Thyroid hormone beta receptor activation has additive cholesterol lowering activity in combination with atorvastatin in rabbits, dogs and monkeys.

BACKGROUND AND PURPOSE: Thyroid hormone receptor (TR) agonists are in clinical trials for the treatment of hypercholesterolaemia. As statins are the standard of clinical care, any new therapies must have adjunctive activity, when given in combination with statins. As already known for the statins, the cholesterol lowering effect of TR activation involves increased expression of the low-density lipoprotein receptor. Using animal models, we tested whether TR activation would have additive cholesterol lowering activity in the presence of effective doses of a statin. EXPERIMENTAL APPROACH: We evaluated the activity of a liver-targeted prodrug, MB07811, of a novel TH receptor beta agonist, MB07344, as monotherapy and in combination with atorvastatin in rabbits, dogs and monkeys. KEY RESULTS: In rabbits, MB07344 (i.v.) decreased total plasma cholesterol (TPC) comparable to that achieved with a maximally effective dose of atorvastatin (p.o.). The addition of MB07344 to atorvastatin resulted in a further decrease in TPC. Similarly, the addition of MB07811 (p.o.) to atorvastatin treatment decreased TPC beyond the level achieved with either agent as monotherapy. In dogs and monkeys, atorvastatin and MB07811 were administered as monotherapy or in combination. Consistent with the rabbit studies, the combination treatment caused a greater decrease in TPC than either MB07811 or atorvastatin administered as monotherapy. CONCLUSIONS AND IMPLICATIONS: We conclude that the effects of MB07811 and atorvastatin in lowering cholesterol are additive in animals. These results would encourage and support the demonstration of similarly improved efficacy of combination versus monotherapy with such agents in the clinic.

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes.

Gene delivery to differentiated hepatocytes is notoriously difficult. Hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented medium acquire paracellular junctions, arrange themselves in multicellular islands and are an excellent in vitro model for studying liver function. Baculovirus-mediated gene delivery to hepatocytes in this culture system is restricted to peripheral cells of the islands. However, this limitation can be overcome by transient calcium depletion of the cells prior to and during baculovirus infection. Examination of the mechanism underlying this process revealed that calcium depletion was accompanied by a transient loss of intercellular contacts and paracellular junction complex integrity, increased distance between adjoining cells, and internalization of the tight junction protein, zona occludens ZO-1. Internalization of ZO-1 was accompanied by baculovirus infection of internal cells of hepatocyte islands. When calcium levels were restored, paracellular junction complex integrity returned to normal by 12 h. No permanent alterations in hepatocyte ultrastructure and albumin mRNA, and protein expression were caused by this gene transfer method. Loss in paracellular junction complex integrity exposes the basolateral (sinusoidal) surface of hepatocytes resulting in homogeneous baculovirus-mediated gene delivery to approximately 75% of the cells in long-term DMSO culture. We conclude that the use of recombinant baculovirus as a vector in combination with transient calcium depletion is a highly efficient method for delivering exogenous genes to hepatocytes without loss of hepatic differentiation.

Regulation of heme metabolism in rat hepatocytes and hepatocyte cell lines: delta-aminolevulinic acid synthase and heme oxygenase are regulated by different heme-dependent mechanisms.

