Novel NBQ-48 as marker of hypoxic cells in 2D and 3D colon cancer cells.

This study presents the applicability of a novel nitro-substituted heterocyclic compound NBQ48, member of the family of compounds identified as 3 nitrobenzazolo[3, 2-a] quinolinium chloride salts (NBQS) as a screening tool to identify hypoxic tumor cells. The applicability was tested on COLO 205 colon cancer cells 2D and 3D cultures treated with NBQ48 to assess the formation of a bio-reduction fluorescent metabolite under hypoxic conditions in contrast, to those under aerobic environment. Hypoxic environment was created applying a controlled hypoxic gas chamber. Prior to testing the applicability of NBQ48 as a hypoxic fluorescent marker, cytotoxic studies where performed to identify a low-toxicity dose at 24 hours under aerobic and hypoxic environments that would allow the bio-reduction process with little toxicity. The differences in fluorescence emission after treatment was measured by fluorometer and fluorescence microscopy. Results indicated that cell treatments up to 24 hours with NBQ48 under aerobic environment did not reach an IC50 dose in COLO205 cells, however under hypoxic environment the IC50 reached at 100µM. The significant fluorescence increment after 24 and 48 hrs in 2D and 3D cultures treated with NBQ48 (75uM) at hypoxic in contrast to aerobic environments clearly demonstrated the need of a low oxygen content for the bio-reduction of the parent NBQ48. This study confirms the applicability of NBQ48 as markers for hypoxia in metabolically active 2D and 3D cultures. This hydrophilic hypoxic marker could additionally aid researchers in testing hypoxia activated pro-drugs for therapeutic applications in cancer as well as on other diseases where cellular hypoxia is a significant risk factor.

Synthesis of novel 4-Boc-piperidone chalcones and evaluation of their cytotoxic activity against highly-metastatic cancer cells.

In this study, six curcuminoids containing a tert-butoxycarbonyl (Boc) piperidone core were successfully synthesized, five of them are novel compounds reported here for the first time. These compounds were prepared through an aldolic condensation by adding tetrahydropyranyl-protected benzaldehydes or substituted benzaldehyde to a reaction mixture containing 4-Boc-piperidone and lithium hydroxide in an alcoholic solvent. A 44-94% yield was obtained supporting the developed methodology as a good strategy for the synthesis of 4-Boc-piperidone chalcones. Cytotoxic activity against LoVo and COLO 205 human colorectal cell lines was observed at GI50 values that range from 0.84 to 34.7 µg/mL, while in PC3 and 22RV1 human prostate cancer cell lines, GI50 values ranging from 17.1 to 22.9 µg/mL were obtained. Results from biochemical assays suggest that the cytotoxicity of the 4-Boc-piperidone chalcones can be linked to their ability to induce apoptosis, decrease the activity of NFκB and cellular proliferation. Our findings strongly support the potential of Boc-piperidone chalcones as novel cytotoxic agents against highly-metastatic cancer cells.

Cell Death Effects of the Phthalate 2-Ethyl-1-Hexanol on Human Linfoblast Cells.

Phthalates have been used in a wide variety of consumer goods. Their versatility as plasticizers has translated into worldwide use in a vast array of consumer products. These compounds can leach into matrices, such as food and liquids that can be routed for human exposure. One of the most used phthalates is Diethylhexyl phthalate (DEHP). Diethylhexyl phthalate and its metabolite 2-ethyl-1-hexanol (2-EH) have demonstrated biological effects which merit further evaluation. In this work, we expand on our previous work with DEHP and screen the 2-EH metabolite for different cell death endpoints such as growth inhibition, apoptosis, autophagy, caspase activation, DNA fragmentation, and cell cycle arrest using fluorophores and the NC3000 instrument. Significant results (p < 0.05) revealed higher toxicity for the 2-EH metabolite when compared to DEHP. Also, 2-EH presented apoptosis induction with characteristic hallmarks, such as loss of mitochondrial membrane potential, caspase activation, DNA fragmentation and cell cycle arrest at the S phase. In addition, the presence of autophagosome was detected through L3CB protein staining. We conclude that 2-EH presents differences in cell death endpoints that interestingly differ from the DEHP parent compound. Further studies are needed to establish the molecular pathways responsible for the observed effects.

