Beatriz Zayas

Mitochondrial permeability and toxicity of diethylhexyl and monoethylhexyl phthalates on TK6 human lymphoblasts cells

Phthalates are ubiquitous compounds used in the manufacturing industry. Some are known endocrine disruptors, acting as xenoestrogens, others induce reproductive toxicity and damage to DNA among other effects. Studies on apoptosis induction and mitochondrial damage capacity of phthalates on the immune system are limited. This study aims to determine cell viability inhibition and apoptosis induction of diethylhexyl phthalate (DEHP) and monoethylhexyl phthalate (MEHP) on the human TK6 lymphoblast cell line at concentrations found in the environment. Key hallmark events, such as mitochondrial membrane permeability, generation of reactive oxygen species (ROS) and activation of caspase 3 and 7 were measured. Concentrations that inhibit viability of 50% (IC50) of the cells were determined at 24, 48 and 72 h with doses ranging from 10 to 500 μM. Changes in mitochondrial membrane permeability, ROS generation and activation of caspases 3 and 7, were measured as part of the cell death mechanism. The IC50 at 24 h was approximately 250 μM for both phthalates; at 48 h were 234 and 196 μM for DEHP and MEHP, respectively and at 72 h IC50s were 100 and 80 μM for DEHP and MEHP, respectively. Overall the longer the time of exposure the lower the IC50s for both compounds. Both compounds affected mitochondrial membrane potential, promoted ROS generation and activated caspases 3 and 7. MEHP is more toxic, promotes higher level of ROS production and caspases activation. Our findings suggest that DEHP and MEHP have the capacity to induce apoptosis in cells of the immune system at concentrations found in the environment.

HPLC–MS/MS method for the intracellular determination of ribavirin monophosphate and ribavirin triphosphate in CEMss cells

A sensitive and specific method using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the determination of ribavirin monophosphate (RBV-MP) and ribavirin triphosphate (RBV-TP) in cells has been developed and validated. In this method, ribavirin phosphorylated metabolites were extracted and separated by anion exchange solid phase extraction (SPE). The RBV-MP and RBV-TP fractions were dephosphorylated using acid phosphatase and further purified by phenyl boronate SPE prior to HPLC-MS/MS analysis. (13)C(5)-uridine was added as internal standard to obtain better accuracy and precision of the analysis. The MS/MS detector was optimized at multiple reaction monitoring (MRM) using positive electrospray ionization to detect 245-->113 and 250-->133 transitions for ribavirin and internal standard, respectively. The calibration curve was linear over a concentration range of 0.01-10 microg/mL with a limit of quantitation of 0.01 microg/mL. Mean inter-assay accuracy and precision for RBV-MP and RBV-TP quality control samples at 0.03, 0.3 and 8 microg/mL were 5% and 10%, respectively. This method was successfully used for the in vitro determination of RBV-MP and RBV-TP in CEM(ss) cells cultured with RBV.

DNA adducts formed by a novel antitumor agent 11β-dichloro in vitro and in vivo

The multifunctional molecule 11β-dichloro consists of a ligand for the androgen receptor linked to a bifunctional alkylating group, permitting it to create DNA adducts that bind the androgen receptor. We propose that binding of the androgen receptor to 11β-DNA adducts acts to both shield damaged sites from repair and disrupt the expression of genes essential for growth and survival. We investigated the formation 11β-DNA adducts in tumor xenograft and nontumor tissues in mice. Using [14C]-11β-dichloro, we show that the molecule remains intact in blood and is widely distributed in mouse tissues after i.p. injection. Covalent 11β-guanine adducts identified in DNA that had been allowed to react with 11β-dichloro in vitro were also found in DNA isolated from cells in culture treated with 11β-dichloro as well as in DNA isolated from liver and tumor tissues of mice treated with the compound. We used accelerator mass spectrometry to determine the levels of [14C]-11β-DNA adducts in LNCaP cells treated in culture as well as in liver tissue and LNCaP xenograft tumors in treated mice. The level of DNA adducts in tumor tissue was found to be similar to that found in LNCaP cells in culture treated with 2.5 μmol/L 11β-dichloro. Our results indicate that 11β-dichloro has sufficient stability to enter the circulation, penetrate tissues, and form DNA adducts that are capable of binding the androgen receptor in target tissues in vivo. These data suggest the involvement of our novel mechanisms in the antitumor effects of 11β-dichloro. [Mol Cancer Ther 2006;5(4):977–84]

Anticancer activity of 4-aminoquinoline-triazine based molecular hybrids

In this study the potential for anticancer activity of 4-aminoquinoline-triazine based hybrids has been investigated on 60 human cancer cell lines (NCI 60). The representative compounds show activity on a range of cell lines and apoptosis as the mode of growth inhibition.

Imidazolium-derived ionic salts induce inhibition of cancerous cell growth through apoptosis

A study of the effects of imidazolium-based ionic liquids on 60 human cancer cell lines representing diverse histologies has identified four compounds which show potency at a nanomolar dose. Their effects on annexin V, DNA fragmentation, and the cell cycle, together with mitochondrial membrane permeabilization tests, provide insights into their mechanism of action. Also, experiments with A431 human epidermoid carcinoma cells suggest the activation of an apoptotic pathway, due to activity of the initiator caspase 8 and effector caspase 3.

Novel 3,5-bis(arylidiene)-4-piperidone based monocarbonyl analogs of curcumin: anticancer activity evaluation and mode of action study

A series of eighteen novel 3,5-bis(arylidiene)-4-piperidone based symmetrical monocarbonyl analogs of curcumin were synthesised and a subset was screened by National Cancer Institute (NCI), USA for their anticancer activity. Dose–response studies and the mechanism of action investigation suggest that most active compounds are apoptosis inducers.