Regulation of delta-aminolevulinic acid (ALA) synthase and heme oxygenase was analyzed in primary rat hepatocytes and in two immortalized cell lines, CWSV16 and CWSV17 cells. ALA synthase was induced by 4,6-dioxohepatnoic acid (4,6-DHA), a specific inhibitor of ALA dehydratase, in all three systems; however, the induction in CWSV17 cells was greater than in either of the other two systems. Therefore, CWSV17 cells were used to explore the regulation of both enzymes by heme and 4,6-DHA. Data obtained from detailed concentration curves demonstrated that 4,6-DHA induced the activity of ALA synthase once ALA dehydratase activity became rate-limiting for heme biosynthesis. Heme induced heme oxygenase activity with increases occurring at concentrations of 10 microM or greater. Heme blocked the 4,6-DHA-dependent induction of ALA synthase with an EC50 of 1.25 microM. Heme-dependent decreases of ALA synthase mRNA levels occurred more quickly and at lower concentrations than heme-dependent increases of heme oxygenase mRNA levels. ALA synthase mRNA remained at reduced levels for extended periods of time, while the increases in heme oxygenase mRNA were much more transient. The drastic differences in concentrations and times at which heme-dependent effects were observed strongly suggest that two-different heme-dependent mechanisms control the ALA synthase and heme oxygenase mRNAs. In CWSV17 cells, heme decreased the stability of ALA synthase mRNA from 2.5 to 1.3 h, while 4,6-DHA increased the stability of the mRNA to 5.2 h. These studies demonstrate that regulation of ALA synthase mRNA levels by heme in a mammalian system is mediated by a change in ALA synthase mRNA stability. The results reported here demonstrate the function of the regulatory heme pool on both ALA synthase and heme oxygenase in a mammalian hepatocyte system.

Hepatic iron concentration: noninvasive estimation by means of MR imaging techniques.

PURPOSE: To identify a magnetic resonance (MR) imaging method sufficiently sensitive and specific in the estimation of hepatic iron content to obviate liver biopsy. MATERIALS AND METHODS: Thirty-eight patients underwent percutaneous needle biopsy of the liver with chemical measurement of the hepatic iron concentration and hepatic MR imaging with several spin-echo and gradient-recalled-echo (GRE) techniques. Correlations between MR imaging parameters and the hepatic iron concentration were determined. RESULTS: Inverse curvilinear relationships were noted between several MR parameters and hepatic iron concentrations. GRE sequences with short repetition and echo times were more accurate and precise than spin-echo sequences for the estimation of hepatic iron concentration. A GRE sequence with a repetition time of 18 msec, an echo time of 5 msec, and a flip angle of 10 degrees showed close correlation between the hepatic iron concentration and the natural logarithm of the ratio of the signal intensity of liver to the SD of background noise (r = -0.94) and low coefficient of variation (12%). CONCLUSION: MR imaging with these parameters is a rapid, noninvasive, and accurate modality for estimation of hepatic iron concentration; it is sufficiently accurate and precise to obviate liver biopsy for the purpose of measuring hepatic iron concentration.

Gamma delta intraepithelial lymphocytes drive tumor necrosis factor-alpha responsiveness to intestinal iron challenge: relevance to hemochromatosis.

The dependence of intestinal epithelial cell (IEC) growth and differentiation on intraepithelial lymphocytes (IELs) expressing the gamma/delta (gamma delta) T-cell receptor (TCR), suggested a potential role for gamma delta + IELs in the regulation of iron absorption. We therefore examined the levels of hepatic iron and the IEL cytokine responses in C57BL/6J control and class I and TCR knockout lines (placed on a C57BL/6J genetic background) following the administration of supplemental dietary iron. The highest level of liver iron was found in the beta 2-microglobulin knockout (beta 2m-/-) mice followed by the TCR-delta knockout (TCR delta-/-) animals. TCR-alpha knockout (TCR alpha-/-) and control animals did not differ in their iron levels. Liver iron loading correlated inversely with the ability of the mice to generate an IEL tumor necrosis factor (TNF)-alpha response. These observations suggest a model in which IEC iron loading is communicated to IELs via the HFE class I protein. The result of this communication is the initiation of TNF-alpha release by gamma delta + IELs (sustained by macrophages and dendritic cells) contributing to the upregulation of ferritin expression and possibly to the normal maintenance of the IEC apoptotic pathway.

Metabolism of 3,5,5-trimethylhexanoyl-ferrocene by rat liver: release of iron from 3,5,5-trimethylhexanoyl-ferrocene by a microsomal, phenobarbital-inducible cytochrome P-450.

