarkable selectivity that may be useful for biomonitoring purposes. Likewise, the NVP adduct with the terminal valine of hemoglobin, detected by LC-ESI-MS/MS after N-alkyl Edman degradation, appears as an easily assessed marker of NVP binding to proteins. Our results demonstrate the merits and complementarity of the two MS-based methodologies for the characterization of protein binding by NVP and suggest a series of plausible biomarkers of NVP toxicity that should be useful in the monitoring of toxicity effects in patients administered NVP.

Amino acid adduct formation by the nevirapine metabolite, 12-hydroxynevirapine--a possible factor in nevirapine toxicity.

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type-1 (HIV-1), mostly to prevent mother-to-child transmission of the virus in developing countries. However, reports of severe NVP-induced hepatotoxicity and serious adverse cutaneous effects have raised concerns about its use. NVP metabolism involves oxidation of the 4-methyl substituent to 4-hydroxymethyl-NVP (12-hydroxy-NVP) and the formation of phenolic derivatives. Further metabolism, through either oxidation to quinoid derivatives or phase II esterification, may produce electrophilic derivatives capable of reacting with bionucleophiles to yield covalent adducts. These adducts could potentially be involved in the initiation of toxic responses. To gain insight into potentially reactive sites in proteins and prepare reliable and fully characterized NVP-amino acid adduct standards for subsequent assessment as biomarkers of NVP toxicity, we have used the model electrophile, 12-mesyloxy-NVP, as a synthetic surrogate for the NVP metabolite, 12-sulfoxy-NVP. Reactions of this model ester were conducted with glutathione and the nucleophilic amino acids arginine, cysteine, histidine, and tryptophan. Moreover, because adducts through the N-terminal valine of hemoglobin are convenient biomarkers of exposure to electrophilic toxicants, we also investigated the reaction with valine. We obtained very efficient (>80%) binding through the sulfur of both glutathione and N-acetylcysteine and moderate yields (10-14%) for binding through C2 of the indole ring of tryptophan and N1 of the imidazole ring of histidine. Reaction with arginine occurred through the alpha-amino group, possibly due to the high basicity of the guanidino group in the side chain. Reaction at the alpha-amino group of valine occurred to a significant extent (33%); the resulting adduct was converted to a thiohydantoin derivative, to obtain a standard useful for prospective biomonitoring studies. All adducts were characterized by a combination of (1)H and (13)C NMR spectroscopy and mass spectrometry techniques. The NVP conjugates with glutathione and N-acetylcysteine identified in this work were previously reported to be formed in vivo, although the corresponding structures were not fully characterized. Our results support the validity of 12-mesyloxy-NVP as a surrogate for 12-sulfoxy-NVP and suggest that NVP metabolism to 12-hydroxy-NVP, and subsequent esterification, could potentially be a factor in NVP toxicity. They further imply that multiple sites in proteins may be targets for modification by 12-hydroxy-NVP-derived electrophiles in vivo. Additionally, we obtained reliable, fully characterized standards for the assessment of protein modification by NVP in vivo, which should help clarify the potential role of metabolism in NVP-induced toxicity.

Evidence for nevirapine bioactivation in man: searching for the first step in the mechanism of nevirapine toxicity.

