Metal ion interactions in the control of haem oxygenase induction in liver and kidney.

Mn2+ and Zn2+ exhibit a striking ability to block the induction by Sn2+ and Ni2+ of haem oxygenase (EC 1.14.99.3) in kidney. The blocking effects of Mn2+ and Zn2+ were found to be greatest on simultaneous administration, time-dependent when administered up to 8 h before the inducing metal ions, and ineffective when administered as little as 10 min after the inducing metal ions. The decreases in cytochrome P-450 and haem contents and the sequential changes in delta-aminolaevulinate synthase (EC 2.3.1.37) activity that occur concomitant with haem oxygenase induction were largely eliminated with simultaneous or prior treatment with Mn2+ or Zn2+, but not when Mn2+ or Zn2+ was administered after Sn2+ or Ni2+. Mn2+ and Zn2+ did not increase the catabolism of the enzyme in vivo. Zn2+ on simultaneous administration was also able substantially to block the induction of haem oxygenase by Co2+, Cd2+ and Ni2+ in liver. The Zn2+ blockade of Cd2+ induction was examined in detail, and prior or simultaneous administration of Zn2+ was found to be effective in blocking the induction of haem oxygenase and the concomitant decreases in cytochrome P-450 and haem contents, ethylmorphine demethylase activity and the sequential changes in delta-aminolaevulinate synthase activity. Zn2+ administration 10 min or more after Cd2+ was ineffective in preventing the occurrence of these perturbations in haem metabolism. These findings describe a new and striking biological property of Mn2+ and Zn2+, and indicate the existence of significant metal ion interactions in the control of haem metabolism.     

Regulation of hepatic haem metabolism. Disparate mechanisms of induction of haem oxygenase by drugs and metals.

We studied drug- and metal-mediated increases in activity of haem oxygenase, the rate-controlling enzyme for haem breakdown, in chick-embryo hepatocytes in ovo and in primary culture. Phenobarbitone and phenobarbitone-like drugs (glutethimide, mephenytoin), which are known to increase concentrations of an isoform of cytochrome P-450 in chick-embryo hepatocytes, were found to increase activities of haem oxygenase as well. In contrast, 20-methylcholanthrene, which increases the concentration of a different isoform of cytochrome P-450, had no effect on activity of haem oxygenase. Inhibitors of haem synthesis, 4,6-dioxoheptanoic acid or desferrioxamine, prevented drug-mediated induction of both cytochrome P-450 and haem oxygenase in embryo hepatocytes in ovo or in culture. Addition of haem restored induction of both enzymes. These results are interpreted to indicate that phenobarbitone and its congeners induce haem oxygenase by increasing hepatic haem formation. In contrast, increases in haem oxygenase activity by metals such as cobalt, cadmium and iron were not dependent on increased haem synthesis and were not inhibited by 4,6-dioxoheptanoic acid. We conclude that (1) induction of hepatic haem oxygenase activity by phenobarbitone-type drugs is due to increased haem formation, and (2) induction of haem oxygenase by drugs and metals occurs by different mechanisms.     

Induction of liver cell haem oxygenase in iron-overloaded rats.

Rats were chronically iron-overloaded by intraperitonel injections of iron-dextran. Electron microscopy revealed that the excess iron was deposited in ferritin-like particles packed in lysosomes and scattered in hepatic cytoplasm. No mitochondrial iron deposition or damage was seen. Furthermore, mitochondrial preparations from chronically iron-overloaded animals were found to be contaminated with lysosomes, which could explain previously reported increases in mitochondrial iron by chemical analysis. Mitochondrial function, as measured by cytochromes a-a3, b and c concentrations as well as activity of the rate-limiting enzyme of haem synthesis, delta-aminolaevulinate synthetase, was not diminished by chronic iron-overloading. Microsomal haem was decreased by 30% at the time that haem oxygenase, the rate-limiting enzyme of haem degradation, was increased approx. 3-fold. Animals were given a single intraperitoneal injection of iron-dextran and the activities of delta-aminolaevulinate synthetase and haem oxygenase were measured over 24 h. delta-Aminolaevulinate synthetase activity increased approx. 2-fold in these acutely iron-overloaded rat livers, but at a time after the increase in haem oxygenase. These results suggest that an early consequence of excess iron in liver is acceleration of the rate of haem degradation, possible by haem oxygenase.     

Electron-transport pathway of the NADH-dependent haem oxygenase system of rat liver microsomal fraction induced by cobalt chloride.

The hepatic microsomal haem oxygenase activity of rats treated with CoCl2 was studied kinetically by measuring biliverdin, the immediate product of the reaction. Biliverdin was extracted with diethyl ether/ethanol mixture, and was determined by the difference between A690 and A800. The apparent Km value for NADPH (at 50 microM-haematin) was about 0.2 microM when an NADPH-generating system was used, whereas that for NADH was about 630 microM. Essentially the same Vmax. values were obtained for both the NADH- and NADPH-dependent haem oxygenase reactions. No synergism was observed with NADH and NADPH. The NADH-dependent reaction was competitively inhibited by NADP+, with a Ki of about 10 microM. The inhibitoin of the NADH-dependent reaction by the antibody against rat liver microsomal NADPH-cytochrome c reductase was essentially complete, with a pattern similar to that of the NADPH-dependent reaction. The immunochemical experiment and the comparison of the kinetic values with the reported data on isolated NADH-cytochrome b5 reductase and NADPH--cytochrome c reductase indicated the involvement of the latter enzyme in NADH-dependent haem oxygenation by microsomal fraction in situ.     