3,5,5-Trimethylhexanoyl (TMH)-ferrocene has been used to produce iron loading in whole animals and in cultured hepatocytes. Iron loading produced by TMH-ferrocene is highly efficient and, of the compounds used to produce iron loading in experimental systems, most closely mimics the loading patterns observed in the human disease hemochromatosis. Previous work with TMH-ferrocene has shown that TMH-ferrocene is degraded in vivo because the iron is released from the ferrocene nucleus. Because TMH-ferrocene is highly lipophilic and stable chemically, we hypothesize that this molecule indeed could be degraded enzymatically. To measure the breakdown of TMH-ferrocene, iron release from the molecule was analyzed using a Ferrochem II analyzer, which uses constant potential coulometry to measure the amount of ionic iron within a biological sample. In this study, we show that TMH-ferrocene is degraded by a microsomal enzyme that requires NADPH and molecular oxygen. The TMH-ferrocenase activity is heat labile, requires a physiologic temperature, is induced by phenobarbital, and is inhibited by carbon monoxide and piperonyl butoxide but not by dicoumarol. The enzyme follows Michaelis-Menten kinetics and has a Km of 58.5 microM and a Vmax of 57.5 nmol Fe released/mg protein/min. We conclude that TMH-ferrocene is degraded by a phenobarbital-inducible cytochrome P-450.

Accumulation of iron by primary rat hepatocytes in long-term culture: changes in nuclear shape mediated by non-transferrin-bound forms of iron.

We have previously shown that hepatocytes in long-term dimethylsulfoxide (DMSO) culture, fed a chemically defined medium, are highly differentiated and an excellent in vitro model of adult liver. Hepatocytes in long-term DMSO culture can be iron loaded by exposure to non-transferrin-bound iron (NTBI) in the form of ferrous sulfate (FeSO4), ferric nitrilotriacetate, or trimethylhexanoyl (TMH)-ferrocene. Holotransferrin, at equivalent times and concentrations, was unable to load hepatocytes. Of the iron compounds tested, TMH-ferrocene most accurately simulated the morphological features of iron-loaded hepatocytes in vivo. When exposed to 25 micromol/L TMH-ferrocene, hepatocytes loaded increasing amounts of iron for 2 months before the cells died. When exposed to lower concentrations of TMH-ferrocene (as low as 2.5 micromol/L), hepatocytes continuously loaded iron and remained viable for more than 2 months. The cellular deposition of iron was different in hepatocytes exposed to TMH-ferrocene compared with those exposed to FeSO4; exposure to TMH-ferrocene resulted in the presence of more ferritin cores within lysosomes than were seen with FeSO4. When the concentration of TMH-ferrocene was increased, a greater number of ferritin cores were observed within the lysosome, and total cellular ferritin, as assessed by Western blot, increased. The formation of hemosiderin was also observed. Furthermore, nuclear shape was distorted in iron-loaded hepatocytes. The extent of deviation from circularity in the nucleus correlated with increasing concentrations of TMH-ferrocene and was greater in hepatocytes exposed to FeSO4 than an equivalent concentration of TMH-ferrocene. The deviation from circularity was smallest in hepatocytes that contained well formed ferritin cores and increased in hepatocytes that contained greater amounts of hemosiderin. Furthermore, in hepatocytes treated with FeSO4, a large amount of cell-associated iron was detected but without a significant increase in the total amount of ferritin. The deviation from circularity was the largest in FeSO4-treated hepatocytes, indicating that iron not properly incorporated into ferritin caused more cellular damage. We conclude that iron-loaded hepatocytes in long-term DMSO culture represent a flexible system for studying the effects of chronic iron loading on hepatocytes.

Exposure of primary rat hepatocytes in long-term DMSO culture to selected transition metals induces hepatocyte proliferation and formation of duct-like structures.

We previously showed that primary rat hepatocytes plated on a rat-tail collagen coated dish and fed a chemically-defined medium supplemented with 2% dimethylsulfoxide (DMSO) can be maintained in a well-differentiated, non-replicating state for periods of several months. In this study, we show that the addition of copper, iron, and zinc to the DMSO-containing chemically defined medium induced DNA synthesis and cell replication during the first two months in culture without loss of hepatic differentiation. DNA synthesis occurred throughout the hepatocyte population without regard to cellular size. No changes were observed in properties indicative of well-differentiated hepatocytes, including cellular morphology, ultrastructure, albumin, or cytokeratin-8 expression. During the third month in culture, after the hepatocytes had become confluent, pseudoduct structures became apparent. Examination of cells lining the ducts by immunohistochemistry showed that these cells lost the ability to express albumin and stained more intensely for cytokeratin 19 and laminin. The ultrastructure of the cells lining the ducts was altered and became more characteristic of bile duct cells. Immunoelectron microscopy revealed that connexin 43, a marker of bile-duct proliferation, was expressed in the duct-like cells. We conclude that under these specific nutritive conditions, primary rat hepatocytes proliferate and, with time, begin to form duct-like structures with altered gene expression and ultrastructural properties.

