Reactions of Chlorambucil and its main metabolite, Phenylacetic Acid Mustard, with 2’-deoxyribonucleosides and Calf Thymus DNA

Chlorambucil is an anticancer agent used in the treatment of a variety of cancers, especially in chronic lymphocytic leukemia, and autoimmune diseases. Nevertheless, chlorambucil is potentially mutagenic, teratogenic and carcinogenic. The high antitumor activity and high toxicity of chlorambucil and its main metabolite, phenylacetic acid mustard, to normal tissues have been known for a long time. Despite this, no detailed chemical data on their reactions with biomolecules in aqueous media have been available. The aim of the work described in this thesis was to analyze reactions of chlorambucil with 2’-deoxyribonucleosides and calf thymus DNA in aqueous buffered solution, at physiological pH, and to identify and characterize all adducts by using modern analyzing methods. Our research was also focused on the reactions of phenylacetic acid mustard with 2’-deoxynucleosides under similar conditions. A review of the literature consisting of general background of nucleic acids, alkylating agents and ultraviolet spectroscopy used to identify the purine and pyrimidine nucleosides, as well as the results from experimental work are presented and discussed in this doctoral thesis.

Urinary 2,5-hexanedione in workers exposed to n-hexane: influence of the sample treatment

The aim of the present study was to determine 2,5-hexanedione (2,5-HD), a metabolite of n-hexane, by gas chromatography/flame ionization detection in 31 workers exposed to n-hexane after two types of sample pretreatment, i.e., with (total 2,5-HD) and without (free 2,5-HD) acid hydrolysis. The mean urinary 2,5-HD was 0.52 mg/L (free) and 2.88 mg/L (total), this difference being significant (Student t-test, p < 0.05). The differences in the results according to the sample treatment support the need to modify the current Brazilian legislation, which proposes the analysis of 2,5-HD without indicating whether it is the free or total metabolite.

Enhanced fatty acid oxidation in adipocytes and macrophages reduces lipid-induced triglyceride accumulation and inflammation

Lipid overload in obesity and type 2 diabetes is associated with adipocyte dysfunction, inflammation, macrophage infiltration, and decreased fatty acid oxidation (FAO). Here, we report that the expression of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme in mitochondrial FAO, is higher in human adipose tissue macrophages than in adipocytes and that it is differentially expressed in visceral vs. subcutaneous adipose tissue in both an obese and a type 2 diabetes cohort. These observations led us to further investigate the potential role of CPT1A in adipocytes and macrophages. We expressed CPT1AM, a permanently active mutant form of CPT1A, in 3T3-L1 CARΔ1 adipocytes and RAW 264.7 macrophages through adenoviral infection. Enhanced FAO in palmitate-incubated adipocytes and macrophages reduced triglyceride content and inflammation, improved insulin sensitivity in adipocytes, and reduced endoplasmic reticulum stress and ROS damage in macrophages. We conclude that increasing FAO in adipocytes and macrophages improves palmitate-induced derangements. This indicates that enhancing FAO in metabolically relevant cells such as adipocytes and macrophages may be a promising strategy for the treatment of chronic inflammatory pathologies such as obesity and type 2 diabetes.

Enhanced fatty acid oxidation in adipocytes and macrophages reduces lipid-induced triglyceride accumulation and inflammation

Lipid overload in obesity and type 2 diabetes is associated with adipocyte dysfunction, inflammation, macrophage infiltration, and decreased fatty acid oxidation (FAO). Here, we report that the expression of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme in mitochondrial FAO, is higher in human adipose tissue macrophages than in adipocytes and that it is differentially expressed in visceral vs. subcutaneous adipose tissue in both an obese and a type 2 diabetes cohort. These observations led us to further investigate the potential role of CPT1A in adipocytes and macrophages. We expressed CPT1AM, a permanently active mutant form of CPT1A, in 3T3-L1 CARΔ1 adipocytes and RAW 264.7 macrophages through adenoviral infection. Enhanced FAO in palmitate-incubated adipocytes and macrophages reduced triglyceride content and inflammation, improved insulin sensitivity in adipocytes, and reduced endoplasmic reticulum stress and ROS damage in macrophages. We conclude that increasing FAO in adipocytes and macrophages improves palmitate-induced derangements. This indicates that enhancing FAO in metabolically relevant cells such as adipocytes and macrophages may be a promising strategy for the treatment of chronic inflammatory pathologies such as obesity and type 2 diabetes.