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.     

Heme oxygenase induction by menadione bisulfite adduct-generated oxidative stress in rat liver

The in vivo effect of menadione bisulfite adduct on both hepatic oxidative stress and heme oxygenase induction was studied. A marked increase in lipid peroxidation was observed 1 h after menadione bisulfite adduct administration. To evaluate liver antioxidant enzymatic defenses, superoxide dismutase, catalase and glutathione peroxidase activities were determined. Antioxidant enzymes significantly decreased 3 h after menadione bisulfite adduct injection. Heme oxygenase activity appeared 6 h after treatment, peaking 9 h after menadione bisulfite adduct administration. Such induction was preceded by a decrease in the intrahepatic GSH pool and an increase in hydrogen peroxide steady-state concentration, both effects taking place some hours before induction of heme oxygenase. Iron ferritin levels and ferritin content began to increase 6 h after heme oxygenase induction, and these increases were significantly higher 15 h after treatment and remained high for at least 24 h after menadione bisulfite adduct injection. Administration of bilirubin entirely prevented heme oxygenase induction as well as the decrease in hepatic GSH and the increase in lipid peroxidation when administered 2 h before menadione bisulfite adduct treatment. These results indicate that the induction of heme oxygenase by menadione bisulfite adduct may be a general response to oxidant stress, by increasing bilirubin and ferritin levels and could therefore provide a major cellular defense mechanism against oxidative damage.     

Prolonged induction of hepatic haem oxygenase and decreases in cytochrome P-450 content by organotin compounds.

The administration of organotin compounds to rats in single doses causes a significant and prolonged induction of haem oxygenase and a sustained decrease in haemoprotein content in the liver. The extent of induction of hepatic haem oxygenase varied between 3 and 5-fold at 72h after a single injection of water-insoluble organotins of differing structure. The alterations in haem metabolism produced by tricyclohexyltin hydroxide were studied in detail. The effects were dose-dependent, with doses as low as 3.75 mg/kg body wt. resulting in significant induction of haem oxygenase and a decrease in cytochrome P-450 and cytochrome b5 contents at 72h in the liver. The effects with time of a single dose of tricyclohexyltin on various parameters of liver haem metabolism were also examined. The organotin produced a substantial and very prolonged induction of haem oxygenase accompanied by a steady decline in cytochrome P-450 content for periods up to 8 days. The long duration of action of these organotins with respect to induction of haem oxygenase and depletion of cellular haemoprotein content provides a highly sensitive metabolic system with which to define further the toxic potential of organometals as well as to study the adaptive responses in liver to long-term perturbations of haem metabolism by foreign chemicals.     

Inhibition of haem synthesis caused by cobalt in rat liver. Evidence for two different sites of action.

Cobalt inhibits liver haem synthesis in vivo by acting at least two different sites in the biosynthetic pathway: (1) synthesis of 5-aminolaevulinate and (2) conversion of 5-amino-laevulinate into haem. The first effect is largely, if not entirely, due to inhibition of the activity of 5-aminolaevulinate synthase, rather than to inhibition of the formation of the enzyme. The second effect results from diversion of 5-aminolaevulinate into an unidentified liver pool with solubility properties similar to those of cobalt protoporphyrin.     

Inhibition of rat liver and kidney arginase by cadmium ion

Cadmium ion activates arginase from many species of organisms but is an inhibitor of arginase from many other species. The purpose of this study was to investigate the inhibition of rat liver and kidney arginase by cadmium ion. Rat kidney arginase was inhibited by much lower concentrations of cadmium ion than rat liver arginase. Cadmium ion was a mixed noncompetitive inhibitor of both rat liver and kidney arginase. Cadmium ion enhanced the substrate activation of rat kidney arginase while still inhibiting the enzyme. Cadmium ion prevented the substrate inhibition of rat kidney arginase by fluoride while still inhibiting the enzyme. Cadmium ion also inhibited rat kidney arginase in the presence of manganese ion.     

Inhibition of rat liver and kidney arginase by cadmium ion

Cadmium ion activates arginase from many species of organisms but is an inhibitor of arginase from many other species. The purpose of this study was to investigate the inhibition of rat liver and kidney arginase by cadmium ion. Rat kidney arginase was inhibited by much lower concentrations of cadmium ion than rat liver arginase. Cadmium ion was a mixed noncompetitive inhibitor of both rat liver and kidney arginase. Cadmium ion enhanced the substrate activation of rat kidney arginase while still inhibiting the enzyme. Cadmium ion prevented the substrate inhibition of rat kidney arginase by fluoride while still inhibiting the enzyme. Cadmium ion also inhibited rat kidney arginase in the presence of manganese ion.     

Defective utilization of haem in selenium-deficient rat liver.

We have previously suggested that an inherent defect in hepatic haem utilization was responsible for the rapid stimulation of hepatic microsomal haem oxygenase activity observed in selenium-deficient rats given phenobarbital, a well known inducer of haem formation. To test this hypothesis, hepatic haem content was deliberately raised in selenium-deficient rats by administration of either tryptophan or allylisopropylacetamide, or by injecting haem itself. We now report that selenium-deficient rats are apparently relatively less efficient in utilizing hepatic haem than normal controls. The findings detailed in the present paper thus indicate that stimulation of hepatic microsomal haem oxygenase activity is indeed a manifestation of abnormal haem utilization in selenium deficiency. This suggests a novel role for selenium in hepatic haem metabolism.