Cyclodiene organochlorine insecticide-induced alterations in the sulfur-redox cycle in CHO-K1 cells.

The effect of the cyclodiene organochlorine pesticides aldrin, dieldrin and endosulfan was assessed on CHO-K1 cultures at fractions of their lethal doses, determined by the neutral red (NRI) incorporation assay (NRI6.25, NRI12.5 and NRI25). Glutathione peroxidase, reductase and S-transferase, and total and oxidised glutathione were evaluated along the standard growth curve of the cultures. After a 24-h incubation with each insecticide, glutathione peroxidase incurred a large increase, while glutathione reductase and S-transferase activities were slightly higher than untreated controls. Unlike oxidised glutathione, the content of total glutathione declined significantly after exposure to cyclodiene insecticides. Changes in cell membrane integrity were assessed by the lactate dehydrogenase (LDH) release assay and lipid peroxidation for a wide range of pesticide concentrations. Membrane leakage and peroxide production were significantly enhanced at concentrations of aldrin and as low as 12.5 microg/ml, whereas dieldrin and endosulfan increased membrane fragility at much higher concentrations.

Muscle and serum changes with salbutamol administration in aerobically exercised rats.

Treatment of experimental animals subjected to 90 days physical training programme plus repeated doses of salbutamol, a beta-adrenergic agonist, administered under two different regimes: therapeutic (16 microg/kg body weight, twice a day) and doping (3 mg/kg body weight, twice a day), caused a marked increase in size of skeletal (soleus, gastrocnemius and plantaris) leg muscles. Adrenergic involvement of salbutamol-linked hypertrophy was demonstrated by co-administration of the non-specific beta-adrenergic antagonist D,L-propranolol (10 mg/kg body weight twice a day). The salbutamol-induced muscle hypertrophy was associated with an early increase in creatine phosphokinase (CK) and its myocardial isozyme (CKmb), without significant changes in lactate dehydrogenase (LDH), alanine aminotransferase (AAT) and aspartate aminotransferase (DAT). The induction of muscle-injury biomarkers was completely abolished by co-administration of propranolol, thus suggesting the adrenergic involvement of these alterations.

Alterations on polyamine content and glutathione metabolism induced by different concentrations of paraquat in CHO-K1 cells.

The effect of herbicide paraquat has been assessed on CHO-K1 cultures at different concentrations. Glutathione peroxidase, reductase and S-transferase, as well as total and oxidized glutathione, were evaluated along the standard growth curve of the cultures. Paraquat was then administered during mid-log phase at concentrations that produced a calculated lethality of 6.25%, 12.5% and 25%, using the lysosomal dye assay, neutral red. After 24hr of incubation with paraquat, glutathione peroxidase suffered a large dose-response increase, unlike glutathione reductase and S-transferase, the activities of which were lower than untreated controls. The profile of total glutathione content was similar to that found for glutathione peroxidase, increasing with the administered doses of the herbicide. Polyamine content has been also studied at the same concentrations of paraquat, showing that intracellular spermidine and spermine pools were negatively affected with paraquat in a dose-response manner, unlike putrescine, which maintained elevated pools at the three concentrations assayed.

Early alterations of polyamine metabolism induced after acute administration of clenbuterol in mouse heart.

An acute treatment of mice with clenbuterol, a beta-adrenergic agonist, produced a marked increase of polyamines levels in heart, particularly during the early phase of administration of the drug. A single dose of 1.5 mg/kg caused as much as a 10 fold induction in activity of ornithine decarboxylase (ODC) and 3 to 4 fold increase in levels of putrescine, spermidine and spermine in mouse heart. Maximum changes were observed 3 to 4 hours post-administration of clenbuterol. This treatment did not produce any change in S-adenosylmethionine decarboxylase activity. The induction of cardiac ODC by clenbuterol was also dose dependent with a peak at about 5 micromol/kg. Co-administration of difluoromethylornithine, an irreversible inhibitor of ODC, or propranolol, a nonspecific beta-antagonist, with clenbuterol completely prevented the induction of ODC activity as well as the increase in polyamine levels in heart. However, pretreatment with alprenolol or metoprolol, the specific beta1 and beta2-antagonists, respectively, produced only partial prevention. The cardiac ODC from controls as well as clenbuterol treated mice exhibited similar affinity (Km) for its substrate, ornithine, while maximum enzyme activity (Vmax) was about 14 fold higher in clenbuterol treated mouse heart than in the control. Clenbuterol produced no change in the level of specific ODC mRNA or the protein, but the enzyme from the drug-treated mouse heart was considerably more stable than the control. Pretreatment of mice with either cycloheximide or actinomycin D followed by administration of clenbuterol could not prevent the induction in ODC activity suggesting that de novo biosynthesis of the enzyme protein or ODC mRNA was not responsible for induction of ODC activity. Post-translational changes in ODC may be responsible for an early increase of ODC activity due to clenbuterol treatment.