The importance of pre-treatment of spent hydrotreating catalysts on metals recovery

This work describes a three-step pre-treatment route for processing spent commercial NiMo/Al2O3 catalysts. Extraction of soluble coke with n-hexane and/or leaching of foulant elements with oxalic acid were performed before burning insoluble coke under air. Oxidized catalysts were leached with 9 mol L-1 sulfuric acid. Iron was the only foulant element partially leached by oxalic acid. The amount of insoluble matter in sulfuric acid was drastically reduced when iron and/or soluble coke were previously removed. Losses of active phase metals (Ni, Mo) during leaching with oxalic acid were compensated by the increase of their recovery in the sulfuric acid leachate.

Formulation and characterization of poloxamer 407®: thermoreversible gel containing polymeric microparticles and hyaluronic acid

The influence of the composition and preparation method on the sol-gel transition temperature (Tsol-gel) and rheological response of poloxamer-based formulations was determined. Manual and more complex mechanical stirring were found to provide similar results. In addition, a linear dependence of Tsol-gel on the poloxamer content was observed in the range of concentrations analyzed, and a Poloxamer 407® concentration of 18% was selected. The addition of hyaluronic acid did not lead to significant changes in the Tsol-gel values. In contrast, the addition of microparticles caused a reduction in Tsol-gel without a significant reduction in gel strength, and pseudoplastic characteristics were observed, indicating that a thermoreversible gel was obtained with a rheology suitable for application in the treatment of burn wounds.

A study on the inhibition of mild steel corrosion in hydrochloric acid by pyridoxol hydrochloride

The inhibition of the corrosion of mild steel in 2M hydrochloric acid solutions by Pyridoxol hydrochloride (PXO) has been studied using weight loss and hydrogen evolution techniques. The inhibitor (PXO) exhibited highest inhibition efficiency of 71.93% at the highest inhibitor concentration of 1.0 x 10-2M investigated and a temperature of 303K from weight loss result. Also, inhibition was found to increase with increasing concentration of the inhibitor and decreasing temperature. A first order type of mechanism has been deduced from the kinetic treatment of the weight loss results and the process of inhibition attributed to physical adsorption. The results obtained from the two techniques show that pyridoxol hydrochloride could serve as an effective inhibitor of the corrosion of mild steel in HCl acid solution. The compound obeys the Langmuir adsorption isotherm equation.

Kainic acid-induced seizures: inflammation and excitotoxic neuronal damage in the developing rat hippocampus

Epileptic seizures are harmful to the developing brain. During epileptic seizures, overactivation of glutamate receptors (GluR) leads to neuronal degeneration, defined as excitotoxicity. The hippocampus is especially vulnerable to excitotoxic neuronal death, but its mechanism has remained incompletely known in the developing brain. Recently, signs of activation of inflammatory processes after epileptic seizures have been detected in the hippocampus. The purpose of this thesis was to study the inflammatory reaction and death mechanisms in excitoxic neurodegeneration induced by the glutamate analogue kainic acid (KA) in the developing hippocampus. Organotypic hippocampal slice cultures (OHCs), prepared from 6-7-day-old rats (P6-7) and treated with KA, served as an in vitro model. KA-induced status epilepticus in P9 and P21 rats was used as an in vivo model. The results showed that the pyramidal cell layers of the hippocampus were the most susceptible to irreversible and age-specific neurodegeneration, which occurred in the juvenile (P21), but not in the immature (P9), rat hippocampus. The primary death mechanism was necrosis as there were no significant changes in the expression of selected apoptosis markers and morphological cellular features of necrosis were found. Inflammatory response was similarly age-dependent after KA treatment as a rapid, fulminant and wide response was detected in the juvenile, but not in the immature, rat brain. An anti-inflammatory drug treatment, given before KA, was not neuroprotective in OHCs, possibly because of the timing of the treatment. In summary, the results suggest that KA induces an age-dependent inflammatory response and necrotic neurodegeneration, which may cause disturbances in hippocampal connectivity and promote epileptogenesis.

