Abstract
The administration of cobalt to rats caused a marked increase in the oxidative degradation of heme (hematin, iron protoporphyrin-IX) BY HEPATIC MICROSOMAL ENZYMES. The onset of this enzyme stimulation was very rapid, beginning within 2 hours after injection of the metal and reaching its maximum in 16 to 24 hours. During the rapid phase of stimulation, i.e. the first 2 to 4 hours, when heme oxidation was 450% above control values, there was a significant decrease in microsomal oxidative N-demethylation activity and in microsomal oxidative Ndemethylation activity and in microsomal content of heme with an insignificant decrease in cytochrome P-450 content. Within 24 hours the oxidative activity of the microsomal electron transport chain for drugs was decreased to about 30% of the control. However, during the same period the oxidation of heme approached levels 800% above control. During this period there was a further decrease in the microsomal content of heme with a significant decrease in cytochrome P-450 content and an increase in the activity of delta-aminolevulinate synthetase. The activity of delta-aminolevulinate synthetase reached its maximum within 8 hours after cobalt treatment. Repeated injections (at 24-hour intervals) of cobalt were necessary to maintain these changes in microsomal enzyme activities since, after single injections of the metal, these parameters returned to normal within 72 hours. The inducing effect of cobalt on the oxidation of heme could be inhibited by the administration of actinomycin D and puromycin. Furthermore, this stimulatory effect could not be elicited by in vitro treatment of microsomes with cobalt nor could the effect be attributed to any soluble components of the cytoplasm. Cobalt protoporphyrin-IX was less effective than cobalt chloride in stimulating heme oxidation. 3-Amino-1, 2, 4-triazole did not enhance hepatic heme oxidation activity, while allylisopropylacetamide decreased this activity. The oxidative degradation of heme was found not to be cytochrome P-450 dependent since the highly increased levels of heme oxidation in microsomes from cobalt-treated animals could be retained despite the fact that the cytochrome P-450 content of such microsomes was decreased to spectrally undetectable amounts and drug oxidation was eliminated by treatment of the microsomes with 4 M urea. These findings exclude an obligatory role for cytochrome P-450 in the oxidation of heme compounds, although the possibility that this process is a heme-dependent oxidation is not ruled out.
Highlights
450% above control values, there was a significant decrease in microsomal oxidative N-demethylation activity and in microsomal content of heme with an insignificant decrease in cytochrome P-450 content
During this period there was a further decrease in the microsomal content of heme with a significant decrease in cytochrome P-450 content and an increase in the activity of &aminolevulinate synthetase
Within 2 hours following the first injection of cobalt chloride the heme oxidation activity of microsomes was increased to about twice that of control values; during the same period of time, all other measured parameters of the microsomal membranes were essentially unchanged
Summary
Sigma Chemical Co. with the exceptions of: cobalt protoheme (cobalt protoporphyrin-IX). Products), glucose-6-phosphate dehydrogenase (Boehringer), and cobalt (cobaltous) chloride (Fisher Scientific Co.). Solutions of iron protoporphyrin-IX (hematin, heme), cobalt protoporphyrin-IX, and protoporphyrin-IX were prepared by dissolving the porphyrins in a small volume of 0.1 N sodium hydroxide and adjusting the pH to. 7.4, these solutions were used within 15 min after preparation. D and puromycin dihydrochloride were dissolved in buffered 0.9% NaCl solution (saline) [9] and injected intraperitoneally. Cobalt chloride (60 mg/kg) and allylisopropylacetamide (400 mg/kg) were dissolved in 0.9% NaCl and injected subcutaneously [8, 10]. 3-Amino-1,2,4-triazole (3 g/kg) was dissolved in saline and administered by intraperitoneal injection [11]. Tissue Preparations-Rats were fasted for 24 hours and killed by decapitation Cobalt chloride (60 mg/kg) and allylisopropylacetamide (400 mg/kg) were dissolved in 0.9% NaCl and injected subcutaneously [8, 10]. 3-Amino-1,2,4-triazole (3 g/kg) was dissolved in saline and administered by intraperitoneal injection [11].
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