Mechanism of induction of heme oxygenase by metalloporphyrins in primary chick embryo liver cells: evidence against a stress-mediated response.

Heme oxygenase catalyzes the first and rate-controlling step in heme catabolism. One of the two forms of heme oxygenase (heme oxygenase-1) has been shown to be increased by heme, metals, and in some systems, by certain environmental stresses. However, it remains uncertain whether heme induces hepatic heme oxygenase-1 by a general stress response, or a specific heme-dependent cellular response. The work communicated here explores this issue by examining possible mechanisms whereby heme and other metalloporphyrins induce heme oxygenase-1 in normal liver cells. Primary cultures of chick embryo liver cells were tested for their ability to increase heme oxygenase mRNA after exposure to selected metalloporphyrins (heme, chromium mesoporphyrin, cobalt protoporphyrin and manganese protoporphyrin). The ability of antioxidants to decrease metalloporphyrin-mediated induction of heme oxygenase-1 mRNA was also tested. Our results indicate that: 1) the increase in heme oxygenase-1 mRNA mediated by heme or other metalloporphyrins may involve a short-lived protein(s) since the increase was prevented by several inhibitors of protein synthesis; and 2) in normal liver cells, heme-dependent oxidative stress does not play a key role in the heme-mediated induction of heme oxygenase-1. We conclude that heme and other non-heme metalloporphyrins induce heme oxygenase-1 through a mechanism requiring protein synthesis, not because metalloporphyrins increase cellular oxidative or other stress.

Hepatic 5-aminolevulinic acid synthase mRNA stability is modulated by inhibitors of heme biosynthesis and by metalloporphyrins.

Hepatic 5-aminolevulinic acid synthase, the first and normally rate-controlling enzyme of heme biosynthesis, is regulated by heme. One of the known mechanisms whereby increased cellular heme regulates 5-aminolevulinic acid synthase is by decreasing the stability of its mRNA. In primary cultures of chick embryo liver cells, we tested whether a decrease in cellular heme might increase 5-aminolevulinic acid synthase mRNA stability and whether heme or other metalloporphyrins could reverse this stabilization. We found that: (a) The stability of 5-aminolevulinic acid synthase mRNA was markedly increased by inhibitors of heme biosynthesis, namely, 4,6-dioxoheptanoic acid or deferoxamine; (b) This increased stability of 5-aminolevulinic acid synthase mRNA was reversed by the addition of heme (10 microM) or by the combination of zinc mesoporphyrin (50 nM), an inhibitor of heme oxygenase, and heme (200 nM); (c) Repression of 5-aminolevulinic acid synthase mRNA levels by zinc mesoporphyrin (10 microM) was due to inhibition of heme oxygenase, rather than a direct, heme-like, effect of zinc mesoporphyrin on 5-aminolevulinic acid synthase mRNA; (d) Among the several non-heme metalloporphyrins tested, only zinc mesoporphyrin and chromium mesoporphyrin significantly decreased 5-aminolevulinic acid synthase mRNA without increasing heme oxygenase mRNA.

A detailed analysis of the Knodell score and other histologic parameters as predictors of response to interferon therapy in chronic hepatitis C.