Anticoccidial effects of acetic acid on performance and pathogenic parameters in broiler chickens challenged with Eimeria tenella

The objective of the present study was to evaluate the anticoccidial effect of the different concentrations of the acetic acid in the broiler chickens in comparison with the amprolium anticoccidial. A total of 198 chicks were placed 11 per pen with three pens per treatment. The different concentrations (1%, 2% and 3%) of acetic acid and amproilum (at the dose rate of 125ppm) were given to the experimental groups in drinking water from 10-19th days of age. One group was kept as infected non medicated control and one as non infected non medicated control. All the groups were inoculated orally with 75,000 sporulated oocysts at the 12th day of age except non infected non medicated control. Anticoccidial effect was evaluated on the basis of performance (weight gain, feed conversion ratio) and pathogenic (oocyst score, lesion score and mortality %age) parameters. Among acetic acid medicated groups, the maximum anticoccidial effect was seen in the group medicated with 3% acetic acid followed by 2% and 1% acetic acid medicated groups. Amprolium and 3% acetic acid were almost equivalent in suppressing the negative performance and pathogenic effects associated with coccidiosis (Eimeria tenella) challenge. In summary, acetic acid has the potential to be used as alternative to chemotherapeutic drugs for Eimeria tenella control. Concentration-dependent anticoccidial effect of acetic acid suggests that further studies should be carried out to determine the possible maximum safe levels of acetic acid with least toxic effects to be used as anticoccidial.

Anticoccidial activity of hydrochloric acid (HCl) against Eimeria tenella in broiler chickens

The present study was planned to evaluate the anticoccidial activity of the different concentrations of the HCl against Eimeria tenella infection in broiler chickens in comparison with the amprolium anticoccidial. For this purpose, a total of 198 chicks were placed 11 per pen with three pens per treatment. The different concentrations of HCl (1000ppm, 2000ppm and 3000ppm) and amproilum (at the dose rate of 125ppm) were given to the experimental groups in drinking water from 10 to 19th days of age. One group was kept as infected non medicated control and one as non infected non medicated control. At the 12th day of age, all the groups were inoculated orally with 75,000 sporulated oocysts except non infected non medicated control. Anticoccidial activity was evaluated on the basis of performance (weight gain, feed conversion ratio) and pathogenic (oocyst score, lesion score and mortality %age) parameters. Among HCl medicated groups, the maximum anticoccidial effect was seen in the group medicated with 1000ppm HCl followed by 2000ppm and 3000ppm HCl medicated groups. Amprolium and 1000ppm HCl were almost equivalent in suppressing the negative performance and pathogenic effects associated with coccidiosis (Eimeria tenella) challenge. In summary, the lower doses of HCl have the potential to be used as alternative to chemotherapeutic drugs for Eimeria tenella control. It is therefore suggested that further studies should be carried out to determine the possible minimum safe levels of HCl with least toxic effects to be used as anticoccidial.

Treatment of NI-containing acidic mine waters with calcite side-stones

Acid mine drainage (AMD) presents a serious problem for the environment for the massive formation of acidic leachates containing heavy metals. The present work deals with the AMD treatment using neutralizing limestone side-products. The conventional methods for prevention, mitigating and control of AMD formation are described. The experimental testing of Nordkalk Oy calcite-containing side-stones for acid neutralizing and removal of nickel from solutions presents the research objective. The batch experiments in acid neutralizing with subsequent metal content analysis were carried out. The results showed the dependence of pH on the dose of neutralizing material and the exposure time. The nickel removal, unlike iron, within the pH range from 1.2 to 6.0 appeared to be inadequate. The further research on nickel co-precipitation with iron and aluminium may appear to be necessary together with testing of alkalinity strengthening materials.

Microwave-assisted solvent extraction and analysis of shikimic acid from plant tissues

A better method for determination of shikimate in plant tissues is needed to monitor exposure of plants to the herbicide glyphosate [N-(phosphonomethyl)glycine] and to screen the plant kingdom for high levels of this valuable phytochemical precursor to the pharmaceutical oseltamivir. A simple, rapid, and efficient method using microwave-assisted extraction (MWAE) with water as the extraction solvent was developed for the determination of shikimic acid in plant tissues. High performance liquid chromatography was used for the separation of shikimic acid, and chromatographic data were acquired using photodiode array detection. This MWAE technique was successful in recovering shikimic acid from a series of fortified plant tissues at more than 90% efficiency with an interference-free chromatogram. This allowed the use of lower amounts of reagents and organic solvents, reducing the use of toxic and/or hazardous chemicals, as compared to currently used methodologies. The method was used to determine the level of endogenous shikimic acid in several species of Brachiaria and sugarcane (Saccharum officinarum) and on B. decumbens and soybean (Glycine max) after treatment with glyphosate. The method was sensitive, rapid and reliable in all cases.

