Biomedical subjects

M D Maines

Publications and source records attributed to M D Maines.

At least 145 records · Page 8Linked to original sources

PubMed1977

Enzymatic oxidation of cobalt protoporphyrin IX: observations on the mechanism of heme oxygenase action.

Studies on the enzymatic mechanism of microsomal heme oxygenase were made utilizing various porphyrins and metalloporphyrins of different ring substituents and central metal ions. Co-heme (cobalt protoporphyrin IX) was shown to be a substrate for the enzyme and the product of its oxidative metabolism was identified as the natural bile pigment, biliverdin IXalpha isomer. Metalloporphyrins, which do not bind molecular oxygen (Ni, Mn, and Sn protoporphyrin IX), were not substrates for heme oxygenase, although they could competitively inhibit oxidation of reactive substrates for the enzyme. The presence of lipophilic substitutents on pyrrole rings I and II, as well as a central metal atom, were required for the heme oxidation reaction to occur. The oxidative cleavage of Co-heme displayed typical characteristics of an enzyme-mediated reaction, and the oxidation of this substrate, as well as that of Fe-heme (iron protoporphyrin IX), could be supported with either reduced nicotinamide adenine dinucleotide phosphate or reduced nicotinamide adenine dinucleotide. A hypothesis is proposed on the mode of action of heme oxygenase in which the enzyme and its substrate are considered to form a "transitory" hemoprotein which can activate molecular oxygen for cleavage of the heme tetrapyrrole ring. In this formulation, heme as substrate for heme oxygenase is synonymous with heme as prosthetic group for the enzyme.

Animals↗

PubMed1977

Cobalt regulation of heme synthesis and degradation in avian embryo liver cell culture.

Inorganic cobalt was found to induce heme oxygenase activity in primary cultures of embryonic chick liver cells and to inhibit the induction of delta-aminolevulinate synthetase by the porphyrinogenic compounds allylisopropylacetamide, dicarbethoxy-1,4-dihydrocollidine, etiocholanolone, phenobarbital, Aroclor (R)1254, and secobarbital. Much smaller concentrations of Co2+ (5 muM) were required to inhibit delta-aminolevulinate synthetase than to induce heme oxygenase activity (50 muM). These effects of Co2+ on heme synthesis and heme degradation were potentiated by depletion of cellular glutathione content as a result of treatment with diethyl maleate. Cobalt inhibition of the induction of delta-aminolevulinate synthetase was of the same magnitude and probably involved the same mechanism as that produced by cobalt heme dimethyl ester and iron heme. The induction of heme oxygenase by cobalt could be blocked by cycloheximide. Plasma protein synthesis was not inhibited in the presence of concentrations of Co2+ which produced inhibition of delta-aminolevulinate synthetase or induction of heme oxygenase. Other metals such as Cd2+ and Cu2+ also inhibited the induction of delta-aminolevulinate synthetase by allylisopropylacetamide. These findings indicate that Co2+ can regulate heme metabolism directly in liver cells without intermediate actions on extrahepatic tissues. It is suggested that regulation of production of delta-aminolevulinate synthetase and heme oxygenase is mediated through the action of the metal ion rather than the metal in the form of a tetrapyrrole chelate.

Animals↗

PubMed1977

Regulation of cytochrome P-450-dependent microsomal drug-metabolizing enzymes by nickel, cobalt, and iron.

The effects of metals as modifiers of the activity of microsomal drug-metabolizing enzymes were studied with the use of nickel, cobalt, and iron. These metals were found to impair cellular heme-dependent metabolism by affecting both the heme biosynthetic and heme degradative pathways, inhibiting the former and inducing the latter. As powerful cellular toxins, metals depress respiratory activity and indirectly reduce drug-detoxifying ability of cells. Metals also perturb cellular glutathione content and thus may alter the activity of glutathione-dependent enzymes. The toxicity of metals is cumulative depending on concentration and degree of cellular exposure to one or to several closely related metals. On the other hand, these metal effects on cellular heme metabolism could also have selective therapeutic application in circumstances in which it may be desirable to suppress heme synthesis in order to decrease drug biotransformation, i.e., when a certain drug metabolite is more toxic than the parent compound.

