Structural requisites of 2-(p-chlorophenoxy)propionic acid analogues for activity on native rat skeletal muscle chloride conductance and on heterologously expressed CLC-1.
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(1) The 2-(p-chlorophenoxy)propionic acid (CPP) modulates in a stereoselective manner the macroscopic chloride conductance (gCl), the electrical parameter sustained by the CLC-1 channel, of skeletal muscle. In order to determine the structural requirements for modulating native gCl and to identify high-affinity ligands, the effects of newly synthesised CPP analogues have been evaluated on gCl of rat EDL muscle fibres by means of the two-microelectrode current-clamp technique. (2) Each type of the following independent modification of CPP structure led to a three- to 10-fold decrease or to a complete lack of gCl-blocking activity: replacement of the electron-attractive chlorine atom of the aromatic ring, substitution of the oxygen atom of the phenoxy group, modification at the chiral centre and substitution of the carboxylic function with a phosphonate one. (3) The analogues bearing a second chlorophenoxy group on the asymmetric carbon atom showed a significant gCl-blocking activity. Similar to racemate CPP, the analogue with this group, spaced by an alkyl chain formed by three methylenic groups, blocked gCl by 45% at 100 micro M. (4) These latter derivatives were tested on heterelogously expressed CLC-1 performing inside-out patch-clamp recordings to further define how interaction between drug and channel protein could take place. Depending on the exact chemical nature of modification, these derivatives strongly blocked CLC-1 with K(D) values at -140 mV ranging from about 4 to 180 micro M. (5) In conclusion, we identified four molecular determinants pivotal for the interaction with the binding site on muscle CLC-1 channels: (a) the carboxylic group that confers the optimal acidity and the negative charge; (b) the chlorophenoxy moiety that might interact with a hydrophobic pocket; (c) the chiral centre that allows the proper spatial disposition of the molecule; (d) an additional phenoxy group that remarkably stabilises the binding by interacting with a second hydrophobic pocket. (+info)
Investigations of pharmacologic properties of the renal CLC-K1 chloride channel co-expressed with barttin by the use of 2-(p-Chlorophenoxy)propionic acid derivatives and other structurally unrelated chloride channels blockers.
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CLC-K chloride channels are expressed in the kidney, where they play a pivotal role in the mechanisms of urine concentration and Na(+) reabsorption. The identification of barttin as an essential beta-subunit of CLC-K channels allowed performance of a pharmacologic characterization of wild-type CLC-K1 expressed in Xenopus oocytes. To this end, a series of 2-(p-chlorophenoxy)propionic acid (CPP) derivatives were screened using the two-microelectrode voltage-clamp technique. Several chemical modifications regarding the phenoxy group of the side chain (elimination of the oxygen atom or of methylenic groups, substitutions of the chlorine atom) did not alter the drug blocking activity, with five different derivatives showing a similar potency. Among these, a derivative of CPP carrying a benzyl group on the chiral center in the place of the methyl group represented the minimal structure for blocking CLC-K1. It inhibited the channel from the extracellular side with an affinity in the 150 micro M range. The blocking potency of this compound is fourfold increased by lowering the extracellular chloride concentration, suggesting that the drug interacts with the channel pore. Concomitantly, the effect of some "classical" Cl(-) channel blockers (9-anthracenecarboxylic acid, 2-(phenylamino)benzoic acid, iminodibenzoic acid, niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt, and 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid disodium salt) was screened. 4,4'-Diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt was the only one capable of blocking CLC-K1 with a potency similar to the CPP derivative, although in an irreversible manner. The newly identified substances provide a useful tool to investigate the biophysical and physiologic role of these renal channels and a starting point for the development of therapeutic drugs with diuretic action. (+info)
Molecular determinants of differential pore blocking of kidney CLC-K chloride channels.
