Célia Martins

Coffee is one of the world’s most widely consumed beverages but intake it is not encouraged in consumers with some health conditions. In this way, with the growing interest in developing healthier substitutes, a coffee-like beverage obtained from Quercus ilex and Quercus suber acorn´s species, was elaborates maintaining the flavor. Although, this beverage is a promising coffee alternative, little is known about effects of roasting process in its composition. To that end the antioxidant capacity and toxicity of the developed coffee were analyzed and phenolic compounds and fatty acids (esterified and free forms) were characterized through HPLC-DAD and GC-FID, respectively. The results showed that Quercus based beverages presented antioxidant capacity related to their phenolic content, mainly to ellagic acid as the primary phenolic compound identified. Due to this composition, the beverage also presented antimutagenic activity. The main fatty acids in the esterified lipids were mainly oleic, linoleic, palmitic, stearic and cis vaccenic. In the free fatty acids fraction (FFA) they were oleic, linoleic and palmitic acids. Heat processing produced a reduction in total fatty acid concentration in TG and FFA fraction of Q. ilex. Nevertheless, for Q. suber alterations were only found for FFA. Coffee production did not form genotoxic or cytotoxic compounds. Overall, these results show the feasibility of Quercus acorn-based foodstuffs and its potential to produce a functional coffee-like beverage.

The cell membrane P-glycoprotein (P-gp; MDR1, ABCB1) is an energy-dependent efflux pump that belongs to the ATP-binding cassette (ABC) family of transporters, and has been associated with drug resistance in eukaryotic cells. Multidrug resistance (MDR) is related to an increased expression and function of the ABCB1 (P-gp) efflux pump that often causes chemotherapeutic failure in cancer. Modulators of this efflux pump, such as the calcium channel blocker verapamil (VP) and cyclosporine A (CypA), can reverse the MDR phenotype but in vivo studies have revealed disappointing results due to adverse side effects. Currently available methods are unable to visualize and assess in a real-time basis the effectiveness of ABCB1 inhibitors on the uptake and efflux of ABCB1 substrates. However, predicting and testing ABCB1 modulation activity using living cells during drug development are crucial. The use of ABCB1-transfected mouse T-lymphoma cell line to study the uptake/efflux of fluorescent probes like ethidium bromide (EB), rhodamine 123 (Rh-123), and carbocyanine dye DiOC2, in the presence and absence of potential inhibitors, is currently used in our laboratories to evaluate the ability of a drug to inhibit ABCB1-mediated drug accumulation and efflux. Here we describe and compare three in vitro methods, which evaluate the permeability, transport kinetics of fluorescent substrates, and inhibition of the ABCB1 efflux pump by drugs of chemical synthesis or extracted from natural sources, using model cancer cell lines overexpressing this transporter, namely (1) real-time fluorimetry that assesses the accumulation of ethidium bromide, (2) flow cytometry, and (3) fluorescent microscopy using rhodamine 123 and DiOC2.

The interaction of ACMA (9-amino-6-chloro-2-methoxy acridine) (D) with DNA (P) has been studied by absorbance, fluorescence, circular dichroism, spectrophotometry, viscometry and unwinding electrophoresis. A T-jump kinetic study has also been undertaken. The experimental data show that, totally unlike other drugs, ACMA is able to form with DNA three complexes (PD(I), PD(II), PD(III)) that differ from each other by the characteristics and extent of the binding process. The main features of PD(I) fulfil the classical intercalation pattern and the formation/dissociation kinetics have been elucidated by T-jump techniques. PD(II) and PD(III) are also intercalated species but, in addition to the dye units lodged between base pairs, they also bear dye molecules externally bound, more in PD(III) relative to PD(II). A reaction mechanism is put forward here. Comparison between absorbance, fluorescence and kinetic experiments has enabled us to determine the binding constants of the three complexes, namely (6.5 ± 1.1) × 10(4) M(-1) (PD(I)), (5.5 ± 1.5) × 10(4) M(-1) (PD(II)) and (5.7 ± 0.03) × 10(4) M(-1) (PD(III)). The Comet assay reveals that the ACMA binding to DNA brings about genotoxic properties. The mutagenic potential studied by the Ames test reveals that ACMA can produce frameshift and transversion/transition mutations. ACMA also is able to produce base-pair substitution in the presence of S9 mix. Moreover, the MTT assays have revealed cytotoxicity. The biological effects observed have been rationalized in light of these features.