Animals↗

PubMed1977

Regulation of heme pathway enzymes and cellular glutathione content by metals that do not chelate with tetrapyrroles: blockade of metal effects by thiols.

The trace metals nickel and platinum, which are not substrates for ferrochelatase and thus do not form heme in biological systems, were found to act similaryl to cobalt, and heme itself, in regulating heme metabolism in liver and kidney. These metals induced heme oxygenase activity in both organs with the peak of induced enzyme activity reached approximately 16 hr after single injections in rats. Both metals caused transient depression of cellular glutathione content followed by increases above normal after 12 hr in liver. Nickel and platinum were more potent inducers of heme oxygenase in kidney than in liver (10-13 times normal versus 5-6 times normal). At high concentrations, they inhibited heme oxygenase [heme, hydrogen-donor:oxygen oxidoreductase (alpha-methene-oxidizing, hydroxylating), EC 1.14.99.3] in vitro. Both were active in regulating heme metabolism only when administered in the ionic form. Complexing of the metals with sulfhydryl agents completely blocked their actions on heme metabolism. Administration of cysteine orally prior to or shortly after administration of the metals had a similar blocking effect. Nickel and platinum produced depression of delta-aminolevulinate synthase [succinyl-CoA:glycine c-succinyltransferase (decarboxylating), EC 2.3.1.37] activity in liver, but neigther inhibited this rate-limiting ennzyme for heme synthesis in vitro. Furthermore, despite the substantial decreases in cellular heme and hemoprotein contents mediated by the metal, production of delta-amimolevulinate synthase did not undergo the compensatory increase that would be expected if there were a direct reciprocal feedback relationship between cellular heme level and synthesis of this enzyme. These findings indicate that it is not necessary for metal ions to be chelated in the porphyrin ring in order to regulate the enzymes of heme synthesis and heme oxidation. Accordingly, it is suggested that the iron atom of heme is the proximately active regulator of delta-aminolevulinate synthase and heme oxygenase--actions generally ascribed to the iron-tetrapyrrole complex itself--and that the tetrapyrrole moiety of the complex functions primarily as a means of transport of the metal to regulatory sites in cells.

5-Aminolevulinate Synthetase↗

PubMed1976

Evidence for the catabolism of polychlorinated biphenyl-induced cytochrome P-448 by microsomal heme oxygenase, and the inhibition of delta-aminolevulinate dehydratase by polychlorinated biphenyls.

Polychlorinated biphenyls (PCB) are potent inducers of hepatic microsomal CO-binding hemoprotein P-448 (P1-450) and of delta-aminolevulinate synthetase (ALAS) activity. Inorganic cobalt was able to block PCB induction of cytochrome P-448 and to modify the PCB effect on ALAS activity in a time-dependent manner. PCB were also found to decrease the activity of delta-aminolevulinic acid dehydratase (ALAD) in liver. Pretreatment of rats with cobalt (30 min) produced the following changes in PCB actions on heme metabolism in liver: (a) augmentation of the porphyrinogenic effect of PCB, as determined by the total porphyrin content and ALAS activity; (b) augmentation of PCB inhibition of ALAD activity; and (c) blockade of induction of microsomal hemoprotein (cytochrome P-448). PCB did not interfere with cobalt induction of hepatic heme oxygenase activity. The sequence of administration of the metal and the PCB was important in relation to the changes produced in hepatic ALAS activity and microsomal hemoprotein and heme contents. When cobalt was administered 24 h after PCB treatment, the magnitude of induction of ALAS by PCB was lowered, and there was a great reduction in microsomal hemoprotein and heme contents. The renal response to PCB was different than that of the liver. In the kidney, PCB blocked the induction of heme oxygenase and depletion of cellular heme produced by cobalt. Furthermore, renal microsomal heme content was increased by PCB treatment alone or in combination with cobalt. It is concluded that (a) the heme moiety of microsomal cytochrome P-448 is metabolized by the heme oxygenase system, and it is suggested that for this catabolism to take place, the hemoprotein must be first converted to the denatured form of the hemoprotein, cytochrome P-420; (b) that the synthesis of heme in the kidney and the liver are regulated through different mechanisms; and (c) that ionic cobalt controls activity of ALAS by first inhibiting synthesis of the enzyme followed by the indirect induction of the enzyme as a result of the catabolism of heme, the physiological repressor of ALAS, by the metal-induced heme oxygenase. Thus microsomal heme oxygenase may be viewed as having an overall regulatory role in relation to mictochondrial ALAS by virtue of its ability to catabolize endogenous heme.