Toxicity and Apoptosis Related Effects of Benzimidazo [3,2-α] Quinolinium Salts Upon Human Lymphoma Cells.

OBJECTIVES: The present study evaluates novel cationic quinoline derivatives known as benzimidazo[3,2-a]quinolinium salts (BQS) named NBQ-48 and ABQ-48 that have structural similarities to known anti-cancer substances such as ellipticine and berberine. METHODS: Toledo human lymphoma (ATCC CRL2631) cells were treated for 24 to 48 hours. Apoptosis related endpoints such as cell cycle arrest, mitochondrial damage, RNS and ROS generation and the activity of several apoptosis related proteins such as caspases and apoptosis inducing factor (AIF) were studied using fluorescence staining and western blot respectively. RESULTS: Results indicated a higher toxicity from the amino substituted ABQ-48 versus the NBQ-48 (GI50's of 50uM versus 100uM respectively). Both compounds induced cell death through various apoptosis related endpoints including a decrease in mitochondrial membrane potential with an increase in ROS and activation of the effector caspase 3. Interestingly, AIF release was observed on cells treated with the amino substituted ABQ-48 but not on the nitro substituted NBQ-48 samples suggesting a caspase independent mechanism for ABQ-48. CONCLUSIONS: The results obtained presents the toxic effects of two novel benzimidazo[3,2-a]quinolinium salts in human lymphoma tumor cells. The identified mechanism of action includes multiple apoptosis related effects. Furthermore the data presents a clear variation in caspase dependent or independent mechanism for each compound.

Immunomodulatory Response Triggered by the Alkaloids, 3-Amino-7-Benzylbenzimidazo[3,2-a] Quinolinium Chloride (ABQ-48) and 3-Nitro-7-Benzylbenzimidazo [3,2-a] Quinolinium Chloride (NBQ-48).

ABQ-48 (3-amino-7-benzylbenzimidazo[3,2-a]quinolinium chloride) and NBQ-48 (3-nitro-7-benzylbenzimidaw[3,2-a] quinolinium chloride) are un-natural alkaloids containing a planar heteroaromatic systems characterized by quaternized nitrogen fused to benzothiazole nucleus. Both compounds are structurally related to naturally occurring substances such as elliptine (from Ochrosia), and berberine (from Berberis). Previous in vitro studies have shown these agents to control tumor-cell proliferation indicating that both BQS are active but especially ABQ-48 at a 1 OuM dose with over 80% control of the proliferation of multiple cancer cell lines from various etiologies including colon, melanoma, CNS and ovarian cells. Mechanism of action studies have also been conducted however this is the first approach to evaluate immune modulatory activity of these novel BQS. Immune-based therapy is an increasing field in which scientists identify how the immunomodulatory activity of known and newly discovered compounds elicits an immune response that could be used against diseases. In this study, our main objective was to apply an in vitro model to show the immunomodulatory effects of ABQ-48 and NBQ-48 by analyzing the cytokine profile resulting after extracted murine spleen cells were treated with both BQS using a fluorescence-based multiplex ELISA approach. Screened cytokines included: G-CSF, GM-CSF, IL-1a, IL-2, IL-3, IL-5, IL-6, IL-7, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-21, IL-23, IFN-γ, and TNF-α. Our study results show ABQ 48 and NBQ-48 to stimulate the release of G-CSF, IL-2, IL-6, and, IFN-γ when mouse splenocytes are incubated with serial dilutions of these agents. Our finding opens new possibilities of potentially using ABQ-48 and NBQ-48 as immunomodulatory agents; with intend to activate the immune system such as the production of neutrophils against cancer or reducing chemotherapy side effects.

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.