S-adenosylmethionine synthesis in Leishmania infantum promastigotes.

Methionine adenosyltransferase (MAT), S -adenosylmethionine (AdoMet), and S -adenosylhomocysteine (AdoHcy), have been analysed at different time-points during the growth curve of Leishmania infantum. MAT activity and AdoMet content peaked in the lag and early log phases, whereas higher levels of AdoHcy were found in stationary phase cells. MAT activity of cell extracts displayed hyperbolic kinetics for both its substrates, l -methionine and ATP, with km values of 35 microm and 5 m m, respectively. MAT has an absolute requirement for divalent cations, and is dependent on sulfydryl protective agents. Unlike other sources, L. infantum MAT activity seems to be transcriptionally regulated, with an accumulation of MAT-mRNA during rapid growth periods of promastigotes.

Plasma and muscle polyamine levels in aerobically exercised rats treated with salbutamol.

The induction of hypertrophy of cardiac and skeletal muscles has been studied after treatment with two different salbutamol dosages, therapeutic and doping. Treatment of rats subjected to a physical training schedule with repeated doses (16 microg kg(-1) per day or 3 mg kg(-1) per day) of salbutamol, a specific beta-adrenergic agonist, induced a marked increase in both skeletal and heart-muscle weight, whereas total body weight did not change significantly. Adrenergic involvement of salbutamol-linked muscle hypertrophy was demonstrated by co-administration of the non-specific beta-adrenergic antagonist, propranolol (20 mg kg(-1) per day). Salbutamol-induced muscle hypertrophy was associated with an increase in serum, skeletal-muscle and heart levels of the naturally occurring polyamines putrescine, spermidine and spermine. These observations suggest the involvement of polyamines in muscle hypertrophy and the possible role of blood polyamines as exposure biomarkers in beta-adrenergic-muscle hypertrophy.

Interaction of cationic diamidines with Leishmania infantum DNA.

The interaction of a series of potent leishmanicidal aromatic diamidines resembling pentamidine, was studied with Leishmania infantum DNA and polynucleotides. The diamidines viz., CGP040215A, CGP033829A and CGP039937A, interacted with leishmania DNA as well as with the polynucleotides poly(dA)-poly(dT), poly(dA-dT) and poly(dG-dC). The thermodynamic analysis to determine the association constants and the binding enthalpy pointed toward binding of the diamidines at AT regions of the DNA. The results also indicate that the diamidines bind at the outside of the DNA double helix, probably to the minor groove regions, with hydrogen bonds connecting the amide nitrogen of the diamidine to carbonyl oxygen atoms of thymidine or adenosine bases. However, CGP040215A and CGP033829A, the bisaryl diamidines, showed higher affinity than CGP039937A, the monoaryl diamidine. The spectrophotometric analysis of the interaction of these diamidines to test their effects on the melting temperature of leishmanial DNA suggests non-intercalating binding. The diamidines also showed potent inhibition of DNA polymerase activity of L. infantum extracts in vitro.

Polyamine-mediated heart hypertrophy induced by clenbuterol in the mouse.

The use of beta-agonists as growth-promoting agents in cattle could lead to toxic side-effects in man. One such effect is the accumulation of polyamines which seem to be implicated in muscle and heart hypertrophy. We have studied the induction of cardiac hypertrophy after treatment with clenbuterol and the role of polyamines in this effect. Treatment of mice with repeated doses of clenbuterol, a specific beta-adrenergic agonist, resulted in a marked increase in heart muscle weight whereas total body weight did not change significantly. Clenbuterol-linked cardiac hypertrophy could be prevented by co-administration of either the non-specific beta-adrenergic antagonist, propranolol, or the irreversible inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine. The clenbuterol-induced cardiac hypertrophy was associated with a corresponding increase in the level of the polyamines putrescine, spermidine and spermine. These observations are indicative of the role of polyamines in cardiac hypertrophy induced by clenbuterol.

The pharmacology of leishmaniasis.

The development of new strategies on chemotherapy of parasitic protozoan diseases is one of the most exciting research fields of recent years. World Health Organization (WHO) reports have recognized that the physiology and biochemistry of protozoan parasites and the host-parasite relation are the main targets for the design of new drugs that can be used in the future against these diseases.