UNLABELLED: The Knodell score is inaccurate at predicting response to interferon alpha (IFN-alpha) therapy in patients with hepatitis C. Our aim was to see if specific histologic parameters, including iron deposition in liver biopsies, are better predictors of response to IFN-alpha than the total Knodell score. Thirty-five unselected patients were studied who had hepatitis C treated with IFN-alpha between 1990 and 1993, and pretreatment serum iron indices and liver needle biopsies performed. Biopsies were divided for light microscopy and quantitative iron determination. H&E-stained slides were graded for components I, II, III, IV, and total Knodell score. Quantitative determinations were percentage of portal triads with inflammation, piecemeal necrosis, lymphoid aggregates, and inflamed bile ducts; percentage of lobules with inflammation or acidophilic bodies; and percentage of triads with positive iron stain. Complete responders (CR) to IFN-alpha were defined by normalization of serum alanine aminotransferase (< or = 40 IU/liter), and noncomplete responders (NCR) by partial or no response. Data were analyzed statistically. CR had < 40% of triads positive for iron (P = 0.02) and lower serum ferritin (P = 0.05) and higher scores for lobular necrosis (P = 0.04). The percentage of iron-positive triads correlated only with cirrhosis. Addition of cirrhosis to percentage of iron-positive triads did not improve the predictive power of the portal iron. CR and NCR did not differ with respect to total Knodell score or any of the other individual parameters except Knodell II. CONCLUSIONS: (a) Individual features of lobular necrosis and iron staining in portal triads are better predictors of response to IFN-alpha than the total Knodell score.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of iron in the liver predicts the response of chronic hepatitis C infection to interferon therapy.

Recent evidence suggests that patients with chronic hepatitis C virus (CHCV) who respond to interferon-alpha (IFN) therapy have a lower hepatic iron concentration than those who do not. The object of this study was to assess the concentration and distribution of iron in liver biopsies from 15 patients with CHCV seen at the authors' medical center between June 1992 and March 1993. Patients with complete response to IFN were compared to those with non-complete response with respect to quantitative hepatic iron concentration, serum iron indices, and a detailed analysis of histologic features of hematoxylin-and-eosin and iron-stained pre-IFN biopsies. Patients with non-complete response had significantly higher scores for stainable iron in sinusoidal cells (P = .02) and portal tracts (P = .05) than did patients with complete response. Total hepatic iron scores, mean quantitative hepatic iron, and mean serum ferritin were higher in patients with noncomplete response, but the differences were not significant. In conclusion, iron deposition in sinusoidal cells and portal tracts is significantly less frequent in patients with complete response to IFN than in those with poor or no response, and may be a useful, objective predictor of response to IFN therapy.

High prevalence of serological markers of autoimmunity in patients with chronic hepatitis C.

The advent of specific antiviral therapy for chronic hepatitis C has increased the importance of establishing the correct etiology of chronic hepatitis in patients, especially because interferon alfa (IFN-alpha) has been reported to exacerbate autoimmune hepatitis (AIH), whereas corticosteroids increase viral replication in chronic hepatitis C. In our medical center, we have treated many patients with apparent chronic hepatitis C and serological or clinical evidence of autoimmunity. Our aim was to estimate the prevalence of this association and to learn whether demographic or clinical features distinguished between patients with or without autoimmune markers. We performed a retrospective review of the records of 244 unselected patients seen at the Clinics and Hospital of the University of Massachusetts between May 1991 and November 1993, who had elevated serum aminotransferases. One hundred seventeen patients had chronic hepatitis C defined by elevations of serum alanine transaminase (ALT) for at least 6 months, positive serum antibodies to hepatitis C virus (HCV; second-generation enzyme immunoassay [EIA2] or recombinant immunoblot assay [RIBA]), and absence of hepatitis B surface antigen in the serum. Records were reviewed for results of autoimmune markers in sera, including anti-nuclear antibodies (ANAs), anti-smooth muscle antibodies (SMAs), rheumatoid factor (RF), antimitochondrial antibodies (AMAs), anti-liver and kidney microsomal (LKM) antibodies, and cryoglobulins. We found a high prevalence of positivity, particularly for anti-SMAs (66%) and RF (76%) in both men and women. Forty of 41 patients tested negative for anti-LKM antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue distribution of zinc-mesoporphyrin in rats: relationship to inhibition of heme oxygenase.

