Givosiran to treat acute porphyria.

Porphyrias are a family of rare diseases chiefly due to inborn errors of heme biosynthesis. The porphyrias are generally characterized either by the main site of overproduction of heme precursors (hepatic or erythropoietic) or the main clinical manifestations (acute or cutaneous). The regulation of 5- (or δ)-aminolevulinic acid synthase 1 (ALAS1) plays a key role in the pathway of normal hepatic heme synthesis, providing insight into the pathophysiologic mechanisms and potential therapeutic targets for the treatment of the porphyrias. Givosiran (Givlaari; Alnylam Pharmaceuticals) is an ALAS1-directed small interfering RNA (siRNA) which has been developed for the treatment of acute hepatic porphyria (AHP). It was first approved in 2019 by the U.S. Food and Drug Administration (FDA) for the treatment of adult patients with AHP, and it received also approval in the E.U. in 2020 for the treatment of AHP in adults and adolescents aged 12 years and older.

Comparative analysis of portal hepatic infiltrating leucocytes in acute drug-induced liver injury, idiopathic autoimmune and viral hepatitis.

Drug-induced liver injury (DILI) is often caused by innate and adaptive host immune responses. Characterization of inflammatory infiltrates in the liver may improve understanding of the underlying pathogenesis of DILI. This study aimed to enumerate and characterize leucocytes infiltrating liver tissue from subjects with acute DILI (n = 32) versus non-DILI causes of acute liver injury (n = 25). Immunostains for CD11b/CD4 (Kupffer and T helper cells), CD3/CD20 (T and B cells) and CD8/CD56 [T cytotoxic and natural killer (NK) cells] were evaluated in biopsies from subjects with acute DILI, either immunoallergic (IAD) or autoimmune (AID) and idiopathic autoimmune (AIH) and viral hepatitis (VH) and correlated with clinical and pathological features. All biopsies showed numerous CD8(+) T cells and macrophages. DILI cases had significantly fewer B lymphocytes than AIH and VH and significantly fewer NK cells than VH. Prominent plasma cells were unusual in IAD (three of 10 cases), but were associated strongly with AIH (eight of nine) and also observed in most with AID (six of nine). They were also found in five of 10 cases with VH. Liver biopsies from subjects with DILI were characterized by low counts of mature B cells and NK cells in portal triads in contrast to VH. NK cells were found only in cases of VH, whereas AIH and VH both showed higher counts of B cells than DILI. Plasma cells were associated most strongly with AIH and less so with AID, but were uncommon in IAD.

Antigen-specific T lymphocyte proliferation decreases over time in advanced chronic hepatitis C.

To evaluate T cell immunity in advanced liver disease, antigen-specific lymphoproliferative (LP) responses were prospectively studied in the context of the Hepatitis C Antiviral Long-term Treatment against Cirrhosis trial. Peripheral blood responses to hepatitis C virus (HCV), tetanus and Candida protein antigens were measured at baseline, month 12 (M12), M24, M36 and M48 in 186 patients randomized to either low-dose peginterferon-alfa-2a (PEG-IFN) only or observation. Liver histology was evaluated at baseline, M24 and M48. Patients with cirrhosis (Ishak 5-6) were less likely to have positive LP responses to HCV at baseline than patients with fibrosis (15%vs 29%, P = 0.03) and had lower levels of HCV c100 responses at baseline, M24 and M48 (P = 0.11, P = 0.05, P = 0.02, respectively). For 97 patients with complete longitudinal data, the frequency of positive LP responses to HCV, tetanus and Candida antigens declined over time (P < 0.003), and the slope of this decline was greater in the PEG-IFN treatment group than the observation group (P < 0.02). Lower levels of tetanus LP responses were associated with fibrosis progression and clinical outcomes (P = 0.009). Poorer CD4+ T cell proliferative function was associated with more advanced liver disease in chronic hepatitis C and may be further affected by long-term PEG-IFN treatment.

Serum proteomic profiling in patients with drug-induced liver injury.

