ABSTRACT
The reactions between superoxide free radical anion (.O2-) with the halocarbons CCl4. CHCl3, BrCH2CH2Br(EDB), decachlora-biphenyl (DCBP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in dimethyl sulphoxide (DMSO) results in the emission of chemiluminescence (CL). The chemiluminescence reactions are characterized as having biphasic second order kinetics, CL wavelengths between 350 nm and 650 nm, and exhibiting perturbation by chemicals reactive with singlet oxygen. These data suggest that singlet oxygen species are the excited state responsible for the light emissions. Polarographic studies confirm .O2- consumption and halide release in the reactions, while gas liquid chromatography and NBT reduction demonstrate the decomposition of the halocarbons into products. A chemiluminescent reaction mechanism is proposed involving reductive dehalogenation of the halocarbons and the generation of singlet oxygen. The significance of singlet oxygen generation is discussed with respect to a general mechanism for explaining the rapid initiation of lipid peroxidative membrane damage in halocarbon toxigenicity in animal and plant tissues.
Subject(s)
Carbon Tetrachloride/chemistry , Chloroform/chemistry , Polychlorinated Biphenyls/chemistry , Polychlorinated Dibenzodioxins/chemistry , Superoxides/chemistry , Animals , Dimethyl Sulfoxide , Kinetics , Lipid Peroxidation , Luminescent Measurements , Nitroblue Tetrazolium , Oxygen , Plants , Polarography/methods , Singlet OxygenABSTRACT
Extraction of whole lobes of normal rat liver with dimethyl sulphoxide (DMSO) under N2 gives extracts which contain 5-10 mumol/l.O2- (50-100 nmol.O2- per 10 ml extract per 4 g liver; 1.25-2.50 nmol.O2- per millilitre per gram liver). Evidence for .O2- in the extracts is given by: (1) electron spin resonance signals (ESR), (2) differential pulse polarography (DPP), (3) chemiluminescence (CL), and (4) nitroblue tetrazolium reduction (NBT). All tests yield results identical with those obtained with authentic .O2-. Extraction of .O2- is enhanced by tetrabutyl ammonium ion, and is maximal at 1-3 min. These results raise the possibility that substantial amounts of .O2- are normally sequestered in protective membranous sites in vivo.
Subject(s)
Liver/chemistry , Superoxides/analysis , Animals , Dimethyl Sulfoxide , Electron Spin Resonance Spectroscopy , Free Radicals , Luminescent Measurements , Male , Nitroblue Tetrazolium , Oxidation-Reduction , Polarography , Rats , Rats, Inbred StrainsABSTRACT
The effect of the heavy metal toxicants HgCl2, CH3HgCl, and CdCl2 on the acetylating activity of membranous carnitine acetyltransferase (CarAc) in membrane vesicles from the maternal surface of human placental syncytiotrophoblast has been investigated. CarAc was inhibited by inorganic and organic mercury and cadmium. Carnitine acetylation was inhibited by as little as 5 microM mercury, with complete inhibition at 50 microM inorganic and organic mercury. Inhibition by cadmium was incomplete (less than 60%) at 500 microM CdCl2. Kinetic studies using Hanes plots revealed a mixed type of inhibition of CarAc by the metals. Cysteine preincubation decreased the amount of inhibition of CarAc by the metals. These results indicate that the inhibition of CarAc by heavy metals occurs by binding of the sulfhydryl on the enzyme by the metals. This interaction may be a mechanism of the heavy metal-induced fetotoxicity.
Subject(s)
Acetyltransferases/antagonists & inhibitors , Cadmium/pharmacology , Carnitine O-Acetyltransferase/antagonists & inhibitors , Mercury/pharmacology , Trophoblasts/enzymology , Cadmium/antagonists & inhibitors , Cadmium Chloride , Cysteine/pharmacology , Humans , In Vitro Techniques , Kinetics , Mercuric Chloride/pharmacology , Mercury/antagonists & inhibitors , Methylmercury Compounds/pharmacologyABSTRACT
Microvillous membrane vesicle preparations from the maternal surface of human placental syncytiotrophoblast were examined for the presence of carnitine and choline acetyltransferase activity. Radiometric assay for acetylcholine employed butyronitrile-tetraphenylboron extraction of the quaternary ions. Acetylcarnitine was assayed by anion exchange chromatography. The data reveal that carnitine is the primary substrate for the vesicle acetyltransferase enzyme(s), whereas choline appears to be a minor substrate. For acetylcarnitine synthesis, the Km is 0.749 mM carnitine and Vmax is 641 pmol X mg protein-1 X minute-1, respectively; for acetylcholine synthesis, the Km is 0.5 mM choline and Vmax is 53 pmol X mg protein-1 X minute-1, respectively. Approximately ten times more acetylated product was formed with carnitine than with choline. The carnitine-mediated reaction obeyed Michaelis-Menten kinetics, whereas the choline reaction exhibited anomalous behavior. Vesicle preparations were stable for 21 days at -80 degrees C. Preliminary studies on hypotonically lysed vesicles demonstrate that the acetyltransferase is particulate and is bound to the membrane of the vesicle. These findings demonstrate that carnitine acetyltransferase activity is in the plasmalemma membrane of the syncytiotrophoblast and suggest a role for this enzyme, analogous to the mitochondrial fatty acid shuttle system, in the maternofetal translocation of fatty acyl residues.