Despite its efficacy, including in the prevention of vertical transmission, the antiretroviral nevirapine is associated with severe idiosyncratic hepatotoxicity and skin rash. The mechanisms underlying nevirapine toxicity are not fully understood, but drug bioactivation to reactive metabolites capable of forming stable protein adducts is thought to be involved. This hypothesis is based on the paradigm that drug reactive metabolites have the potential to bind to self-proteins, which results in drug-modified proteins being perceived as foreign by the immune system. The aim of the present work was to identify hemoglobin adducts in HIV patients as biomarkers of nevirapine haptenation upon bioactivation. The ultimate goal is to develop diagnostic methods for predicting the onset of nevirapine-induced toxic reactions. All included subjects were adults on nevirapine-containing antiretroviral therapy for at least 1month. The protocol received prior approval from the Hospital Ethics Committees and patients gave their written informed consent. Nevirapine-derived adducts with the N-terminal valine of hemoglobin were analyzed by an established liquid chromatography-electrospray ionization-tandem mass spectrometry method and characterized on the basis of retention time and mass spectrometric fragmentation pattern by comparison with adduct standards prepared synthetically. The nevirapine adducts were detected in 12/13 patient samples, and quantified in 11/12 samples (2.58±0.8 fmol/g of hemoglobin). This work represents the first evidence of nevirapine-protein adduct formation in man and confirms the ability of nevirapine to modify self-proteins, thus providing clues to the molecular mechanisms underlying nevirapine toxicity. Moreover, the possibility of assessing nevirapine-protein adduct levels has the potential to become useful for predicting the onset of nevirapine-induced adverse reactions.

Synthesis and evaluation of diaryl sulfides and diaryl selenide compounds for antitubulin and cytotoxic activity

We have devised a procedure for the synthesis of analogs of combretastatin A-4 (CA-4) containing sulfur and selenium atoms as spacer groups between the aromatic rings. CA-4 is well known for its potent activity as an inhibitor of tubulin polymerization, and its prodrugs combretastatin A-4 phosphate (CA-4P) and combretastatin A-1 phosphate (CA-1P) are being investigated as antitumor agents that cause tumor vascular collapse in addition to their activity as cytotoxic compounds. Here we report the preparation of two sulfur analogs and one selenium analog of CA-4. All synthesized compounds, as well as several synthetic intermediates, were evaluated for inhibition of tubulin polymerization and for cytotoxic activity in human cancer cells. Compounds 3 and 4 were active at nM concentration against MCF-7 breast cancer cells. As inhibitors of tubulin polymerization, both 3 and 4 were more active than CA-4 itself. In addition, 4 was the most active of these agents against 786, HT-29 and PC-3 cancer cells. Molecular modeling binding studies are also reported for compounds 1, 3, 4 and CA-4 to tubulin within the colchicine site.

DNA adduct formation and induction of micronuclei and mutations in B6C3F1/Tk mice treated neonatally with acrylamide or glycidamide†

Acrylamide, a food contaminant, is carcinogenic in experimental animals, with both genotoxic and nongenotoxic pathways being proposed. To obtain information regarding mechanisms of acrylamide tumorigenesis, we compared the extent of DNA adduct formation and induction of micronuclei and mutations in mice treated neonatally with acrylamide and its electrophilic metabolite glycidamide. Male and female B6C3F1/Tk mice were treated intraperitoneally on postnatal days (PNDs) 1, 8 and 15 or PNDs 1–8 with 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. One day after the final dose, B6C3F1/Tk+/+ mice were killed to measure DNA adduct levels and peripheral blood micronuclei. Three weeks after the last treatment, B6C3F1/Tk+/− mice were killed to assess the Hprt and Tk mutant frequencies in spleen lymphocytes. The levels of N7‐(2‐carbamoyl‐2‐hydroxyethyl)guanine, the major glycidamide‐DNA adduct, decreased in the order 0.70 mmol glycidamide > 0.70 mmol acrylamide > 0.14 mmol glycidamide ∼ 0.14 mmol acrylamide. Only glycidamide increased the frequency of micronucleated reticulocytes and normochromatic erythrocytes. In mice treated on PNDs 1, 8 and 15, the Hprt mutant frequency was increased by 0.70 mmol glycidamide. In mice dosed on PNDs 1–8, 0.70 mmol glycidamide caused extensive mortality; each of the other treatments increased the Tk mutant frequency, whereas acrylamide increased the Hprt mutant frequency. These data suggest that the mutagenic response in neonatal mice treated on PNDs 1, 8 and 15 is due to glycidamide, whereas mutations resulting from dosing on PNDs 1–8 are due to another mechanism.

