Covalent alteration of the prosthetic heme of human hemoglobin by BrCCl3. Cross-linking of heme to cysteine residue 93. (1/9)

Recent studies have shown that a protein-bound heme adduct formed from the reaction of BrCCl3 with myoglobin was due to bonding of the proximal histidine residue through the ring I vinyl of a heme-CCl2 moiety. The present study reveals that BrCCl3 also reacts with the heme of reduced human hemoglobin to form two protein-bound heme adducts. Edman degradation and mass spectrometry provided evidence that these protein-bound heme adducts were addition products in which heme-CCL2 or heme-CCl3 were bound to cysteine residue 93 of the beta-chain of hemoglobin. It appeared that the cysteine residue was bonded regiospecifically to the ring I vinyl group of the altered heme moiety, because the nonprotein-bound products of the reaction included the beta-carboxyvinyl and alpha-hydroxy-beta-trichloromethylethyl derivatives of the ring I vinyl moiety of heme. The absorption spectra of the protein-bound adducts in both the oxidized and reduced states were highly similar to those described for hemichromes, which are thought to be involved in the formation of Heinz bodies and subsequent red cell lysis.  (+info)

Bromotrichloromethane hepatotoxicity. The role of stimulated hepatocellular regeneration in recovery: biochemical and histopathological studies in control and chlordecone pretreated male rats. (2/9)

It has been shown that BrCCl3 is a more potent hepatotoxin than CCl4. Pretreatment with nontoxic dietary levels of chlordecone (CD) results in amplification of BrCCl3 hepatotoxicity. The objective of this research was to investigate and compare the histopathological alterations during a time course after a low dose of BrCCl3 alone and in combination with dietary CD. Male Sprague-Dawley rats were maintained on 10 ppm dietary CD or normal diet for 15 days. On day 16, they received a single ip dose (30 microliters/kg) of BrCCl3 in corn oil (CO) vehicle or corn oil alone. Blood and liver samples were collected at 0, 3, 6, 12, 24, 36, 48, 72, 96, and 120 hr for serum enzymes and histopathological examination, respectively. Serum enzymes (SDH, ALT, AST) were significantly (p less than 0.05) elevated in rats receiving the CD + BrCCl3 combination in comparison to BrCCl3 alone. For 48 hr, a continuous increase in serum enzyme activities was detected in rats treated with CD + BrCCl3 combination, but not in the rats receiving other treatments (ND + BrCCl3, ND + CO, or CD + CO). The most extensive hepatolobular necrosis was observed in rats treated with the CD + BrCCl3 combination. Thirty-six hr after the administration of BrCCl3 to rats maintained on normal diet, high mitotic activity was observed, which continued through 72 hr resulting in complete restoration of hepatolobular structure. In contrast, rats receiving the combination of CD + BrCCl3 exhibited minimal and belated hepatomitotic activity for a short period of time, resulting in progressive hepatic failure, culminating in animal death. In conclusion, hepatotoxicity of a low dose of BrCCl3 alone appeared to be overcome via stimulated hepatocellular regeneration and hepatolobular restoration. CD appears to amplify BrCCl3 hepatotoxicity via interference with this hormetic mechanism, permitting a progressive and continued hepatic injury leading to complete hepatic failure, culminating in animal death.  (+info)

Histological evidence for dissociation of lipid peroxidation and cell necrosis in bromotrichloromethane hepatotoxicity in the perfused rat liver. (3/9)

Bromotrichloromethane (CBrCl3)-induced hepatic lipid peroxidation and cell necrosis were studied histologically and biochemically, using isolated perfused livers from phenobarbital-pretreated rats. Lipid peroxidation was assessed by fuchsin staining of the liver slices and release of thiobarbituric acid reactive substances (TBARS) into the perfusate; necrosis was assessed by trypan blue uptake and lactate dehydrogenase (LDH) leakage. A good correlation was observed between the Schiff-positive reaction and TBARS release under various experimental conditions, supporting the validity of the fuchsin staining method for histological detection of lipid peroxidation. Lobular localization of lipid peroxidation and necrosis was as follows: Under high oxygen supply (95% O2-saturated buffer), infusion of CBrCl3 caused the Schiff-positive reaction in the pericentral to midzonal hepatocytes, irrespective of the direction of perfusion, but did not produce necrosis. Under low oxygen supply (20% O2) with retrograde perfusion, dissociation of lipid peroxidation and necrosis was observed, i.e., trypan blue uptake in the periportal zones and Schiff-positive staining in the pericentral hepatocytes. Thus, lipid peroxidation by itself may have a relatively minor role in the development of CBrCl3-induced acute hepatic cell death.  (+info)

Comparative antioxidant effectiveness of dietary beta-carotene, vitamin E, selenium and coenzyme Q10 in rat erythrocytes and plasma. (4/9)