BACKGROUND: Idiosyncratic drug-induced liver injury (DILI) is a complex disorder that is difficult to predict, diagnose and treat. AIM: To describe the global serum proteome of patients with DILI and controls. METHODS: A label-free, mass spectrometry-based quantitative proteomic approach was used to explore protein expression in serum samples from 74 DILI patients (collected within 14 days of DILI onset) and 40 controls. A longitudinal analysis was conducted in a subset of 21 DILI patients with available 6-month follow-up serum samples. RESULTS: Comparison of DILI patients based on pattern, severity and causality assessment of liver injury revealed many differentially expressed priority 1 proteins among groups. Expression of fumarylacetoacetase was correlated with alanine aminotransferase (ALT; r = 0.237; P = 0.047), aspartate aminotransferase (AST; r = 0.389; P = 0.001) and alkaline phosphatase (r = -0.240; P = 0.043), and this was the only protein with significant differential expression when comparing patients with hepatocellular vs. cholestatic or mixed injury. In the longitudinal analysis, expression of 53 priority 1 proteins changed significantly from onset of DILI to 6-month follow-up, and nearly all proteins returned to expression levels comparable to control subjects. Ninety-two serum priority 1 proteins with significant differential expression were identified when comparing the DILI and control groups. Pattern analysis revealed proteins that are components of inflammation, immune system activation and several hepatotoxicity-specific pathways. Apolipoprotein E expression had the greatest power to differentiate DILI patients from controls (89% correct classification; AUROC = 0.97). CONCLUSION: This proteomic analysis identified differentially expressed proteins that are components of pathways previously implicated in the pathogenesis of idiosyncratic drug-induced liver injury.

A naturally occurring human/hepatitis E recombinant virus predominates in serum but not in faeces of a chronic hepatitis E patient and has a growth advantage in cell culture.

Hepatitis E virus is the aetiological agent of acute hepatitis E, a self-limiting disease prevalent in developing countries. Molecular analysis of viral genomic RNA from a chronically infected patient confirmed the recent discovery that chronic infection correlated with extensive diversification of the virus quasispecies: the hypervariable region of some virus genomes in this USA patient contained large continuous deletions and a minor proportion of genomes in faeces and serum had acquired a mammalian sequence that encoded 39 aa of S19 ribosomal protein fused to the virus non-structural protein. Genomes with this insert were selected during virus passage in cultured cells to become the predominant species, suggesting that the inserted sequence promoted virus growth. The results demonstrated that hepatitis E virus can mutate dramatically during a prolonged infection and suggests it may be important to prevent or cure chronic infections before new variants with unpredictable properties arise.

Association of filamin A and vimentin with hepatitis C virus proteins in infected human hepatocytes.

Chronic hepatitis C (CHC) infection caused by hepatitis C virus (HCV) is a major cause of liver disease and remains a major therapeutic challenge. A variety of host proteins interact with HCV proteins. The definitive role of cytoskeletal (CS) proteins in HCV infection remains to be determined. In this study, our aim was to determine the expression profile of differentially regulated and expressed selected CS proteins and their association with HCV proteins in infected hepatocytes as possible therapeutic targets. Using proteomics, qRT-PCR, Western blot and immunofluorescence techniques, we revealed that filamin A (fila) and vimentin (vim) were prominently increased proteins in HCV-expressing human hepatoma cells compared with parental cells and in liver biopsies from patients with CHC vs controls. HCV nonstructural (NS) 3 and NS5A proteins were associated with fila, while core protein partially with fila and vim. Immunoprecipitation showed interactions among fila and NS3 and NS5A proteins. Cells treated with interferon-α showed a dose- and time-dependent decrease in CS and HCV proteins. NS proteins clustered at the perinuclear region following cytochalasin b treatment, whereas disperse cytoplasmic and perinuclear distribution was observed in the no-treatment group. This study demonstrates and signifies that changes occur in the expression of CS proteins in HCV-infected hepatocytes and, for the first time, shows the up-regulation and interaction of fila with HCV proteins. Association between CS and HCV proteins may have implications in future design of CS protein-targeted therapy for the treatment for HCV infection.

Effect of bovine serum albumin on mitochondrial respiration in the brain and liver of mice and rats.

We studied the effect of BSA (in the isolation medium) on the oxidation rate of succinate, glutamate, pyruvate, and α-ketoglutarate by mitochondria of the brain and liver from C57Bl/6g mice and Taconic Sprague Dawley rats. BSA had no effect on liver mitochondrial respiration, but increased oxidation of substrates (particularly of succinate) in brain mitochondria. Therefore, the major effect of BSA on brain mitochondria is manifested in activation of SDH. The improvement of mitochondrial properties in the brain after treatment with BSA is associated with antioxidant activity of this agent. Our results confirm the hypothesis that inhibition of SDH in brain mitochondria is not the artifact. This process serves as a mechanism protecting neurons from free oxygen radicals during succinate oxidation.