5-Aminolevulinate Synthetase↗

PubMed1976

Tin: a potent inducer of heme oxygenase in kidney.

Tin greatly enhances heme breakdown in kidney, thus impairing heme-dependent cellular functions, such as cytochrome P-450 mediated drug biotransformation. This novel action of the metal results from a potent induction effect on heme oxygenase, the enzyme that catalyzes heme oxidation in microsomes. The possible toxicological implications of this tin effect in the kidney merit further investigation.

5-Aminolevulinate Synthetase↗

PubMed1976

Studies on the mechanism of induction of haem oxygenase by cobalt and other metal ions.

Cobalt ions (Co2+) are potent inducers of haem oxygenase in liver and inhibit microsomal drug oxidation probably by depleting microsomal haem and cytochrome P-450. Complexing of Co2+ ions with cysteine or glutathione (GSH) blocked ability of the former to induce haem oxygenase. When hepatic GSH content was depleted by treatment of animals with diethyl maleate, the inducing effect of Co2+ on haem oxygenase was significantly augmented. Other metal ions such as Cr2+, Mn2+, Fe2+, Fe3+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+ and Pb2+ were also capable of inducing haem oxygenase and depleting microsomal haem and cytochrome P-450. None of these metal ions had a stimulatory effect on hepatic haem oxidation activity in vitro. It is suggested that the inducing action of Co2+ and other metal ions on microsomal haem oxygenase involves either the covalent binding of the metal ions to some cellular component concerned directly with regulating haem oxygenase or non-specific complex-formation by the metal ions, which depletes some regulatory system in liver cells of an essential component involved in controlling synthesis or activity of the enzyme.

5-Aminolevulinate Synthetase↗

PubMed1976

Selenium regulation of hepatic heme metabolism: induction of delta-aminolevulinate synthase and heme oxygenase.

Selenium was found to be a novel regulator of cellular heme methabolism in that the element induced both the mitochondrial enzyme delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating); EC 2-3-1-37] and the microsomal enzyme heme oxygenase [heme, hydrogen-donor:oxygen oxidoreductase(alpha-methene-oxidizing, hydroxylating); EC 1-14-99-3] in liver. The effect of selenium on these enzyme activities was prompt, reaching a maximum within 2 hr after a single injection. Other changes in parameters of hepatic heme metabolism occurred after administration of the element. Thirty minutes after injection the cellular content of heme was significantly increased; however, this value slightly decreased below control values within 2 hr, coinciding with the period of rapid induction of heme oxygenase. At later peroids heme content returned to normal values. Selenium treatment caused only a slight decrease in microsomal cytochrome P-450 content. However, drug-metabolizing activity was severely inhibited by higher doses of the element. Unlike other inducers of delta-aminolevulinate synthase, which as a rule are also porphyrinogenic agents, selenium induction of this enzyme was not accompanied by an increase in the cellular content of prophyrins. When rats were pretreated with selenium 90 min before administration of heme, a potent inhibitor of delta-aminolevulinate synthase production, the inhibitory effect of heme of formation of this mitochondrial enzyme was completely blocked. Selenium, at high concentrations in vitro, was inhibitory to delta-aminolevulinate synthase activity. It is postulated that selenium may not be a direct inducer of heme oxygenase as is the case with trace metals such as cobalt, but may mediate an increase in heme oxygenase through increased production and cellular availability of "free" heme, which results from the increased heme synthetic activity of hematocytes. Subsequently, the increased heme oxygenase activity is in turn responsible for the lack of increase in the microsomal heme content, thus maintaining heme levels at normal values despite the highly increased activities of both heme oxygenase and delta-aminolevulinate synthase. It is further suggested that the increase in delta-aminolevulinate synthase activity is not due to a decreased rate of enzyme degradation or an activation of preformed enzyme, but to increased rate of synthesis of enzyme protein. Although selenium in trace amounts has been postulated to be involved in microsomal electron transfer process, the data from this study indicate that excess selenium can substantially inhibit microsomal drug metabolism.

