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

Abstract

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.

Purification and characterization of heme oxygenase from chick liver

“…The reaction was initiated with the addition of 1 pmol NADPH. As previously described, bovine serum albumin stabilizes heme and biliverdin in aqueous solution and enhances activity of heme oxygenase [21]. One unit of activity of heme oxygenase catalyzes the formation of 1 nmol bilirubin/min at 37 "C. Periodically, equivalence of results with the single-cuvette dual-wavelength assay for heme oxygenase were confirmed by scanning the product (Fig.…”

“…Addition of biliverdin reductase was absolutely required for formation of bilirubin (Table S2), but had no effect on the rate of formation of biliverdin. However, because of the higher sensitivity for measurement of bilirubin formation ( A E 470-540 nm for bilirubin = 66 mM-' cm-' [21]); Ae 680-750 nm for biliverdin = 15 mM-' cm-', as determined with standards in our reaction mixture), bilirubin was the product routinely measured. Optimal concentrations of the co-factors and other enzymes listed in Table S2 were established in initial experiments.…”

“…The rate of formation of bilirubin was estimated by the increase in absorbance at 470 nm with respect to 540 nm using an Aminco DW-2c spectrophotometer (SLM Instruments, Champaign-Urbana, IL) in the dualwavelength mode. An absorption coefficient of 66 mM-l cm-was used for the absorbance at 470 nm with respect to 540 nm [21]. One unit of biliverdin reductase catalyzes the formation of 1 nmol bilirubin/min at 37°C.…”

“…Chick embryo liver cells in culture have proven a useful system for studying the regulation of porphyrin and heme metabolism since they retain normal levels and inducibility (as in ovo) of cytochromes P-450 [21,24,251 and the ratecontrolling enzymes of heme synthesis (5-aminolevulinate synthase) [26] and breakdown (heme oxygenase) [14, 211. Using this culture system, we recently showed that there are at least two disparate mechanisms for induction of hepatic heme oxygenase, one dependent on heme and a second independent of heme, produced by certain transition or heavy metals (notably cadmium or cobalt) [21], extending results obtained in other systems 11 -31. Although evidence for two forms of heme oxygenase (called heme oxygenase-1 and heme oxygenase-2) has been obtained in mammals, only heme oxygenase-1 has been found to be inducible, regardless of the class of inducer used [3, 271. To aid our ongoing studies of heme metabolism in chick embryo liver cells, we have purified and characterized heme oxygenase from chick liver.…”

Hepatic 5‐Aminolevulinic Acid Synthase mRNA Stability is Modulated by Inhibitors of Heme Biosynthesis and by Metalloporphyrins

“…Conversely, if the heme content in the regulatory heme pool is too high, heme oxygenase, the first and rate-con-trolling enzyme in heme degradation , is induced [21, 221, subsequently decreasing the size of the regulatory heme pool. Typically, the concentration of heme required to induce heme oxygenase is greater than the concentration required to reduce Alv synthase [7,15,22,231.…”