Primary chick embryo liver cells were used to explore the regulation of δ-aminolevulinic acid synthase and heme oxygenase, the enzymes that catalyze the rate-limiting reactions of heme anabolism and catabolism, respectively. The general focus of the work was the exploration of the novel observation in which glutethimide and iron synergistically induced both δ-aminolevulinic acid synthase and heme oxygenase, a phenomenon that would not be predicted a priori. The course of events appeared to be: first, that heme synthesis was increased after addition of the glutethimide and that iron potentiated heme synthesis; second, the heme induced heme oxygenase five to ten fold; and third, that heme oxygenase degraded the heme permitting an uncontrolled induction of δ-aminolevulinic acid synthase. This induction of δ-aminolevulinic acid synthase could be prevented by the addition of a metalloporphyrin inhibitor of heme oxygenase. Induced δ-aminolevulinic acid synthase activity could be dramatically reduced by the addition of nanomolar concentrations of a metalloporphyrin, inhibitory for heme oxygenase, and heme. Specific observations related to the synergistic induction of heme oxygenase by glutethimide and iron was that the induction of heme oxygenase activity by glutethimide and iron occurred rapidly, with maximal increases occurring four to six hours after original treatment. Induction of heme oxygenase by glutethimide and iron was shown to be dependent on de novoheme synthesis since 4,6-dioxoheptanoic acid, a potent and specific inhibitor of heme biosynthesis, prevented the activity of heme oxygenase from increasing in the presence of glutethimide and iron. Induction of activity was associated with increases in heme oxygenase mRNA and protein; and, when induction was prevented by 4,6-dioxoheptanoic acid, no increase in either mRNA or immunoreactive protein was observed. δ-Aminolevulinic acid synthase activity was also synergistically increased by glutethimide and iron; this increase occurred 4-6 hours after maximal heme oxygenase activity had been attained. The temporal relationship between the induction of δ-aminolevulinic acid synthase and heme oxygenase suggested that the oxygenase depleted a regulatory heme pool that would normally prevent uncontrolled induction of the synthase. When cultures were exposed to tin-mesoporphyrin, a potent inhibitor of heme oxygenase, induction of δ-aminolevulinic acid synthase, normally produced by glutethimide and iron, was prevented. Addition of tin-mesoporphyrin after δ-aminolevulinic acid synthase induction had already been established promptly halted any further induction. When heme or a combination of heme and tin-mesoporphyrin was added after induction of δ-aminolevulinic acid synthase was established, activity of the synthase was rapidly reduced. Finally, experiments in primary chick embryo liver cells with tin-, zinc- and copper- chelated porphyrins were done to assess their effects on activities of δ-aminolevulinic acid synthase, induced by prior treatment of cells with glutethimide and iron. Nanomolar concentrations of zinc- or tin porphyrins reduced δ-aminolevulinic acid synthase activities, while copper-chelated porphyrins did not. When nanomolar concentrations of heme were added with zinc- or tin-porphyrins, δ-aminolevulinic acid synthase activity was further reduced. Effects of the non-heme metalloporphyrins on δ-aminolevulinic acid synthase were closely correlated with their abilities to inhibit heme oxygenase (r=0.78). The largest decrease of δ-aminolevulinic acid synthase (67%) was obtained with zinc-mesoporphyrin and heme. There was a rapid appearance of the cytosolic, precursor form of δ-aminolevulinic acid synthase in the presence of both 10 μM heme or 50 nM zinc-mesoporphyrin and 200 nM heme. Reduction of the half-life of the mRNA from 5.2 hours to 2.2-2.5 hours was observed in the presence of both 10 μM heme or 50 nM zinc-mesoporphyrin and 200 nM heme. In summary, the chick embryo liver cell culture model treated with glutethimide and iron may serve as one experimental model for patients suffering from acute porphyrias, in whom uncontrolled induction of hepatic δ-aminolevulinic acid synthase plays a key role in pathogenesis of disease. The synergistic induction of δ-aminolevulinic acid synthase in the presence of glutethimide and iron may serve as an experimental paradigm for this disease. The reduction of δ-aminolevulinic acid synthase by low doses of zinc-mesoporphyrin and heme may help form the experimental foundation for eventual studies in patients suffering from acute porphyrias.