Duloxetine hepatotoxicity: a case-series from the drug-induced liver injury network.

BACKGROUND: Case reports suggest that duloxetine hepatotoxicity may arise, but risk factors, presenting features and clinical course are not well-described. AIM: To describe the presenting features and outcomes of seven well-characterized patients with suspected duloxetine hepatotoxicity. METHODS: Patients enrolled in the Drug-Induced Liver Injury Network Prospective Study underwent an extensive laboratory and clinical evaluation to exclude competing aetiologies of liver injury as well as a standardized assessment for causality and disease severity. RESULTS: Between 1/2006 and 9/2009, six of the seven cases of DILI attributed to duloxetine were assessed as definite or very likely. Median patient age was 49 years, six (86%) were women and the median latency from drug initiation to DILI onset was 50 days. Six patients developed jaundice and the median peak alanine aminotransferase in the five patients with acute hepatocellular injury was 1633 IU/L. Ascites developed in one patient and acute renal dysfunction in two others (29%). All patients recovered without liver transplantation even though three had pre-existing chronic liver disease. Liver histology in four cases demonstrated varying patterns of liver injury. CONCLUSIONS: Duloxetine hepatotoxicity developed within 2 months of drug intake and led to clinically significant liver injury. A spectrum of laboratory, histological and extra-hepatic features were noted at presentation.

Changes in quality of life and sexual health are associated with low-dose peginterferon therapy and disease progression in patients with chronic hepatitis C.

BACKGROUND: Primary analysis of the Hepatitis C Antiviral Long-Term Treatment against Cirrhosis (HALT-C) Trial showed long-term peginterferon therapy did not reduce complications in patients with chronic hepatitis C and advanced fibrosis or cirrhosis. AIM: To assess the effects of long-term peginterferon therapy and disease progression on health-related quality of life (HRQOL), symptoms and sexual health in HALT-C patients. METHODS: A total of 517 HALT-C patients received peginterferon alfa-2a (90 microg/week); 532 received no additional treatment for 3.5 years. Patients were followed up for outcomes of death, hepatocellular carcinoma and hepatic decompensation. Sexual health, SF-36 scores and symptoms were serially assessed by repeated-measures analyses of covariance. RESULTS: Patients with cirrhosis (n = 427) reported lower general well-being and more fatigue (P < 0.001) than patients with fibrosis (n = 622). Physical scores declined significantly over time, independent of treatment, and patients with cirrhosis reported lower scores. Vitality scores were lower in those with cirrhosis, and treated patients experienced a greater decline over time than untreated patients; HRQOL rebounded after treatment ended. Patients with a clinical outcome had significantly greater declines in all SF-36 and symptom scores. Among men, Sexual Health scores were significantly worse in treated patients and in those with a clinical outcome. CONCLUSION: Clinical progression of chronic hepatitis C and maintenance peginterferon therapy led to worsening of symptoms, HRQOL and, in men, sexual health in a large patient cohort followed up over 4 years (NCT00006164).

Extrahepatic manifestations of infection with hepatitis C virus.

Chronic hepatitis C is associated with, and may trigger or exacerbate, an extraordinary variety of extrahepatic manifestations. Most of these manifestations affect the skin, the most frequent and important of which are the leukocytoclastic vasculitis of MC type II and PCT. The former is an example of an autoimmune disorder triggered by HCV infection, whereas PCT is a skin disease caused by hepatic overproduction of uro- and 7-carboxyl porphyrins caused by increased oxidative stress in hepatocytes. Currently available effective therapies of CHC (IFN, ribavirin) may also trigger or exacerbate extrahepatic manifestations, especially including autoimmune thyroiditis, skin rashes, and hemolytic anemia.

Comparative effects of different doses of ribavirin plus interferon-alpha2b for therapy of chronic hepatitis C: results of a controlled, randomized trial.