5-Aminolevulinate Synthetase↗

PubMed1976

Cobalt inhibition of synthesis and induction of delta-aminolevulinate synthase in liver.

Cobalt has complex actions on the metabolism of heme in the liver. In this organ the metal potently induces heme oxygenase (EC 1.14.99.3), and decreases cellular heme and hemoprotein content. The metal also displays biphasic effects on hepatic heme synthesis. These effects are reflected in the ability of cobalt to initially inhibit synthesis of delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating) EC 2.3.1.37], the rate limiting enzyme of the heme pathway, following which a later enhanced rate of formation of this enzyme occurs. In this study, cobalt was shown to block almost entirely the ability of the barbiturate analogue allylisopropylacetamide to induce delta-aminolevulinate synthase in liver. The blocking effect of cobalt on the otherwise potent enzyme inducing action of this drug was time-dependent; if the metal was injected 30 min prior to allylisopropylacetamide, inhibition of enzyme induction was complete. When the metal was administered 1.5 or more hours after allylisopropylacetamide, inhibition of enzyme induction was incomplete. Cobalt did not block the ability of the drug to directly degrade heme to "green pigment" thus the enzyme inducing action of allylisopropylacetamide and its degradative action on heme are separately mediated.

5-Aminolevulinate Synthetase↗

PubMed1976

The induction of heme oxidation in various tissues by trace metals: evidence for the catabolism of endogenous heme by hepatic heme oxygenase.

Cobalt is a potent inducer of hepatic heme oxygenase and concomitantly decreases microsomal cytochrome P-450 content in liver cells. Studies in which microsomal heme was labelled with 14C-beta-aminolevulinic acid showed that the decline observed in P-450 follows the induction of heme oxygenase indicating that endogenous heme is a substrate for this enzyme. The inducing effect of cobalt on heme oxygenase in liver extended to other organs such as heart, renal cortex and medulla, lungs and intestinal mucosa but not to the spleen and brain. In kidney and heart, cobalt resulted in decreases in microsomal and mitochondrial contents of hemoproteins. In addition ALAS activity of kidney was depressed by the metal. Other metals which chelate with sulfhydryl groups (e.g., Cr, Fe, Ni, Cu, Zn, Cd, Hg, Pb) were also shown to induce heart and renal heme oxygenase activities. Covalent binding of cobalt with mercaptans inactivated the metal with respect to its induction of heme oxygenase and degradation of hemoproteins.

Aminolevulinic Acid↗

PubMed1975

Cobalt stimulation of heme degradation in the liver. Dissociation of microsomal oxidation of heme from cytochrome P-450.

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.

5-Aminolevulinate Synthetase↗

PubMed1975

Study of the developmental pattern of heme catabolism in liver and the effects of cobalt on cytochrome P-450 and the rate of heme oxidation during the neonatal period.

The comparative development patterns of heme oxidation andof cytochrome P-450 dependent drug oxidation in rat liver were examined. High levels of heme oxygenase activity were present in whole embryo preparations at day 13 of gestation. At birth this enzyme activity in liver was approximately equal to that of normal adult liver. In the immediate postnatal period the rate of hepatic heme oxidation increased sharply, reaching levels 3-5 times normal during the first week postpartum. Thereafter, this enzyme activity progressively decreased and returned to normal adult levels by the 28th postpartum day. The development of microsomal heme oxidation and of P-450 dependent drug oxidation exhibited reciprocal patterns, with the latter being at low levels of activity during the immediate postnatal period and reaching adult activity only 4 or more wk after birth. Cobalt injected into pregnant animals or in to nursing mothers did not induce heme oxygenase in the fetus or suckling neonate. However, when treated directly with the metal, 4-day old neonates exhibited a small induction response of this enzyme; and the inducibility of heme oxygenase increased gradually to fully adult levels by the end of the 4th postpartum week. Cobalt at all postnatal developmental stages was capable of diminishing hepatic contents of total microsomal heme and P-450; however this effect of the metal was small in the immediate period after birth and increased progressively with maturation. These findings demonstrate that the patterns of development of hepatic capacity for carrying out the oxidation of heme and the P-450 dependent oxidation of drugs are different and thus provide further evidence that these microsomal enzyme systems are distinct from each other and under separate regulatory mechanisms. The degree of induction response for hepatic heme oxygenase evoked by the trace metal, cobalt, was also shown to have developmental determinants as did the susceptibility of hepatic cytochrome P-450 to degradation by this metal. The very high levels of hepatic heme oxygenase activity which characterize neonates during the first week of life indicate that over-production of bilirubin contributes significantly to the mechanism of neonatal jaundice.