Prevalencia de desnutrición en una unidad de media y larga estancia hospitalaria / Prevalence of malnutrition in a mind-long term stay unit

Introducción: La desnutrición es un problema de salud frecuente, especialmente en el ámbito hospitalario donde se asocia con estancias más prolongadas y mayor tasa de morbi-mortalidad. Por otro lado, los avances científicos actuales y la mayor expectativa de vida, han producido un aumento progresivo de unidades de media y larga estancia (UMLE). Objetivos: Determinar la prevalencia de desnutrición en una UMLE mediante el uso del MNA y la VGS, así como analizar los factores/características clínicas asociadas a la misma y sus repercusiones. Métodos: Estudio transversal, descriptivo de una cohorte formada por 201 pacientes ingresados en la UMLE de un Hospital universitario de forma consecutiva durante 12 meses. Se recogieron variables clínicas, antropométricas, bioquímicas e historia nutricional, así como escala de comorbilidad de Charlson, escala cognitiva de Pfeiffer, escala funcional de Barthel y presencia de edemas, ascitis y úlceras por presión. El estado nutricional fue evaluado en las primeras 24-72 horas de ingreso mediante el MNA y la VGS. La presencia de disfagia se evaluó mediante el cuestionario EAT-10 y el MECV-V. Resultados: La prevalencia global de desnutrición fue del 76,6%, siendo la desnutrición mixta grave la más prevalente (20.4%). Los pacientes desnutridos tenían más edad (p=0,002), mayor grado de dependencia (p<0,0001), mayor deterioro cognitivo (p<0,0001) y mayor prevalencia de infecciones urinarias (p=0,026) y presencia de escaras (p=0,005). En el 43.6% de los pacientes se diagnosticó disfagia (MECV-V patológico). Conclusiones: La prevalencia de desnutrición es muy elevada en unidades médicas de larga estancia, generalmente es grave y se asocia con mayor comorbilidad. Casi la mitad de los pacientes presentaban disfagia. El cribaje y valoración nutricional son imprescindibles para el adecuado diagnóstico y tratamiento del estado nutricional en estas unidades (AU)

Introduction: Malnutrition is a common health problem, especially in hospitalized patients, where it’s associated with longer hospital stays and higher rates of morbidity and mortality. Furthermore, current scientific advances and life expectancy increase, have produced a progressive increase of mid- to long-term stay units (UMLE). Aims: To determinate the prevalence of malnutrition on admission to a mid- to long-term stay unit, using MNA and VGS and to analyze the possible factors/clinical features associated with malnutrition and its consequences. Methods: Descriptive and transversal study conducted with 201 patients admitted consecutively for 12 months in an Universitary Hospital mid- to long-term stay unit (Valencia). Clinical, anthropometric, biochemical and nutritional history data were registered, as well as Charlson comorbidity scale, Pfeiffer cognitive scale, Barthel functional scale and presence of edema, ascitis and pressure ulcers. Nutritional status was evaluated in the first 24-72 hours of admission using MNA and VGS. Dysphagia was evaluated using EAT-10 and MECV-V questionaires. Results: The overall rate of malnutrition was 76,6%, being severe protein energy malnutrition the most common type (20,4%). Malnourished patients were older (p=0,002), presented greater dependence (p<0,0001) and greater cognitive impairment (p<0,0001) and they had higher prevalence of urinary tract infections (p=0,026) and presence of pressure ulcer (p=0,005). Dysphagia was diagnosed in 43.6% of the patients. Conclusions: The prevalence of malnutrition is higher Correspondencia: María Argente Pla. in a mid to long-term stay unit, is usually severe and as sociated with greater comorbidity. Almost half of the patients had dysphagia. Nutritional assessment is essential for establishing the correct diagnosis and treatment of the nutritional status in mid to - long term stay unit (AU)

Cytocompatibility of direct water synthesized cadmium selenide quantum dots in colo-205 cells.