Metalloporphyrins, including heme and others that inhibit heme oxygenase, are agents with expanding therapeutic potential. Recent results from our laboratory showed that a combination of heme and zinc-mesoporphyrin was remarkably effective in ameliorating biochemical features of acute porphyria. The aim of this study was to assess plasma clearance, tissue distribution and persistence, stability, toxicology and metabolic effects of zinc-mesoporphyrin, after its i.v. administration to rats. After administration of 15 mumol/kg b.wt. of zinc-mesoporphyrin (bound to serum albumin in a 1:1 molar ratio) the metalloporphyrin was rapidly cleared from plasma (half-life 3.6 h) with uptake primarily into liver and spleen, considerably less into the kidney and none detectable into the heart or brain. Hepatic heme oxygenase activity was undetectable for 4 days and less than 50% of control 1 week later. Inhibition of splenic heme oxygenase activity was also substantial but less marked than in the liver. No mortality was observed in any of the treated animals, and there was no detectable effect on gross or microscopic appearance of the liver, spleen, kidneys, heart, lungs or brain. Blood counts and chemistries remained within normal limits. We conclude that single doses of ZnMP-serum albumin are nontoxic, rapidly cleared from the plasma and persist primarily in the liver and spleen where heme oxygenase is inhibited for prolonged periods.

Effects of mifepristone (RU-486) on heme metabolism and cytochromes P-450 in cultured chick embryo liver cells, possible implications for acute porphyria.

Mifepristone (RU-486), a potent progesterone receptor antagonist and inducer of cytochromes P-450, is currently in use in Europe, particularly as a post-coital oral contraceptive. Soon it will be available in the United States, as well. Since progesterone has been implicated in the pathogenesis of acute attacks of porphyria, the use of RU-486 or related compounds might be considered in porphyric patients. However, as with other cytochrome P-450 inducers, RU-486 may have the ability to precipitate or exacerbate attacks of acute porphyria. The acute porphyrias in relapse are associated with an increase in activity of delta-aminolevulinic acid synthase, the first and normally rate-controlling enzyme in heme biosynthesis. We have used primary cultures of chick embryo liver cells to test the ability of RU-486 to induce delta-aminolevulinic acid synthase activity and mRNA, cytochromes P-450, porphyrin accumulation, and heme oxygenase. We found that RU-486, at concentrations observed in human plasma after a single oral dose, induced the mRNA and activity of delta-aminolevulinic acid synthase, both by itself and in the presence of deferoxamine, a potent iron chelator that inhibits ferrochelatase. RU-486 and deferoxamine together also produced significant accumulations of protoporphyrin. These results indicate that RU-486 may pose a risk in patients with known acute porphyria and should be used with caution. RU-486 increased the concentration of total cytochrome P-450, and the activity of erythromycin demethylase, an activity specifically catalyzed by cytochrome P-450 3A. Unlike several other porphyrogens (e.g. hydantoins, barbiturates), RU-486 does not produce accumulation of uroporphyrin or induction of heme oxygenase in chick embryo liver cells.

Differential effects of metalloporphyrins on messenger RNA levels of delta-aminolevulinate synthase and heme oxygenase. Studies in cultured chick embryo liver cells.

The acute porphyrias in relapse are commonly treated with intravenous heme infusion to decrease the activity of delta-aminolevulinic acid synthase, normally the rate-controlling enzyme in heme biosynthesis. The biochemical effects of heme treatment are short-lived, probably due in part to heme-mediated induction of heme oxygenase, the rate-controlling enzyme for heme degradation. In this work, selected nonheme metalloporphyrins were screened for their ability to reduce delta-aminolevulinic acid synthase mRNA and induce heme oxygenase mRNA in chick embryo liver cell cultures. Of the metalloporphyrins tested, only zinc-mesoporphyrin reduced delta-aminolevulinic acid synthase mRNA without increasing heme oxygenase mRNA. The combination of zinc-mesoporphyrin and heme, at nanomolar concentrations, decreased delta-aminolevulinic acid synthase mRNA in a dose-dependent manner. The combination of zinc-mesoporphyrin (50 nM) and heme (200 nM) decreased the half-life of the mRNA for delta-aminolevulinic acid synthase from 5.2 to 2.5 h, while a similar decrease was produced by heme (10 microM) alone (2.2 h). The ability of zinc-mesoporphyrin to supplement the reduction of delta-aminolevulinic acid synthase mRNA by heme, in a process similar to that observed with heme alone, provides a rationale for further investigation of this compound for eventual use as a supplement to heme therapy of the acute porphyrias and perhaps other conditions in which heme may be of benefit.