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The highly homologous Cl(-) channels CLC-Ka and CLC-Kb are important for water and salt conservation in the kidney and for the production of endolymph in the inner ear. Mutations in CLC-Kb lead to Bartter's syndrome and mutations in the small CLC-K subunit barttin lead to Bartter's syndrome and deafness. Here we show that CLC-Ka is blocked by the recently identified blocker 2-(p-chlorophenoxy)-3-phenylpropionic acid of the rat channel CLC-K1 with an apparent K(D) approximately 80 microM. We also found that DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid), a generic Cl(-) channel blocker, inhibits CLC-Ka (K(D) approximately 90 microM). Surprisingly, the highly homologous channel CLC-Kb is fivefold to sixfold less sensitive to both compounds. Guided by the crystal structure of bacterial CLC proteins, we identify two amino acids, N68/D68 and G72/E72, in CLC-Ka and CLC-Kb, respectively, that are responsible for the differential drug sensitivity. Both residues expose their side chains in the extracellular pore mouth, delineating the probable drug binding site. These novel CLC-K channel blockers are promising lead compounds for the development of new diuretic drugs. (+info)
Genetic and phenotypic diversity of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy)propionic acid]-degrading bacteria isolated from soils.
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Twelve mecoprop-degrading bacteria were isolated from soil samples, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genus Sphingomonas. Ten different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 12 isolates. The isolates were found to be able to utilize the chiral herbicide mecoprop as a sole source of carbon and energy. While seven of the isolates were able to degrade both (R)- and (S)-mecoprop, four isolates exhibited enantioselective degradation of the (S)-type and one isolate could degrade only the (R)-enantiomer. All of the isolates were observed to possess plasmid DNAs. When certain plasmids were removed from isolates MP11, MP15, and MP23, those strains could no longer degrade mecoprop. This compelling result suggests that plasmid DNAs, in this case, conferred the ability to degrade the herbicide. The isolates MP13, MP15, and MP24 were identified as the same strain; however, they exhibited different plasmid profiles. This indicates that these isolates acquired different mecoprop-degradative plasmids in different soils through natural gene transfer. (+info)
Genetic analysis of phenoxyalkanoic acid degradation in Sphingomonas herbicidovorans MH.
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Phenoxyalkanoic acid degradation is well studied in Beta- and Gammaproteobacteria, but the genetic background has not been elucidated so far in Alphaproteobacteria. We report the isolation of several genes involved in dichlor- and mecoprop degradation from the alphaproteobacterium Sphingomonas herbicidovorans MH and propose that the degradation proceeds analogously to that previously reported for 2,4-dichlorophenoxyacetic acid (2,4-D). Two genes for alpha-ketoglutarate-dependent dioxygenases, sdpA(MH) and rdpA(MH), were found, both of which were adjacent to sequences with potential insertion elements. Furthermore, a gene for a dichlorophenol hydroxylase (tfdB), a putative regulatory gene (cadR), two genes for dichlorocatechol 1,2-dioxygenases (dccA(I/II)), two for dienelactone hydrolases (dccD(I/II)), part of a gene for maleylacetate reductase (dccE), and one gene for a potential phenoxyalkanoic acid permease were isolated. In contrast to other 2,4-D degraders, the sdp, rdp, and dcc genes were scattered over the genome and their expression was not tightly regulated. No coherent pattern was derived on the possible origin of the sdp, rdp, and dcc pathway genes. rdpA(MH) was 99% identical to rdpA(MC1), an (R)-dichlorprop/alpha-ketoglutarate dioxygenase from Delftia acidovorans MC1, which is evidence for a recent gene exchange between Alpha- and Betaproteobacteria. Conversely, DccA(I) and DccA(II) did not group within the known chlorocatechol 1,2-dioxygenases, but formed a separate branch in clustering analysis. This suggests a different reservoir and reduced transfer for the genes of the modified ortho-cleavage pathway in Alphaproteobacteria compared with the ones in Beta- and Gammaproteobacteria. (+info)
Review of the genotoxicity of 4-chloro-2-methylphenoxyacetic acid.