Cadmium selenide quantum dots (CdSe QDs), inorganic semiconducting nanocrystals, are alluring increased attraction due to their highly refined chemistry, availability, and super tunable optical properties suitable for many applications in different research areas, such as photovoltaics, light-emitting devices, environmental sciences, and nanomedicine. Specifically, they are being widely used in bio-imaging in contrast to organic dyes due to their high brightness and improved photo-stability, and their ability to tune their absorption and emission spectra upon changing the crystal size. The production of CdSe QDs is mostly assisted by trioctylphosphine oxide compound, which acts as solvent or solubilizing agent and renders the QDs soluble in organic compounds (such as toluene, chloroform, and hexane) that are highly toxic. To circumvent the toxicity-related factor in CdSe QDs, we report the synthesis of CdSe QDs capped with thioglycolic acid (TGA) in an aqueous medium, and their biocompatibility in colo-205 cancer cells. In this study, the [Cd2+]/[TGA] ratio was adjusted to 11:1 and the Se concentration (10 and 15 mM) was monitored in order to evaluate its influence on the optical properties and cytocompatibility. QDs resulted to be quite stable in water (after purification) and RPMI cell medium and no precipitation was observed for long contact times, making them appealing for in vitro experiments. The spectroscopy analysis, advanced electron microscopy, and X-ray diffractometry studies indicate that the final products were successfully formed exhibiting an improved optical response. Colo-205 cells being exposed to different concentrations of TGA-capped CdSe QDs for 12, 24, and 48 h with doses ranging from 0.5 to 2.0 mM show high tolerance reaching cell viabilities as high as 93 %. No evidence of cellular apoptotic pathways was observed as pointed out by our Annexin V assays at higher concentrations. Moreover, confocal microscopy analysis conducted to evaluate the intracellular uptake of TGA-CdSe QDs reveal that the TGA-CdSe QDs were uniformly distributed within the cytosolic side of cell membranes. Our results also suggest that under controlled conditions, direct water-soluble TGA-CdSe QDs can be potentially employed for bio-imaging colo-205 cancer cells with minimal adverse effects.

Xylazine as a drug of abuse and its effects on the generation of reactive species and DNA damage on human umbilical vein endothelial cells.

Human xylazine (XYL) abuse among addicts has received great interest due to its potential toxic effects upon addicts and the need to understand the mechanism of action associated with the potential health effects. XYL is an alpha-2 agonist restricted to veterinarian applications, without human medical applications. Our previous work demonstrated that XYL and its combination with cocaine (COC) and/or 6-monoacetylmorphine (6-MAM) induce cell death through an apoptotic mechanism. The aim of this study was to determine the effect of xylazine on the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) as well as DNA damage on endothelial cell. Human umbilical vein endothelial cells (HUVEC) were treated with XYL (60 µM), COC (160 µM), 6-MAM (160 µM), camptothecin (positive control, 50 µM), XYL/COC (50 µM), XYL/6-MAM (50 µM), and XYL/COC/6-MAM (40 µM) for a period of 24 hours. Generation of intracellular ROS, RNS, and DNA fragmentation were analyzed using a fluorometric assay. Results reveal that XYL and 6-MAM increase levels of ROS; no induction of RNS production was observed. The combination of these drugs shows significant increase in DNA fragmentation in G2/M phase, while XYL, COC, and 6-MAM, without combination, present higher DNA fragmentation in G0/G1 phase. These findings support that these drugs and their combination alter important biochemical events aligned with an apoptotic mechanism of action in HUVEC.

[Prevalence of malnutrition in a mid-long term stay unit]. / Prevalencia de desnutrición en una unidad de media y larga estancia hospitalaria.

INTRODUCTION: Malnutrition is a common health problem, especially in hospitalized patients, where it's associated with longer hospital stays and higher rates of morbidity and mortality. Furthermore, current scientific advances and life expectancy increase, have produced a progressive increase of mid- to long-term stay units (UMLE). AIMS: To determinate the prevalence of malnutrition on admission to a mid- to long-term stay unit, using MNA and VGS and to analyze the possible factors/clinical features associated with malnutrition and its consequences. METHODS: Descriptive and transversal study conducted with 201 patients admitted consecutively for 12 months in an Universitary Hospital mid- to long-term stay unit (Valencia). Clinical, anthropometric, biochemical and nutritional history data were registered, as well as Charlson comorbidity scale, Pfeiffer cognitive scale, Barthel functional scale and presence of edema, ascitis and pressure ulcers. Nutritional status was evaluated in the first 24-72 hours of admission using MNA and VGS. Dysphagia was evaluated using EAT-10 and MECV-V questionnaires. RESULTS: The overall rate of malnutrition was 76,6%, being severe protein energy malnutrition the most common type (20,4%). Malnourished patients were older (p=0,002), presented greater dependence (p.