Repression of ALA synthase by heme and zinc-mesoporphyrin in a chick embryo liver cell culture model of acute porphyria.

We characterize a liver cell culture model for acute hepatic porphyrias that recapitulates the biochemical features of the human syndrome. In chick embryo liver cells in primary culture exposed to glutethimide and 4,6-dioxoheptanoic acid, heme alone produced a transient dose-dependent decrease in delta-aminolevulinate synthase and a concomitant increase in heme oxygenase. The addition of low concentrations of zinc-mesoporphyrin (50-200 nM), an inhibitor of heme oxygenase, led to more prolonged decreases in activity of the synthase and to an additive effect with heme. These effects of zinc-mesoporphyrin were associated with prolonged inhibition of heme oxygenase. These results suggest that the treatment of choice of acute porphyric syndromes may be the combination of low doses of heme and zinc-mesoporphyrin or another similarly non-toxic inhibitor of heme oxygenase.

Induction of heme oxygenase in intestinal epithelial cells: studies in Caco-2 cell cultures.

Enterally administered, heme is a good source of iron in humans and other animals, but the metabolism of heme by enterocytes has not been fully characterized. Caco-2 cells in culture provide a useful model for studying cells that resemble small intestinal epithelium, both morphologically and functionally. In this paper we show that heme oxygenase, the rate-controlling enzyme of heme catabolism, is present in abundance in Caco-2 cells, and that levels of its mRNA and activity can be increased by exposure of the cells to heme or metal ions (cadmium, cobalt). Caco-2 cells also contain biliverdin reductase activity which, in the basal state, is similar to that of heme oxygenase (approximately 40 pmole of product per mg protein per minute); however, when heme oxygenase is induced, biliverdin reductase may become rate-limiting for bilirubin production.

Repression of hepatic delta-aminolevulinate synthase by heme and metalloporphyrins: relationship to inhibition of heme oxygenase.

Heme- and tin-chelated metalloporphyrins are known to decrease the activity of hepatic delta-amino-levulinate synthase, the rate-controlling enzyme of heme synthesis. We performed experiments in primary chick embryo liver cells with tin-, zinc- and copper-chelated porphyrins to assess their effects on activities of delta-aminolevulinate synthase induced by prior treatment of cells with glutethimide and ferric nitrilotriacetate. These different metalloporphyrins were tested to form the experimental foundation for eventual studies in patients with acute porphyrias, in which uncontrolled induction of hepatic delta-amino-levulinate synthase, which plays a key role in pathogenesis of disease. Zinc and tin porphyrins reduced delta-aminolevulinate synthase activities, whereas copper-chelated porphyrins did not. When heme (iron protoporphyrin) was added with zinc or tin porphyrins, delta-aminolevulinate synthase activity was further reduced. Effects of the nonheme metalloporphyrins on delta-aminolevulinate synthase were closely correlated with their abilities to inhibit heme oxygenase (r = 0.78). The largest decrease of delta-aminolevulinate synthase (67%) was obtained with zinc mesoporphyrin and heme. Dose-response data indicated that only nanomolar concentrations of zinc mesoporphyrin and heme are required to obtain this effect. We found no effect of exposure to heme (10 mumol/L) or heme (200 nmol/L) plus zinc mesoporphyrin (50 nmol/L) on the half-life of activity of delta-aminolevulinate synthase (1.9 to 2.1 hr, regardless of treatment). This result suggests that the repressive effect of heme is directed toward decreasing synthesis, increasing breakdown or decreasing the translation of the messenger RNA of delta-aminolevulinate synthase.(ABSTRACT TRUNCATED AT 250 WORDS)