Putrescine active uptake system in the Trypanosomatid Crithidia fasciculata.

Using the insect Trypanosomatid Crithidia fasciculata as a model parasite of mammalian pathogenic flagellates, i.e. Leishmania and Trypanosoma spp., we have studied the kinetic and regulatory characteristics of the polyamine uptake system. Putrescine transport was age-dependent with maximum expression values at the proliferative logarithmic phase. Putrescine transport in Crithidia fasciculata was energy-dependent and against a putrescine concentration gradient. The integrity of the membrane sulfhydryl groups was absolutely required for optimum transport rates. The specificity of this mechanism was studied in the presence of a series of different chain length aliphatic diamines, showing the high specificity for putrescine and the poor effect of this series at the highest concentration analyzed as well as the higher polyamines spermidine and spermine. Finally, the well-known inhibitor of polyamine biosynthesis, DFMO, led to an upward regulation of putrescine uptake correlating with the depletion of intracellular polyamine pool. In addition, the presence of high concentrations of putrescine in the culture medium produced a downward regulation of this system.

Effects of cationic diamidines on polyamine metabolism in the trypanosomatid Crithidia fasciculata.

The effect of a series of five recently synthesized cationic diamidines on cell proliferation and polyamine metabolism was studied on cultures of the model Trypanosomatid Crithidia fasciculata. Compounds displaying two arylic moieties (CGP039937A and CGP040215A) were ten fold more cytostatic than those displaying only one arylic residue (CGP033829A, CGP035753A and CGP036958A). The depletion of intracellular polyamine, putrescine and spermidine, pools and the effect of these compounds on S-adenosylmethionine decarboxylase and putrescine uptake suggest the requirement of two arylic groups in their chemical structure to obtain measurable effects on both polyamine metabolism and cell growth.

Fluorinated analogues of L-ornithine are powerful inhibitors of ornithine decarboxylase and cell growth of Leishmania infantum promastigotes.

Fluorinated analogues of L-ornithine have been tested on growth and ornithine decarboxylase arising from L.infantum cytosolic extracts. EC50 values estimated from dose/response curves were 38 microM, 2.62 microM and 4.64 microM for alpha-DFMO, delta-MFMO and delta-MFMOme respectively. Also the inhibition produced by all three compounds was effectively reverted by exogenous putrescine, pointing towards the inhibition of L.infantum ODC. ODC from logarithmic phase cytosolic extracts was physicochemically and kinetically characterized, showing a long half-life (more than 24 h) and a km value for L-ornithine of 98 microM. Finally, the inhibitory effect of fluorinated analogues of L-ornithine was analysed on L.infantum ODC showing a time-dependent irreversible behavior, with Ki values estimated on 125 microM, T1/2 3.5 min for alpha-DFMO; 13.3 microM, T1/2 1.8 min for delta-MFMO and 4.3 microM, T1/2 4 min for delta-MFMOme.

Putrescine uptake inhibition by aromatic diamidines in Leishmania infantum promastigotes.

The effect of a series of aromatic diamidines has been tested on Leishmania infantum promastigotes in both culture growth and putrescine uptake. The EC50 values calculated by means of dose-response curves were 45, 80, 165, 259 and 600 microM for 4', 6-diamidino-2-phenylindole (DAPI), dibromo propamidine, pentamidine 2-hydroxy stilbamidine and stilbamidine, respectively, although no inhibitory effects on cell growth were found at 1 mM propamidine, phenamidine and amicarbalide. When these compounds were kinetically analysed for putrescine uptake using Lineweaver-Burk plots, the Ki values reached were: DAPI, 15 microM; pentamidine, 3 microM; dibromo propamidine, 7 microM; 2-hydroxy stilbamidine, 21 microM; stilbamidine, 20 microM; propamidine, 25 microM; and phenamidine, 95 microM. Amicarbalide, however, was not able to reduce putrescine uptake to a significant extent, even at the highest concentration studied of 1 mM.

Pharmacokinetics of triclabendazole in rabbits.