Thiols oxidation and covalent binding of BSA by cyclolignanic quinones are enhanced by the magnesium cation

A novel cyclolignanic quinone, 7-acetyl-3′,4′-didemethoxy-3′,4′-dioxopodophyllotoxin (CLQ), inhibits topoisomerase II (TOPO II) activity. The extent of this inhibition was greater than that produced by the etoposide quinone (EQ) or etoposide. Glutathione (GSH) reduces EQ and CLQ to their corresponding semiquinones under anaerobic conditions. The latter were detected by EPR spectroscopy in the presence of MgCl2 but not in its absence. Semiquinone EPR spectra change with quinone/GSH mol ratio, suggesting covalent binding of GSH to the quinones. Quinone-GSH covalent adducts were isolated and identified by ESI-MS. These orthoquinones also react with nucleophilic groups from BSA to bind covalently under anaerobic conditions. BSA thiol consumption and covalent binding by these quinones are enhanced by MgCl2. Complex formation between the parent quinones and Mg+2 was also observed. Density functional calculations predict the observed blue-shifts in the absorption spectra peaks and large decreases in the partial negative charge of electrophilic carbons at the quinone ring when the quinones are complexed to Mg+2. These observations suggest a possible role of Mg+2 chelation by these quinones in increasing TOPO II thiol and/or amino/imino reactivity with these orthoquinones.

C5-curcuminoid-4-aminoquinoline based molecular hybrids: design, synthesis and mechanistic investigation of anticancer activity

The privileged scaffolds of curcumin and 4-aminoquinolines are extensively used in the design and synthesis of biodynamic agents having remarkable efficacy against diseases like cancer and malaria. Therefore, we anticipated that covalent hybridization of these two pharmacophores via the triazole linker may lead to molecules with better anticancer activity. The synthesized hybrid compounds were tested for their anti-cancer activity on 60 human cancer cell lines, which represent diverse histologies. Our study has identified a set of these hybrids that showed excellent growth inhibition at nano-molar concentrations. The mechanistic investigations through a series of assays showed apoptotic induction as a cause for their displayed anticancer activity.

Highly Organized Nanofiber Formation from Zero Valent iron Nanoparticles After Cadmium Water Remediation

Many studies have used nanoscale zero valent iron (nZVI) nanoparticles to remove redox-sensitive metals (e.g., As, Cr, U, Se, Ni, Cu) from aqueous systems by absorption or reduction processes. However, very few investigations present a detailed study of the product formed after the remediation process. In order to quantify the efficiency of nZVI particles as a possible cadmium remediation agent, we prepared nZVI by sodium borohydride reduction of an iron complex, FeCl3·6H2O, at room temperature and ambient pressure. Fe0 and nanocrystalline structures of iron oxides and oxyhydroxides were obtained with this method. We exposed the nZVI to 6 ppm of Cd2+ and characterized the products with X-ray diffraction, X-ray absorption and X-ray photoelectron spectroscopy. Inductively coupled plasma analysis showed that the nZVI remediation efficiency of cadmium ions was between 80% and 90% in aqueous media. All of the physical characterization results confirmed the presence of Fe0, α-Fe2O3 and FeOOH. High resolution transmission electron microscopy images showed nanofiber formation of a mixture of Fe0, oxyhydroxides and oxides iron formed after interacting with cadmium ions, possibly forming CdFe2O4. These results suggest that the FeOOH shell and other iron oxides in nZVI could enhance Cd2+ removal. This removal is observed to cause a change of the initial structure of nZVI to nanofibers due to possible formation of CdFe2O4 as a waste product.

Synthesis of novel C5-curcuminoid-fatty acid conjugates and mechanistic investigation of their anticancer activity

The first synthesis of C5-curcumin-fatty acid (C5-Curc-FA) conjugates was successfully performed. Through a two-step synthetic route, 10 analogs were synthesized for a structure-activity relationship (SAR) study. It was found that C5-Curc-FA conjugates containing either decanoic acid or palmitic acid moieties were cytotoxic against colorectal adenocarcinoma cell (CCL-229) at IC50s ranging from 22.5 to 56.1μg/mL, being 5c the most active C5-Curc-FA conjugate. Our results strongly suggests that a decanoic acid moiety at the meta position in C5-Curc-FA conjugates is important for their anticancer activity effect. Possible mechanisms for the anticancer activity of C5-Curc-FA conjugates were also investigated including apoptosis induction, mitochondrial damage and caspases activation. It was shown that 5c inhibited the luminescence activity of NFκB, a key signaling molecule involved in cell apoptosis and cell proliferation, at IC50=18.2μg/mL. In addition, it was demonstrated that 5c displayed significant apoptotic effect at GI50=46.0μg/mL in colorectal adenocarcinoma cell line (ATCC CCL-222), which can be explained by the significant mitochondrial membrane permeabilization and caspases 3 and 7 activation effect of 5c. Finally, it was investigated that C5-Curc-FA conjugates can affect the replication process of cancer cells, since compounds 5c, 5e, and 6c inhibited the relaxing activity of the human DNA topoisomerase I at minimum inhibitory concentrations (MICs) that range from 50 to 250μg/mL. Our results strongly support the hypothesis that the inhibition of both NFκB and DNA topoisomerase I by C5-Curc-FA conjugates is associated with their anticancer activity.