5-Aminolevulinic acid synthase-1 (ALAS-1) is the first and normally rate-controlling enzyme for hepatic heme biosynthesis. ALAS-1 is highly inducible, especially in liver, in response to changes in nutritional status, and to drugs that induce cytochrome P-450. The critical biochemical abnormality of the acute porphyrias, a group of disorders of heme synthesis, is an uncontrolled up-regulation of ALAS-1. High intakes of glucose or other metabolizable sugars and intravenous heme are the cornerstones of therapy for acute attacks of porphyrias and both repress the over-expression ALAS-1, although their mechanisms of action have not been fully characterized. In this work, the chick hepatoma cell line, LMH, was characterized with respect to its usefulness in studies of heme biosynthesis and compared with chick embryo liver cells (CELCs), a widely used model for studies of heme metabolism. The inducibility of ALAS-1 mRNA and enzyme activity and accumulation of porphyrins by chemicals were used to evaluate heme biosynthesis in LMH cells. Repression of ALAS-1 mRNA and induced activity by exogenous heme (20 μM) was shown to occur in LMH cells as in CELCs. In addition, a synergistic induction of ALAS-1 enzyme activity was observed in LMH cells, as shown previously in CELCs, by treatment with a barbiturate-like chemical, Glutethimide (Glut), in combination with an inhibitor of heme synthesis, 4,6-dioxoheptanoic acid (DHA). This induction of ALAS-1 enzyme activity is analogous to what occurs in patients with acute hepatic porphyrias and LMH cells were used to further characterize effects of Glut, DHA, glucose, and heme on ALAS-1. A "glucose effect" to decrease Glut and DHA-induced ALAS-1 enzyme activity was obtained in LMH cells and CELCs in the absence of serum or hormones. This "glucose effect" was further characterized in LMH cells using a construct containing approximately 9.1 kb of chick ALAS-1 5'- flanking and 5' -UTR region attached to a luciferase/reporter gene (pGcALAS9.1-Luc). Glut (50 μM) and DHA (250 μM) synergistically induced luciferase activity (5-fold) in LMH cells transiently transfected with pGcALAS9.l-Luc. Addition of glucose (11 or 33 mM), in a dose-dependent manner, decreased the Glut+DHA up-regulation of pGcALAS9.1-Luc activity. Gluconeogenic or glycolytic substrates such as fructose, galactose, glycerol and lactate, but not the non-metabolizable sugar sorbitol, also down-regulated pGcALAS9.1-Luc in LMH cells. The cAMP analog 8-CPT-cAMP, augmented Glut induction of ALAS-1, indicating that the glucose effect may be partly mediated by changes in cAMP levels. The remaining studies focused on delineating the synergistic effect of Glut and DHA, and heme-dependent repression of ALAS-1. The 9.1 kb construct was compared with a construct containing the first 3.5 kb (pGcALAS3.5-Luc). The drug and heme effects were shown to be separate as drug induction was present in -3.4 to +0.082 kb region while the heme responsiveness was present in the -9.1 to -3.4 kb region. Using computer sequence analysis, several consensus activator protein-1 (AP-1) sites were found in the 9.1 kb ALAS-1 sequence but no consensus direct repeat (DR)-4 or DR-5 type recognition sequences for nuclear receptors were identified in the drug-responsive 3.5 kb region. Deletion constructs containing +0.082 to -7.6 kb (pGcALAS7.6-Luc) and +0.082 to -6.2 kb (pGcALAS6.3-Luc) cALAS 5'- flanking and 5' - UTR region were generated and tested and pGcALAS6.3-Luc was shown to have heme-dependent repression of basal and Glut and DHA-induced activity. A recently identified 167 bp chick ALAS-1 drug responsive enhancer (DRE) was PCR amplified and inserted upstream of the 9.1 kb (pGcALAS9.1+DRE), a 0.399 kb (+0.082 to -0.317) (pGcALAS0.3+DRE), and pGL3SV40 construct (pGL3SV40+DRE). DRE mediated the up-regulation of pGL3SV40+DRE construct by Glut was ~ 15-30 fold but interestingly only 3.2 and 3.7-fold for pGcALAS9.l +DRE and pGcALAS0.3+DRE constructs, respectively. In summary, in LMH cells drugs up-regulate ALAS-1 through non-DRE element(s) in the first 3.5 kb of ALAS-1 5'-flanking and 5'-UTR region and heme down-regulates ALAS-1 and determines the extent of the drug response through element(s) in the -6.3 to -3.5 kb region of ALAS-1 5'- flanking region.