Reactions between hydroxylamines and aroyl cyanides

Abstract Aromatic hydroxylamines and sterically bulky aliphatic hydroxylamines react with aroyl cyanides to give exclusively the O -acyl derivatives.

Interactions of D-ribose with polyatomic anions, and alkaline and alkaline-earth cations: possible clues to environmental synthesis conditions in the pre-RNA world

The interaction of some of the most abundant anions in seawater (borate, sulfate and carbonate/bicarbonate) with D-ribose was studied by 1H, 11B and 13C NMR spectroscopy. The results confirmed that only borate improves the stability of D-ribose and favours significant amounts of the ribofuranose isomer, which is the form occurring in present day living organisms. The effect of cations (Na+, K+, Mg2+, Ca2+ and Sr2+) on ribose–borate-bound species was also studied, and it was found that the divalent cations induce a small increase in the relative amounts of ribopyranose isomers in solution and a corresponding decrease in the abundance of the ribofuranose isomers. It was also found that the stability of ribose–borate-bound species, when compared with free ribose over a wide range of temperature and pH values, is higher for compounds with borate, which are stable even at a relatively low pH (6.6) and a relatively high temperature (60 °C). The stability of ribose under moderate pH and relatively high temperature conditions, in the presence of species that occur in seawater, is important for the viability of the early synthetic steps that led to the first nucleotides, which predated the formation of more complex structures, such as RNA.

Tumorigenicity of acrylamide and its metabolite glycidamide in the neonatal mouse bioassay

Acrylamide is a high‐volume industrial chemical, a component of cigarette smoke, and a product formed in certain foods prepared at high temperatures. Previously, we compared the extent of DNA adduct formation and mutations in B6C3F1/Tk mice treated neonatally with acrylamide or glycidamide to obtain information concerning the mechanism of acrylamide genotoxicity. We have now examined the tumorigenicity of acrylamide and glycidamide in mice treated neonatally. Male B6C3F1 mice were injected intraperitoneally on postnatal days 1, 8 and 15 with 0.0, 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day and the tumorigenicity was assessed after 1 year. Survival in each of the groups was >87%, there were no differences in body weights among the groups, and the only treatment‐related neoplasms involved the liver. The incidence of combined hepatocellular adenoma or carcinoma was 3.8% in the control group, 8.3% in the 0.14 mmol acrylamide and glycidamide per kg body weight groups, 4.2% in the 0.70 mmol acrylamide per kg body weight group and 71.4% in the 0.70 mmol glycidamide per kg body weight group. Analysis of the hepatocellular tumors indicated that the increased incidence observed in mice administered 0.70 mmol glycidamide per kg body weight was associated with A → G and A → T mutations at codon 61 of H‐ras. These results, combined with our previous data on DNA adduct formation and mutation induction, suggest that the carcinogenicity of acrylamide is dependent on its metabolism to glycidamide, a pathway that is deficient in neonatal mice.

Arylamine–DNA adduct conformation in relation to mutagenesis

A considerable body of evidence has indicated that local conformational alterations induced by DNA adducts may provide the molecular basis for differences in mutational specificities exhibited by structurally similar adducts. To elucidate the relationships between adduct structure and mutation induction, the ability of several single-ring arylamines present in tobacco smoke (i.e., methylanilines, dimethylanilines, and ethylanilines) to form DNA adducts was investigated. In all cases, the major adducts were C8-substituted deoxyguanosine derivatives, which is consistent with what has been observed with more carcinogenic arylamines, such as 2-aminofluorene and 4-aminobiphenyl. Spectroscopic and theoretical data on the adducts indicated conformational differences depending upon the location of the alkyl substituents. Adducts containing alkyl groups ortho to the amino function (e.g., 2-methylaniline) had a greater percentage of syn conformers about the glycosyl bond than those not bearing such groups. Arylamines with ortho alkyl substituents tend to be more mutagenic and tumorigenic than analogues not containing an ortho alkyl substituent. This increase in biological activity may be due in part to the greater propensity of ortho alkylated adducts to adopt a syn conformation.