Repeated amiodarone exposure in the rat: toxicity and effects on hepatic and extrahepatic monooxygenase activities

Amiodarone is a potent and efficacious antiarrhythmic agent, yet associated with its use are life-threatening pulmonary fibrosis and hepatotoxicity. We have investigated the susceptibility of the male Sprague-Dawley rat to pulmonary and hepatic toxicity after repeated exposure to amiodarone and the effects of such exposure on hepatic and extrahepatic drug metabolizing enzymes. Animals received amiodarone (200 mg.kg-1.day-1 i.p., 5 days/week) for 1 week followed by 150 mg.kg-1.day-1 (5 days/week) for 3 additional weeks. No signs of pulmonary fibrosis or hepatotoxicity were observed, based on histological examination, lung hydroxyproline content, and plasma alanine aminotransferase activity. Analysis of tissues revealed extensive accumulation of amiodarone and desethylamiodarone in lung and liver, but concentrations were significantly lower in animals treated for 4 weeks than for 1 week. In a separate experiment, rats received amiodarone 150 mg.kg-1.day-1 i.p. (5 days/week) for 1 or 4 weeks. No differences in tissue concentrations of amiodarone and desethylamiodarone were detected between animals treated for 1 or 4 weeks. This regimen did not affect hepatic or extrahepatic monooxygenase activities. These results indicate that, in the male Sprague-Dawley rat, there is no observable pulmonary or hepatic toxicity following short-term amiodarone exposure, and there is enhanced elimination of amiodarone and desethylamiodarone when the daily dose of amiodarone is decreased after 1 week from 200 to 150 mg/kg.     

Kinetics of tryptic hydrolysis as a probe of the structure of human plasma apolipoprotein A-II

As a model system to understand apolipoprotein structure-function and their relationships to proteolytic events, the kinetics of tryptic hydrolysis of apolipoprotein A-II (apo A-II) was investigated in solution and in association with phospholipid. The rates of appearance and identities of specific peptides were determined by reversed-phase high-performance liquid chromatography and amino acid analysis, respectively. For the kinetics of hydrolysis of apo A-II in solution, the carboxyl-terminal peptides of residues 55-77 and 56-77 appeared first, followed by peptides of residues 4-23, 29-39, 40-44 and 45-54, which appeared at nearly identical rates. The kinetics of hydrolysis of apo A-II associated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine showed several differences. First, a 100-fold larger amount of trypsin was needed to obtain a similar rate of product formation; second, a new peptide appeared, eluting earlier than apo A-II but having a similar amino acid composition; and third, the relative rates of appearance of peptides were different. The secondary structure surrounding the bonds susceptible to trypsin cleavage was determined by several predictive algorithms. The lysine amino acid bonds were found to be in regions defined by a high helical amphipathic moment. The reduced susceptibility to tryptic hydrolysis of apo-II associated with phospholipid appears to be due to a higher free energy of stabilization of protein secondary structure. As a consequence, the lysine amino acid bonds are in folded regions of the protein where they are conformationally inaccessible to enzymatic hydrolysis. By use of structure-prediction methods, it is possible to designate which regions of apolipoproteins may be important in proteolysis.     

Detection of avian malaria (Plasmodium spp.) in native land birds of American Samoa

This study documents the presence ofPlasmodium spp. in landbirds ofcentral Polynesia. Blood samples collectedfrom eight native and introduced species fromthe island of Tutuila, American Samoa wereevaluated for the presence of Plasmodiumspp. by nested rDNA PCR, serology and/ormicroscopy. A total of 111/188 birds (59%)screened by nested PCR were positive. Detection of Plasmodium spp. was verifiedby nucleotide sequence comparisons of partial18S ribosomal RNA and TRAP(thrombospondin-related anonymous protein)genes using phylogenetic analyses. All samplesscreened by immunoblot to detect antibodiesthat cross-react with Hawaiian isolates of Plasmodium relictum (153) were negative. Lack of cross-reactivity is probably due toantigenic differences between the Hawaiian andSamoan Plasmodium isolates. Similarly,all samples examined by microscopy (214) werenegative. The fact that malaria is present,but not detectable by blood smear evaluation isconsistent with low peripheral parasitemiacharacteristic of chronic infections. Highprevalence of apparently chronic infections,the relative stability of the native land birdcommunities, and the presence of mosquitovectors which are considered endemic andcapable of transmitting avian Plasmodia,suggest that these parasites are indigenous toSamoa and have a long coevolutionary historywith their hosts.     

Spontaneous phospholipid transfer: development of a quantitative model

The effects of lipid structure on the kinetics of spontaneous transfer of a series of phosphatidylcholines have been determined. Donors, which were model-reassembled high-density lipoproteins composed of apo A-I, 1-palmitoyl-2-oleoylphosphatidylcholine, and a trace of a radiolabeled lipid, were mixed with acceptors, which were human low-density lipoproteins. Within a series of phosphatidylcholines, the addition of double bonds and methylene units, respectively, increased and decreased the rate of transfer in a predictable way. An equation that predicts the rates of transfer of a large number of diacylglycerides and phosphoglycerides from any lipoprotein has been empirically derived from these data. The transfers of phosphatidylcholines that contain superpolyunsaturated fatty acids (four or more double bonds) do not obey the derived equation, probably due to limitations on the number of conformational degrees of freedom in these lipids. The range of measured transfer halftimes extends from less than 2 h to more than 12 days. Thus, the spontaneous transfer halftimes of some (but not all) lipids are short compared to the lifetime of lipoproteins in plasma. These results suggest that some lipids transfer among lipoproteins and cells via a spontaneous mechanism while others require specific transfer factors or hydrolysis to achieve this within a physiologically significant time frame.     

Communication between the active sites in dimeric mercuric ion reductase: an alternating sites hypothesis for catalysis

Mercuric reductase, a flavoprotein disulfide oxidoreductase, catalyzes the two-electron reduction of Hg(II) to Hg(0) by NADPH. As with all the members of this class of proteins, the enzyme is a dimer of identical subunits with two active sites per dimer, each composed of one FAD and catalytically essential residues from both subunits. In the enzyme from Tn501, these residues include, at a minimum, FAD and cysteines 135 and 140 from one subunit and cysteines 558' and 559' from the other. With this sort of active site arrangement, the enzyme seems perfectly set up for some type of subunit communication. In this report, we present results from several titrations, as well as kinetics studies, that, taken together, are consistent with the occurrence of subunit communication. In particular, the results indicate that pyridine nucleotide complexed dimers of the enzyme are asymmetric. Since the EH2-NADPH complex of the enzyme is the relevant reductant of Hg(II), these observations suggest that the enzyme may function asymmetrically during catalysis. An alternating sites model is proposed for the catalytic reduction of Hg(II), where both subunits of the dimer function in catalysis, but the steps are staggered and the subunits reverse roles after part of the reaction. An attractive feature of this proposal is that it provides a reasonable solution to the thermodynamic dilemma the enzyme faces in needing to both bind Hg(II) very tightly and reduce it.