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4-Chloro-2-methylphenoxyacetic acid (MCPA) has been examined for genotoxicity in a range of in vitro and in vivo assays, including assays for gene mutation and clastogenicity. MCPA is non-mutagenic in bacterial and mammalian cell gene mutation assays. Increases in percentage aberrant cells were found on analysis of metaphases of human peripheral lymphocytes treated in vitro in the presence of auxiliary metabolic activation (S9), but only at doses approaching 10 mM and causing significant cytotoxicity. These increases may therefore be non-specific. No evidence for clastogenicity in vivo was found in the mouse bone marrow micronucleus assay or the Chinese hamster bone marrow metaphase assay. No evidence for either increases in sister chromatid exchange (SCE) frequency or DNA binding was found in the rat. Very small (less than 1.5 times controls) increases in SCE were observed in vivo in the hamster at toxic or maximum tolerated dose levels. MCPA is not alerting for likely genotoxic activity using established structure-activity relationship principles and it is concluded that, on the weight of evidence from the available data, MCPA is not genotoxic in vivo. This is consistent with its lack of carcinogenicity in rats and mice. (+info)
Effects of ethyl 4-chloro-2-methylphenoxyacetate on bile composition in golden hamsters.
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The effects of ethyl 4-chloro-2-methylphenoxyacetate (MCPA) on the cholesterol levels in the liver, serum and gallbladder bile, the composition of both biliary lipids and bile acids, and the hepatic enzyme activities relating to the bile acid formation were investigated in male golden hamsters. MCPA was administered orally to the animals at the doses of 200, 500 and 1,000 mg/kg/day for 2 weeks. Clofibrate (500 mg/kg/day) was also tested for comparison, because of its similarity to MCPA in chemical structure and pharmacological actions. Body weight gain was slightly decreased, but liver to body weight ratio was increased in the MCPA groups of 500 and 1,000 mg/kg and also in the clofibrate group. In these groups, the cholesterol level in the liver was decreased but that in bile was increased. The composition of biliary bile acids was also changed in both the MCPA groups (500 and 1,000 mg/kg) and the clofibrate group, but in a different manner; the levels and proportions of secondary bile acids were increased in the MCPA groups, while those of cholic acid and deoxycholic acid were decreased in the clofibrate group. These results indicate that MCPA and clofibrate may affect cholesterol catabolism differently, although these two compounds contain the same chlorinated phenoxy acid moiety. The increased level of cholesterol in bile might lead to the formation of cholesterol gallstone, which is frequently found in patients with gallbladder cancer. It is inferred that exposure to MCPA could be a risk factor for gallbladder cancer, which was initiated with some chemical carcinogens, because it increased the levels and proportions of both cholesterol and secondary bile acids in bile. (+info)
Intentional self-poisoning with the chlorophenoxy herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA).
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STUDY OBJECTIVE: Data on poisoning with MCPA (4-chloro-2-methyl-phenoxyacetic acid) are limited to 6 case reports. Our objective is to describe outcomes from intentional self-poisoning with MCPA in a prospective case series of 181 patients presenting to hospitals in Sri Lanka. METHODS: Patient information was collected by on-site study physicians as part of an ongoing prospective cohort study of poisoned patients. Medical history, clinical details, and blood samples were obtained prospectively. RESULTS: Overall clinical toxicity was minimal in 85% of patients, including mild gastrointestinal symptoms in 44% of patients. More severe clinical signs of chlorophenoxy poisoning reported previously, such as rhabdomyolysis, renal dysfunction, and coma, also occurred but were uncommon. Eight patients died (4.4%). Most deaths occurred suddenly from cardiorespiratory arrest within 48 hours of poisoning; the pathophysiological mechanism of death was not apparent. The correlation between admission plasma MCPA concentration and clinical markers of severity of toxicity (physical signs, symptoms, and increased creatine kinase level) was poor. CONCLUSION: Intentional self-poisoning with MCPA generally causes mild toxicity, but cardiorespiratory arrest and death may occur. All patients should receive routine resuscitation and supportive care. It seems reasonable to correct acidosis and maintain an adequate urine output, but there is insufficient evidence to support other specific interventions. Our data do not support a clinical role for measurement of plasma MCPA in the acute management of poisoning, and insufficient data were available to fully examine the utility of measured electrolytes and creatine kinase levels. (+info)