Acrylamide (AA) is a suspected human carcinogen found to be generated during the heating of carbohydrate-rich foodstuffs. AA exhibits 'Michael-type' reactivity towards reduced glutathione (GSH), resulting in vivo in the urinary excretion of mercapturic acid conjugates. GSH is a key factor for mammalian cell homeostasis, with diverse functions that include, among others, the conjugation of electrophilic compounds and the detoxification of products generated by oxidative stress. Therefore, studies focusing on the modulation of GSH are of great importance for the understanding of the mechanisms of AA-induced toxicity. This report addresses this issue by analyzing cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay) and clastogenicity (chromosomal aberrations) as endpoints in V79 cells after exposure to AA. The experiments described herein include the evaluation of the effect of buthionine sulfoximine (BSO), an effective inhibitor of GSH synthesis, GSH-monoethyl ester (GSH-EE), a compound that is taken up by cells and intracellularly hydrolysed to GSH, and also GSH exogenously added to culture medium. Pre-treatment with BSO increased the cytotoxicity and the frequency of aberrant cells excluding gaps (ACEG) induced by AA. While pre-treatment with GSH-EE did not modify the cytotoxicity or the frequency of ACEG induced by AA, co-treatment with AA and GSH decreased both parameters, rendering the cells less prone to the toxic effects of AA. In vitro studies in a cell-free system, using monochlorobimane (MCB), a fluorescent probe for GSH, were also performed in order to evaluate the role of AA in GSH depletion. The results show that spontaneous conjugation of AA with GSH in the extracellular medium is involved in the protection given by GSH. In summary, these results reinforce the role of GSH in the modulation of the cytotoxic and clastogenic effects induced by AA, which may be relevant in an in vivo exposure scenario.

We evaluated the genotoxicity of the food-flavouring agent estragole in V79 cells using the sister chromatid exchange (SCE) assay and the alkaline comet assay. Unexpectedly, we observed an increase in SCE without an exogenous biotransformation system (S9) and a decrease in its presence. Positive results were also observed in the alkaline comet assay without S9, indicating DNA strand breakage. To ascertain repair of damage, we performed the comet assay in V79 cells after two hours of recovery, and observed a reduction of the genotoxic response. Estragole did not produce strand breaks in plasmid DNA in vitro. We then evaluated the formation of DNA adducts in V79 cells by use of the (32)P-postlabelling assay and detected a dose-dependent formation of DNA adducts, which may be responsible for its genotoxicity. We then assayed estragole in the comet assay with two CHO cell lines, a parental AA8 cell line, and an XRCC1-deficient cell line, EM9. Results confirmed the genotoxicity of estragole without biotransformation in both cell lines, although the genotoxicity in EM9 cells compared with that in AA8 cells was not significantly different, suggesting that the XRCC1 protein is not involved in the repair of estragole-induced lesions. Estragole induces apoptosis, but only with high doses (2000μM), and after long treatment periods (24h). Overall, our results suggest that estragole, besides being metabolized to genotoxic metabolites, is a weak direct-acting genotoxin that forms DNA adducts.