Introducción: La desnutrición es un problema de salud frecuente, especialmente en el ámbito hospitalario donde se asocia con estancias más prolongadas y mayor tasa de morbi-mortalidad. Por otro lado, los avances científicos actuales y la mayor expectativa de vida, han producido un aumento progresivo de unidades de media y larga estancia (UMLE). Objetivos: Determinar la prevalencia de desnutrición en una UMLE mediante el uso del MNA y la VGS, así como analizar los factores/características clínicas asociadas a la misma y sus repercusiones. Métodos: Estudio transversal, descriptivo de una cohorte formada por 201 pacientes ingresados en la UMLE de un Hospital universitario de forma consecutiva durante 12 meses. Se recogieron variables clínicas, antropométricas, bioquímicas e historia nutricional, así como escala de comorbilidad de Charlson, escala cognitiva de Pfeiffer, escala funcional de Barthel y presencia de edemas, ascitis y úlceras por presión. El estado nutricional fue evaluado en las primeras 24-72 horas de ingreso mediante el MNA y la VGS. La presencia de disfagia se evaluó mediante el cuestionario EAT-10 y el MECV-V. Resultados: La prevalencia global de desnutrición fue del 76,6%, siendo la desnutrición mixta grave la más prevalente (20.4%). Los pacientes desnutridos tenían más edad (p=0,002), mayor grado de dependencia (p.

Biological Activity of N-Hydroxyethyl-4-aza-2,3-didehydropodophyllotoxin Derivatives upon Colorectal Adenocarcinoma Cells.

Etoposide is a chemotherapy drug derived from the natural lignin podophyllotoxin. Our novel generated Aza-podophyllotoxin compounds (AZP 8a & AZP 9a) are analogues of podophyllotoxin and were previously screened for anti-cancer activity through the NCI 60 cell line screening panel showing activity on various cell types including colon cancer. This study expands the toxicological screening by studying apoptosis and various hallmark events as part of the mechanism of action of these compounds on colon cancer cells. The COLO 205 cell line was selected and exposed to AZP to determine the IC50 doses at 24 hours treatment. Apoptosis hallmark events such as migration of phosphatidylserine (PS) to the cell membrane, DNA fragmentation, cell cycle effects, mitochondrial membrane permeabilization and caspase activation were included. Experiments were performed in triplicates for all tested compounds including AZP 8a, AZP 9a, camptothecin as positive control and vehicle as negative control. Our results present contrasting apoptotic activity between the experimental compounds. Compound 8a presented migration of PS (annexin V assay), DNA fragmentation and cell cycle arrest at S phase. Compound 9a presented PS migration with fragmented DNA, cell cycle arrest at S phase, mitochondrial membrane permeabilization and activation of caspase 3, 8 and 9. Compound 8a without the oxygen atoms in ring A appears to cause effects similarly to autophagy as induced by etoposide, a cancer drug analogue of our heterocyclic compounds. Compound 9a with the oxygen atoms in expanded ring A presented induction of cell death following activation of a classical apoptosis pathway. Our results suggest that minor structural differences among these AZP can account for the difference in biological response and cancer cell toxicity.

Differentiated thyroid carcinoma in a girl with resistance to thyroid hormone management with triiodothyroacetic acid.

Thyroid hormone resistance syndrome is characterized by a reduced target tissue response to the action of thyroid hormone, which leads to high levels of free thyroxine and free triiodothyronine with non-suppressed levels of thyrotropin (TSH). Recently, three cases of papillary thyroid carcinoma associated with thyroid hormone resistance syndrome were published. The main challenge in this situation is the difficulty of maintaining the suppression of TSH levels without producing symptoms of hyperthyroidism. We present another case of an association of thyroid hormone resistance syndrome and papillary thyroid carcinoma, and we share our experience with 3,5,3'-triiodothyroacetic acid, which made possible an easier management of the carcinoma after surgery, maintaining the TSH levels suppressed despite the resistance to thyroid hormones.

Novel Nitrobenzazolo[3,2-a]quinolinium Salts Induce Cell Death through a Mechanism Involving DNA Damage, Cell Cycle Changes, and Mitochondrial Permeabilization.