Animals↗

PubMed1975

The degradative effects of porphyrins and heme compounds on components of the microsomal mixed function oxidase system.

The effects of in vitro treatment of the hepatic microsomal fraction with various porphyrin compounds on the activity and the content of the heme-containing components of the mixed function oxidase system were studied. The compounds examined were hematin, methemalbumin (with heme to protein molar ratio of 13:1 or 1:1), mesohemalbumin, bilirubin, biliverdin, mesoporphyrin IX, and protoprophyrin IX. The activity of the system was monitored by measuring its oxidative activity for the type I and type II substrates, ethylmorphine and aniline, respectively; as well as the microsomal contents of cytochrome P-450 and b5 and 14C-labeled heme, Mesoporphyrin IX was found to be most effective in inhibiting the oxidative activity of the mixed function oxidase system as well as in decreasing the microsomal contents of cytochromes P-450, b5, and heme. Biliverdin exerted no effect on these parameters. Hematin and the other compounds studied exerted variable inhibitory effects on the system. The degradative and inhibitory effects of protoporphvrin IX and mesoporphyrin IS could be blocked significantly by conducting the studies in the dark. The presence of biliverdin decreased the inhibitory effects of the porphyrins on the system; conversely the effects could be magnified in the presence of deuterium oxide. It is suggested that the mechanism by which porphyrins inhibit the mixed function oxidase system is through porphyrin-sensitized photo-oxidation of various constituents of the hepatic microsomal fraction and that the formation of singlet oxygen molecules is most likely involved in this process. Moreover the destructive effects of heme compounds on the microsomal components and activities of the drug-metabolizing mixed function oxidase system raise questions concerning the hypothesis that the components of this system, and specifically cytochrome P-450, are involved in the activity of the heme oxygenase system.

Aminolevulinic Acid↗

PubMed1974

Cobalt induction of hepatic heme oxygenase; with evidence that cytochrome P-450 is not essential for this enzyme activity.

Treatment of rats in vivo with cobalt chloride stimulated heme oxidation by hepatic microsomes to levels up to 800% above controls. This treatment also caused increases in liver weight and in total microsomal protein; in contrast, marked decreases were produced in microsomal oxidation of ethylmorphine (80%), and in cytochrome P-450 (60-70%) and heme (30-50%) contents. Cobalt chloride treatment did not affect heme oxidation by the spleen heme oxygenase system. The rate of heme oxidation by hepatic microsomal enzymes and the microsomal content of cytochrome P-450 were found to be unrelated. This conclusion was reached from studies in which microsomal heme oxygenase activity from cobalt-treated animals could be increased by 900% above control levels in the same microsomal preparation in which cytochrome P-450 content was decreased to spectrally unmeasurable amounts after incubation with 4 M urea. The same treatment eliminated ehtylmorphine demethylation and decreased microsomal NADPH-cytochrome c reductase (EC 1.6.2.4) activity by 75%. It is concluded that (i) the hepatic microsomal enzyme system that oxidizes heme compounds is not the same as that which metabolizes drugs, (ii) cytochrome P-450 is not essential for the oxidation of heme by liver cells, (iii) there is no direct relationship between the rate of heme oxidation and the level of NADPH-cytochrome c reductase activity, and (iv) the oxidation of heme is protein-dependent and the active proteins are inducible, but are different from those involved in drug metabolism.

Animals↗

PubMed1974