Five groups of five weanling rats were each fed a Torula yeast-based diet either unsupplemented or supplemented with 30 mg beta-carotene/kg, 30 IU vitamin E/kg, 1 mg selenium/kg or 30 mg coenzyme Q10/kg. Elevated levels of plasma aspartate aminotransferase and alanine aminotransferase are sensitive indicators of liver damage. The former enzyme was lower (P less than 0.01) in the vitamin E-, selenium- and beta-carotene-supplemented groups than in the unsupplemented control group, and the latter enzyme was lower in the vitamin E- and selenium-supplemented groups, suggesting a relatively equal effectiveness of these three antioxidants against liver damage. Erythrocytes were tested for protection against uninduced oxidative damage or that induced by 1 mmol/L bromotrichloromethane (BrCl3C) by measuring thiobarbituric acid-reactive substances (TBARS), hemoglobin, hemolysis, protein precipitation, alanine release and several enzyme activities. In untreated erythrocytes, selenium, beta-carotene and coenzyme Q10 exhibited protection by lowering (P less than 0.05) TBARS and alanine release, but only vitamin E protected against hemolysis. In BrCl3C-treated erythrocytes, vitamin E, selenium and beta-carotene protected by decreasing (P less than 0.05) protein precipitation, whereas selenium and beta-carotene decreased alanine release. The results of this study suggested that, in a manner analogous to vitamin E and selenium, beta-carotene and coenzyme Q10 function as antioxygenic nutrients.  (+info)

Metabolism-based covalent bonding of the heme prosthetic group to its apoprotein during the reductive debromination of BrCCl3 by myoglobin. (5/9)

The reductive metabolism of BrCCl3 by ferrous myoglobin leads to the alteration of the prosthetic heme to form products that can be dissociated from the protein and to those that are irreversibly bound to the protein. The major dissociable or soluble heme metabolites have recently been characterized. In this study, the irreversibly bound heme product was characterized by Edman degradation, amino acid analysis, and electronic absorption and mass spectrometry of peptides derived from the altered protein. It was found that the prosthetic heme was modified by a CCl2 moiety derived from BrCCl3 and was covalently bound to histidine residue 93, the normal proximal ligand to the heme-iron. The data are consistent with a mechanism by which the trichloromethyl radical reacts with the heme to form an intermediate that either can alkylate the proximal histidine residue or form soluble metabolites. The covalent bonding of the heme prosthetic moiety to the apoprotein likely leads to a change in the tertiary structure of the protein that may be responsible for its altered catalytic activity as well as its enhanced susceptibility to proteolysis. Similar processes may account, at least in part, for the covalent alteration of the heme prosthetic group of other hemoproteins caused by xenobiotics and endogenous substrates.  (+info)

Damage to DNA concurrent with lipid peroxidation in rat liver slices. (6/9)

Lipid peroxidation and DNA damage were evaluated in liver slices incubated for 2 h at 37 degrees C with 1 mM-t-butyl hydroperoxide (t-BOOH), 1 mM-BrCCl3 or 50 microM-ferrous iron. t-BOOH induced the greatest amount of damage to DNA and increased the production of thiobarbituric acid-reactive substances (TBARS). Both phenomena depended on the incubation time. Ferrous iron induced both DNA damage and TBARS production, and BrCCl3 did not induce significant DNA damage and was the weakest TBARS inducer. Butylated hydroxytoluene at 1 mM inhibited both DNA damage and TBARS production. DNA damage and lipid peroxidation in liver slices were correlated, indicating that these events were concurrent.  (+info)

Carbon tetrachloride, bromotrichloromethane and ethanol acute intoxication. New chemical evidence for lipid peroxidation in rat tissue microsomes. (7/9)

Second-derivative spectroscopy was used to determine the conjugated-diene shift that measures the extent of the first step of lipid peroxidation after carbon tetrachloride (CCl4), bromotrichloromethane (BrCCl3) and ethanol intoxication. The conjugated-diene signal was recorded in the second-derivative spectra as a minimum peak at 233 nm. The use of this method enabled us to show that, under our experimental conditions, CCl4- and BrCCl3-dependent conjugated-diene formation in rat liver microsomes is not dose-dependent and increases linearly with time up to 3h. Proportionality was not obtained between the second-derivative-spectroscopy method, and the thiobarbituric acid and difference-spectra methods. In addition, whereas the thiobarbituric acid and difference-spectra methods gave positive results at zero time, second-derivative spectroscopy showed no evidence of formation of conjugated dienes under the same experimental conditions. Intoxication with ethanol was shown by the appearance of the conjugated-diene signal in liver microsomes 24 h after the administration of the toxin. Intoxication with either of the haloalkanes or ethanol did not give rise to any similar peak in lung and brain microsomes. The results obtained are discussed.  (+info)

Structure of the novel heme adduct formed during the reaction of human hemoglobin with BrCCl3 in red cell lysates. (8/9)

It was previously shown that the reductive debromination of BrCCl3 to trichloromethyl radical by human hemoglobin leads to formation of dissociable altered heme products, two of which are identical to those formed from myoglobin and one which is novel. In this study, we have elucidated the structure of this novel adduct with the use of mass spectrometry, as well as 1H and 13C NMR as a substitution product of a -C(Cl) = CCl2 moiety for a beta-hydrogen atom on the prosthetic heme's ring I vinyl group. From studies with the use of 13C-enriched BrCCl3, it was determined that the added carbon atoms were derived from 2 eq of BrCCl3. A mechanism that involves multiple reductive events and a radical cation heme intermediate is proposed. Consistent with this mechanism, cellular reductants were found to selectively enhance the amount of this novel dissociable heme adduct. These studies reveal fine differences between myoglobin and hemoglobin in the accessibility of reactive intermediates to the ring I vinyl group, as well as the potential importance of cellular reductants on the course of heme alteration.  (+info)

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