This study reports the capacity of three nitro substituted benzazolo[3,2-a]quinolinium salts NBQs: NBQ 95 (NSC-763304), NBQ 38 (NSC 763305), and NBQ 97 (NSC-763306) as potential antitumor agents. NBQ's are unnatural alkaloids possessing a positive charge that could facilitate interaction with cell organelles. The anticancer activities of these compounds were evaluated through the National Cancer Institute (NCI) 60 cell line screening which represents diverse histologies. The screening was performed at 10 µM on all cell lines. Results from the NCI screening indicated cytotoxicity activity on six cell lines. In order to explore a possible mechanism of action, a detailed biological activity study of NBQ 95 and NBQ 38 was performed on A431 human epidermoid carcinoma cells to determine an apoptotic pathway involving, cell cycle changes, DNA fragmentation, mutations, mitochondrial membrane permeabilization and caspases activation. DNA fragmentation, cell cycle effects, mutagenesis, mitochondrial permeabilization and activation of caspases were determined by fluorimetry and differential imaging. Our data showed that A431 growth was inhibited with an average IC50 of 30 µM. In terms of the mechanism, these compounds interacted with DNA causing fragmentation and cell cycle arrest at sub G0/G1 stage. Mutagenesis was higher for NBQ 38 and moderate for NBQ 95 Mitochon-drial permeabilization was observed with NBQ 38 and slightly for NBQ 95. Both compounds caused activation of Caspases 3 and 7 suggesting an apoptotic cell death pathway through an intrinsic mechanism. This study reports evidence of the toxicity of these novel compounds with overlapping structural and mechanistic similarities to ellipticine, a known anti-tumor compound.

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 IC50's 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.

Metabolic acidosis as a complication of intravenous dextrose administration in a patient with insulinoma.

There are few cases published in literature in which the use of intravenous dextrose as treatment for an insulinoma resulted in a metabolic acidosis. This is due perhaps to the usual method of administration, which is usually at low concentrations, for limited periods or low volumes. We present the case of a woman with suspected insulinoma by laboratory findings in which an endogenous hyperinsulinism was observed. During hospitalization, the patient required a progressive increase of the glucose infusion to prevent severe hypoglycemia. Two days before surgery, the patient presented symptoms of malaise and muscle weakness and a metabolic acidosis with hypokalemia became apparent in the blood analysis. This metabolic imbalance was attributed to a long period of treatment with high volume of intravenous dextrose infusion. If large doses of dextrose are required in a patient with an insulinoma, then the possibility of a metabolic imbalance must be considered during the follow-up. When the suspicion of an insulinoma is high, and all the attempts of pre-operative localization fail, patients should be derived early to specialized centers with modern imaging techniques, so that surgery is not delayed, and this rare and threatening complication could be avoided.

HPLC-MS/MS method for the intracellular determination of ribavirin monophosphate and ribavirin triphosphate in CEM ss 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.

Role of the nitro functionality in the DNA binding of 3-nitro-10-methylbenzothiazolo[3,2-a]quinolinium chloride.