Resistance to imatinib in patients with chronic myeloid leukemia can lead to advanced disease and blast crisis. Conventional chemotherapy with DNA damaging agents is then used, alone or in combination with other tyrosine kinase inhibitors (TKIs). Our aim was to assess whether imatinib resistant K562 cells were also resistant to DNA damaging agents. After treatment with H(2)O(2) and doxorubicin, but not camptothecin, cell survival was higher in imatinib resistant cells compared to parental cells. DNA damage, measured by comet and γ-H2AX assays, was lower in imatinib resistant cells. mRNA expression levels of 50 genes of the DNA damage response pathway showed increased expression of the base excision repair (BER) genes MBD4 and NTHL1. Knockdown of MBD4 and NTHL1 expression in resistant cells using siRNA decreased cell survival after treatment with H(2)O(2) and doxorubicin. Our results indicate that imatinib resistant cells display cross-resistance to oxidative agents, partly through up-regulation of BER genes. Expression of these genes in imatinib resistant patients was not significantly different compared to sensitive patients. However, the strategy followed in this study could help identify chemotherapeutic agents that are more effective as alternative agents in cases of resistance to TKIs.

Acrylamide (AA) is a probable human carcinogen formed in carbohydrate-rich foodstuffs upon heating. Glycidamide (GA), the AA metabolite formed by epoxidation, is considered the ultimate genotoxic agent. In this study, the in vitro genotoxic potential of AA and GA in human whole blood leukocytes was compared using the alkaline comet assay. Although AA did not induce significant DNA damage in the concentrations tested (up to 1000 μM), GA markedly increased the percentage of tail DNA at concentrations ≥250 μM. Further, this study addressed the role of genetic polymorphisms in key genes involved in metabolism and DNA repair pathways (BER, NER, HRR, and NHEJ) on GA-induced genotoxicity assessed by the alkaline comet assay. The results obtained suggested associations between DNA damage and polymorphisms of BER (MUTYH Gln335His and XRCC1 Gln399Arg) and NER (XPC Ala499Val) genes, either alone or in combination.

The interaction of the Cu(II) drugs CuL(NO(3)) and CuL'(NO(3)) (HL is pyridine-2-carbaldehyde thiosemicarbazone and HL' is pyridine-2-carbaldehyde 4N-methylthiosemicarbazone, in water named [CuL](+) and [CuL'](+)) with [poly(dA-dT)](2), [poly(dG-dC)](2), and calf thymus (CT) DNA has been probed in aqueous solution at pH 6.0, I = 0.1 M, and T = 25 degrees C by absorbance, fluorescence, circular dichroism, and viscosity measurements. The results reveal that these drugs act as groove binders with [poly(dA-dT)](2), with a site size n = 6-7, whereas they act as external binders with [poly(dG-dC)](2) and/or CT-DNA, thus establishing overall electrostatic interaction with n = 1. The binding constants with [CuL'](+) were slightly larger than with [CuL](+). The title compounds display some cleavage activity in the presence of thiols, bringing about the rupture of the DNA strands by the reactive oxygen species formed by reoxidation of Cu(I) to Cu(II); this feature was not observed in the absence of thiols. Mutagenic assays performed both in the presence and in the absence of S9 mix, probed by the Ames test on TA 98, TA 100, and TA 102, were negative. Weak genotoxic activity was detected for [CuL](+) and [CuL'](+), with a significative dose-response effect for…

Some food flavourings, such as safrole and methyleugenol, are known for their genotoxic and hepatocarcinogenic properties whereas for others, such as myristicin, there is less data. Myristicin and eugenol are both alkenylbenzenes, and we compared their direct genotoxicity in repair proficient (AA8) and repair deficient XRCC(-) (EM9) Chinese hamster ovary cells. Cell viability was assessed by the MTT assay. The comet assay was used to evaluate DNA breaks, and the γ-H2AX assay to evaluate induction of double strand breaks. We assessed apoptosis by measuring caspases activation, and the TUNEL assay. Reduction of cell viability was similar in AA8 and EM9 cells, for both compounds. After 1h eugenol produced DNA strand breaks in the comet assay and induced double strand breaks in the γ-H2AX assay in AA8 cells, while myristicin was not genotoxic in both the comet and the γ-H2AX assays. Both flavourings were negative in EM9 cells. After 24h eugenol and myristicin induced DNA fragmentation detected by TUNEL in both cell lines, but only myristicin activated caspases. Myristicin was more apoptotic than eugenol, in both cell lines. The XRCC1 protein does not influence the apoptotic activity of either compound.