1. Pharmacokinetic profiles of triclabendazole (TCBZ) following intravenous (i.v.) and oral administration of the drug in rabbits were carried out. 2. In normal rabbits, TCBZ was metabolized rapidly to its sulphoxide (TCBZ-SO) and sulphone (TCBZ-SO2) derivatives following administration, with undetectable concentrations of unchanged TCBZ in the plasma of the treated animals at any time (detection limit, 10 ng/ml). 3. The disposition kinetics of this drug in rabbits can be described by a two-compartment open model. 4. Mean peak concentrations in plasma of TCBZ-SO and TCBZ-SO2 of 12.41 micrograms/ml and 9.5 micrograms/ml occurred 7.5 and 9.5 hr after oral administration, respectively. 5. Both metabolites were eliminated slowly from plasma with elimination half-lives of 16.86 hr for the sulphoxide and 13 hr for the sulphone. 6. The area under the plasma concentration versus time curve (AUC) was 240 mg hr/l for the sulphoxide, higher than that found for the sulphone, 185 g hr/l.

Aromatic diamidines are reversible inhibitors of porcine kidney diamine oxidase.

The inhibitory ability of aromatic diamidines has been studied on porcine kidney diamine oxidase. The reversibility of drug-protein interactions has been tested by means of exhaustive dialysis experiments, showing in all cases a reversible binding pattern. Ki values obtained by means of Lineweaver-Burk plots were: stilbamidine 12 microM, 2-OH-stilbamide 8.5 microM, phenamidine 4 microM, propamidine 8 microM, dibromopropamidine 4.9 microM and amicarbalide 12 microM.

Selective inhibition of mammalian diamine oxidase by aquatic biotoxins.

The inhibitory activity of saxitoxin and tetrodotoxin on diamine oxidase and S-adenosyl-l-methionine decarboxylase from mammalian sources have been analysed. Unlike tetrodotoxin, saxitoxin was a reversible non-competitive inhibitor of pig kidney diamine oxidase with an estimated K(i) of 140 mum. S-Adenosyl-l-methionine decarboxylase from a highly purified source was not affected by the concentrations tested of either biotoxin. A possible analytical application of this finding is discussed.

Determination of luxabendazole in biological fluids by high-performance liquid chromatography.

Luxabendazole, a new benzimidazole, is a highly potent broad-spectrum anthelmintic. A high-performance liquid chromatographic method has been developed for its determination in serum and urine samples. In order to optimize the clean-up of samples we compared two procedures: C18 Sep-Pak cartridges and ultrafiltration through a cellulose membrane with a 30,000 relative molecular mass cut-off. In order to obtain the most suitable mobile phase, we studied the influence of pH and acetonitrile content on the capacity factor (k'). Chromatographic separation and quantification were performed on a reversed-phase column packed with 5-microns Nucleosil C18. The mobile phase was acetonitrile-0.05 M phosphate buffer (pH 7.0), (40:60, v/v). The column effluent was monitored by ultraviolet-visible spectrophotometry at 290 nm. The method shows good recovery, precision and accuracy. The lower limit of detection for luxabendazole is 15 ng/ml in serum samples and 25 ng/ml in urine samples.

Reversed-phase ion-pair high-performance liquid chromatographic determination of triclabendazole metabolites in serum and urine.

An ion-pair high-performance liquid chromatographic method was developed for measuring the concentrations of triclabendazole metabolites (sulphoxide and sulphone) in plasma and urine samples. The diluted biological fluids are ultrafiltered before chromatography through a 30,000 relative molecular mass cut-off filter and then injected into a C18 column. They are then isocratically eluted with a mobile phase consisting of 0.05 M phosphate buffer (pH 7.0)-acetonitrile (55:45, v/v) with addition of 1.0 mmol/l sodium decanesulphonate and monitored by ultraviolet-visible spectrophotometry at 312 nm. Recoveries over the range 0.01-9.0 micrograms/ml for triclabendazole sulphoxide and sulphone are, respectively, 91.7% and 91.6% in serum and 90.3% and 90.2% in urine. For both metabolites, the limit of detection is 10 ng/ml in both urine and serum.

Inhibition of diamine oxidase from porcine kidney by pentamidine and other aminoguanidine compounds.

1. Three bisguanidine compounds (those of pentamidine, streptidine and phenformin) were compared for their in vitro inhibitory capacity on diamine oxidase activity (EC 1.4.3.6), the first enzyme of putrescine degradation. 2. Pentamidine was the most potent inhibitor, and phenformine the weaker. Two and a half micromoles of pentamidine was enough to reduce the enzyme activity by 50%, while streptidine and phenformin produced the same effect at concentrations greater than 0.90 and 4 mM, respectively. 3. Pentamidine, streptidine and phenformin appeared to be non-competitive inhibitors, and the Ki values calculated by a Dixon plot were 3 microM, 0.95 mM and 4 mM, respectively.