Glyphosate as a tool to produce shikimic acid in plants

Oseltamivir phosphate is a potent viral inhibitor produced from shikimic acid extracted from seeds of Ilicium verum, the most important natural source. With the site of action 5-enolpyruvylshikimate-3-phosphate synthase (EPSP), glyphosate is the only compound capable of inhibiting its activity with the consequent accumulation of shikimic acid in plants. Corn and soybean plants were sprayed with reduced rates of glyphosate (0.0 to 230.4 g a.i. ha¹) and shikimic acid content in the dry mass was determined by HPLC 3, 7 and 10 days after application. Results showed shikimic acid accumulation in dry mass with increases of up to 969% in corn and 33,000% on soybeans, with peak concentrations 3 days after treatment (DAT). Industrial feasibility for shikimic acid production, combined with favorable climatic conditions for growing corn and soybean in virtually all over Brazil, favor the use of reduced rates of glyphosate in shikimic acid biosynthesis, with potential for use as an inducer in exploration of alternative sources for production of oseltamivir phosphate with low environmental impact.

Effect of all-trans retinoic acid on newly diagnosed acute promyelocytic leukemia patients: results of a Brazilian center

Thirty-seven patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (ATRA). Patients received 45 mg m-2 day-1 po of ATRA until complete remission (CR) was achieved, defined as: a) presence of less than 5% blasts in the bone marrow, with b) white blood cells >103/mm3, c) platelets >105/mm3 and d) hemoglobin concentration >8 g/dl, with no blood or platelet transfusions. Thirty-one (83.7%) patients achieved CR by day 50, and 75% of these before day 30. Correction of the coagulopathy, achieved between days 2 and 10 (mean, 3 days), was the first evidence of response to treatment. Only one patient had been previously treated with chemotherapy and three had the microgranular variant M3 form. Dryness of skin and mucosae was the most common side effect observed in 82% of the patients. Thrombosis, hepatotoxicity and retinoid acid syndrome (RAS) were observed in 7 (19%), 6 (16%) and 4 (11%) patients, respectively. Thirteen (35%) patients had to be submitted to chemotherapy due to hyperleukocytosis (above 40 x 103/mm3) and six of these presented with new signs of coagulopathy after chemotherapy. Four (11%) patients died secondarily to intracerebral hemorrhage (IH) and two (5.4%) dropped out of the protocol due to severe ATRA side effects (one RAS and one hepatotoxicity). RAS and IH were related strictly to hyperleukocytosis. The reduced use of platelets and fresh frozen plasma probably lowered the total cost of treatment. We conclude that ATRA is an effective agent for inducing complete remission in APL patients.

Effect of L-arginine, dimercaptosuccinic acid (DMSA) and the association of L-arginine and DMSA on tissue lead mobilization and blood pressure level in plumbism

Lead (Pb)-induced hypertension is characterized by an increase in reactive oxygen species (ROS) and a decrease in nitric oxide (NO). In the present study we evaluated the effect of L-arginine (NO precursor), dimercaptosuccinic acid (DMSA, a chelating agent and ROS scavenger), and the association of L-arginine/DMSA on tissue Pb mobilization and blood pressure levels in plumbism. Tissue Pb levels and blood pressure evolution were evaluated in rats exposed to: 1) Pb (750 ppm, in drinking water, for 70 days), 2) Pb plus water for 30 more days, 3) Pb plus DMSA (50 mg kg-1 day-1, po), L-arginine (0.6%, in drinking water), and the combination of L-arginine/DMSA for 30 more days, and 4) their respective matching controls. Pb exposure increased Pb levels in the blood, liver, femur, kidney and aorta. Pb levels in tissues decreased after cessation of Pb administration, except in the aorta. These levels did not reach those observed in nonintoxicated rats. All treatments mobilized Pb from the kidney, femur and liver. Pb mobilization from the aorta was only effective with the L-arginine/DMSA treatment. Blood Pb concentrations in Pb-treated groups were not different from those of the Pb/water group. Pb increased blood pressure starting from the 5th week. L-arginine and DMSA treatments (4th week) and the combination of L-arginine/DMSA (3rd and 4th weeks) decreased blood pressure levels of intoxicated rats. These levels did not reach those of nonintoxicated rats. Treatment with L-arginine/DMSA was more effective than the isolated treatments in mobilizing Pb from tissues and in reducing the blood pressure of intoxicated rats.