The reactions of two diaminotriazine ligands 2,4-diamino-6-(2-pyridyl)-1,3,5-triazine (2-pydaT) and 6-phenyl-2,4-diamino-1,3,5-triazine (PhdaT) with ruthenium-arene precursors led to a new family of ruthenium(II) compounds that were spectroscopically characterized. Four of the complexes were cationic, with the general formula [(η(6)-arene)Ru(κ(2)-N,N-2-pydaT)Cl]X (X=BF(4), TsO; arene=p-cymene: 1·BF(4), 1·TsO; arene=benzene: 2·BF(4), 2·TsO). The neutral cyclometalated complex [(η(6)-p-cymene)Ru(κ(2)-C,N-PhdaT*)Cl] (3) was also isolated. The structures of complexes 2·BF(4) and 3·H(2)O were determined by X-ray diffraction. Complex 1·BF(4) underwent a partial reversible-aquation process in water. UV/Vis and NMR spectroscopic measurements showed that the reaction was hindered by the addition of NaCl and was pH-controlled in acidic solution. At pH 7.0 (sodium cacodylate) Ru-Cl complex 1·BF(4) was the only species present in solution, even at low ionic strength. However, in alkaline medium (KOH), complex 1·BF(4) underwent basic hydrolysis to afford a Ru-OH complex (5). Fluorimetric studies revealed that the interaction of complex 1·BF(4) with DNA was not straightforward; instead, its main features were closely linked to ionic strength and to the [DNA]/complex ratio. The bifunctional complex 1·BF(4) was capable of interacting concurrently through both its p-cymene and 2-pydaT groups. Cytotoxicity and genotoxicity studies showed that, contrary to the expected behavior, the complex species was biologically inactive; the formation of a Ru-OH complex could be responsible for such behavior.

Acrylamide (AA) is a suspected human carcinogen generated in carbohydrate-rich foodstuff upon heating. Glycidamide (GA), formed via epoxidation presumably mediated by cytochrome P450 2E1, is thought to be the active metabolite that plays a central role in AA genotoxicity. The high levels of AA that are orally consumed could be an additional factor for global cancer risk. However, cancer risk estimation in population is still problematic and the mechanisms of DNA repair triggered by GA are still poorly understood. The aim of this work was to evaluate the role of DNA repair genes in the genotoxicity induced by GA in human cells, using complementary methodologies. For this purpose, the effect of GA (250 µM) in cultured human lymphocytes from healthy non-smoker individuals (n=13) was assessed using the sensitive sister-chromatid exchange assay (SCE, 46 h in vitro exposure). GA was found to be genotoxic, with a large inter-individual variation. Therefore we studied the possible influence...

Acrylamide (AA) has been classified as a probable human carcinogen by IARC. AA is generated in carbohydrate-rich foodstuff upon heating and its discovery in a variety of fried foods has raised public health concerns since oral consumption of AA could be an additional risk factor for cancer. Glycidamide (GA), formed via epoxidation, presumably by cytochrome P450 2E1, is considered the ultimate genotoxic agent playing a central role in AA carcinogenesis. Previous studies done by our group reported a strong correlation between GA-induction of Sister Chromatid Exchanges (SCE) and GA-DNA adducts, suggesting the possible involvement of Base Excision Repair (BER) and Homologous Recombination Repair (HRR) pathways in AA/GA induced DNA damage. Presently there is a lot of information concerning the mechanisms of genotoxicity of AA and GA in mammalian cells, however this is not the case for the role of AA and GA in gene expression. In view of this, we studied gene expression in non-malignant m...