Interest in DNA binding drugs has increased in recent years due to their importance in the treatment of genome-related diseases, like cancer. A new family of water-soluble DNA binding compounds, the benzothiazolo[3,2- a]quinolinium chlorides (BQCls), is studied here as potential candidates for chemical treatment of solid tumor cells that may encounter low-oxygen environments, a condition known as hypoxia. These compounds are good DNA intercalators; however, no studies have been made of these compounds under hypoxic conditions. This work demonstrates the importance of the nitro-functionality in the DNA binding of 3-nitro-10-methylbenzothiazolo[3,2- a]quinolinium chloride (NBQ-91), which possesses nitro-functionality, and 10-methylbenzothiazolo[3,2- a]quinolinium chloride (BQ-106), which does not. Both NBQ-91 and BQ-106 have similar noncovalent binding affinity toward DNA. Dialysis experiments show that NBQ-91 binds DNA under N2-saturated conditions with increasing concentrations of reducing agent, presumably due to reduction of the nitro-functionality. Conversely, because of the lack of nitro-functionality, the presence of a reducing agent had no effect on BQ-106 binding to DNA under both aerobic and N2-saturated conditions. Clonogenic assays were performed to determine the quinolinium chloride cytotoxicities under both aerobic (95% air and 5% CO2) and hypoxic (80% N2 and 20% CO2) conditions. The calculated ratios of cellular toxicity under aerobic to hypoxic conditions caused by the same concentration of test agent (CTR values) show greater levels of cell death under hypoxia than under aerobic conditions for mitomycin C (MC) (CTR = 0.7 at 1 microM) and NBQ-91 (CTR = 0.4 at 200 microM) than for BQ-106 (CTR = 1.0 at 200 microM), which agreed with the previously reported data for MC and confirmed the importance of nitro-functionality for reactivity under hypoxic conditions. There was no correlation between noncovalent binding affinity constants and their cytotoxicity under hypoxic conditions. Adduct formation between the NBQ-91 and 2'-dG was also assessed by reacting 2'-dG or DNA with NBQ-91 and BQ-106 under N2-saturated conditions in the presence of hypoxanthine and xanthine oxidase (HX/XO). DNA covalent adduct formation was analyzed by two techniques: LC-ESI-MS and Sephadex size exclusion chromatography. LC-ESI-MS results clearly indicate the formation of a prominent molecular ion at masses of 266.0 and 530.58 Da, corresponding to the [M + H](+2) and [M](+) molecular ions of the monitored 2'-dG-NBQ-91 adduct. Results from the Sephadex size exclusion chromatography support these findings because the NBQ-91 elution percentage increases in the presence of HX/XO due to the reduction of the nitro-functionality, which results in covalent binding to DNA. This study reports evidence of the DNA binding capacity of this bioreductive drug. The preferential N2-saturated over aerobic conditions for DNA binding makes NBQ-91 a potential bioreductive compound for hypoxic cell killing.

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 MgCl(2) 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 MgCl(2). 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.

Comparison of the nucleic acid covalent binding capacity of two nitro-substituted benzazolo[3,2-a]quinolinium salts upon enzymatic reduction.

The DNA binding capacity of two nitro-substituted benzazolo[3,2-a]quinolinium chlorides (NBQs), NBQ-38 and NBQ-95, was evaluated upon their enzymatic reduction with hypoxanthine (HX)/xanthine oxidase (XO) under anaerobic conditions. In the presence of 2'-deoxyguanosine (2'-dG) or calf thymus DNA, covalent-addition products were monitored. Reactions of each NBQ with 2'-dG or DNA differed in the NBQ to HX molar ratio. Control reactions, one without HX/OX and another under aerobic conditions, were also analyzed. Adducts were isolated and characterized by high performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS). Authentic samples of the reduced forms of these NBQs, identified as ABQ-38 and ABQ-95, were synthesized as standards to monitor bioreduction processes. HPLC analysis showed that the yield of formation of an unknown product (possibly, 2'-dG-NHBQ-38 adduct) from the reaction of NBQ-38 with 2'-dG and DNA was proportional to the HX to NBQ-38 molar ratio. ESI-MS analysis of the DNA hydrolysates showed evidence of an adduct formed upon bioreduction of NBQ-38 by the ions detection at m/z 528.3 and 454.8, consistent with chemical structures of a 2'-dG-NHBQ-38 adduct and a fragment ion. DNA adducts were not observed with NBQ-95, although the corresponding bioreduction product ABQ-95 was detected by ESI-MS. This study provides mechanistic information of these bioreductively-activated pro-drugs with potential therapeutic applications.

Detection and quantification of 4-ABP adducts in DNA from bladder cancer patients.

We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl (dG-C8-ABP) adducts using the liquid chromatography tandem mass spectrometry method with a 700 attomol (1 adduct in 10(9) bases) detection limit. Hemoglobin (Hb) 4-aminobiphenyl (4-ABP) adduct levels were measured by gas chromatography-mass spectrometry. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d9) dG-C8-ABP (MW=443 Da) was added to each sample. Structural evidence and adduct quantification were determined by selected reaction monitoring, based on the expected adduct ion [M+H+]+1, at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444-->328. The method was validated by analysis of DNA (100 microg each) from calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 microg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5-80 dG-C8-ABP adducts per 10(9) bases; only 1 out of 27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12-1960 pg per gram Hb. There was no correlation between levels of DNA and Hb adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.