Compared to either drug alone, therapy with the combination of ribavirin and interferon-alpha leads to improved rates of response in patients with chronic hepatitis C. Side effects often mandate downward dose adjustment or cessation of therapy, and the optimal dose of ribavirin has not been established. The aim of this study was to learn whether 600 mg ribavirin per day would prove as efficacious as 1,000-1,200 mg/day when combined with interferon (3 million units thrice weekly) for therapy of patients previously treated with standard interferon who had failed to respond or who had relapsed. We enrolled 69 patients with chronic hepatitis C and compensated liver disease: 45 were men, 65 were Caucasian, 48 were infected with genotype 1 hepatitis C virus. By random assignment, 35 received 600 mg ribavirin/day (group A), whereas the other 34 received 1,000 mg (< or = 75 kg body wt) or 1,200 mg/day (>75 kg body wt) (group B). At baseline, the two groups were well matched for demographic and laboratory features. In both groups, mean serum levels of alanine aminotransferase (ALT) and hepatitis C viral (HCV) RNA levels fell promptly and remained significantly lower than baseline throughout 24 weeks of therapy. There was no significant difference in mean levels of ALT or HCV RNA during therapy or at the end of follow-up (24 weeks after cessation of therapy). At the end of 24 weeks of posttherapy follow-up, 12 patients in each group had undetectable HCV RNA in serum, whereas 11 (31%) in group A and 9 (26.5%) in group B had normal serum ALT levels. The lower doses of ribavirin (group A) were tolerated better. In conclusion, in previous nonresponders or relapsers to interferon done, combination therapy with interferon-alpha2b (3 MU thrice weekly) + 600 mg ribavirin/day is tolerated better and is as effective as interferon plus higher (standard) doses of ribavirin (1,000-1,200 mg/day).

Urea and hypertonicity increase expression of heme oxygenase-1 in murine renal medullary cells.

Epithelial cells derived from the mammalian kidney medulla are responsive to urea at the levels of signal transduction and gene regulation. Hybridization of RNA harvested from control- and urea-treated murine inner medullary collecting duct (mIMCD3) cells with a cDNA expression array encoding stress-responsive genes suggested that heme oxygenase (HO)-1 mRNA was upregulated by urea. RNase protection assay confirmed this upregulation; hypertonicity also increased HO-1 mRNA expression but neither hypertonic NaCl nor urea were effective in the nonrenal 3T3 cell line. The effect on HO-1 expression appeared to be transcriptionally mediated on the basis of mRNA half-life studies and reporter gene analyses using the promoters of both human and chicken HO-1. Although urea signaling resembles that of heavy metal signaling in other contexts, the effect of urea on HO-1 transcription was independent of the cadmium response element in this promoter. Urea-inducible HO-1 expression was sensitive to antioxidants but not to scavengers of nitric oxide. Urea also upregulated HO-1 protein expression and pharmacological inhibition of HO-1 action with zinc protoporphyrin-sensitized mIMCD3 cells to the adverse effects of hypertonicity but not to urea. Coupled with the prior observation of others that HO-1 expression increases along the renal corticomedullary gradient, these data suggest that HO-1 expression may comprise an element of the adaptive response to hypertonicity and/or urea in renal epithelial cells.

Increased heme oxygenase activity in splanchnic organs from portal hypertensive rats: role in modulating mesenteric vascular reactivity.

BACKGROUND/AIMS: We have recently demonstrated that heme oxygenase-1 is upregulated in splanchnic organs of portal hypertensive rats. In the present study, we assessed whether heme oxygenase enzymatic activity is increased in splanchnic organs of portal hypertensive rats, and the relative contribution of heme oxygenase and nitric oxide synthase to the vascular hyporeactivity in portal hypertension. METHODS: Heme oxygenase activity was measured in splanchnic organs of portal hypertensive and sham-operated rats. The effects of heme oxygenase and nitric oxide synthase inhibition on pressure responses to potassium chloride and methoxamine were assessed in perfused mesenteric vascular beds of portal hypertensive and sham-operated rats. RESULTS: Heme oxygenase activity was increased in the mesentery, intestine, liver, and spleen of portal hypertensive rats. The hyporeactivity to potassium chloride in portal hypertensive rats was overcome after simultaneous inhibition of both heme oxygenase and nitric oxide synthase, but only partially attenuated after nitric oxide synthase inhibition alone. The hyporeactivity to methoxamine was completely reversed after nitric oxide synthase blockade. CONCLUSIONS: These results demonstrate that heme oxygenase activity is increased in splanchnic organs of portal hypertensive rats. They also suggest that heme oxygenase contributes to the hyporeactivity to potassium chloride, but not to methoxamine, in portal hypertensive rats.

Hepatitis C: a review and update.

UNLABELLED: The hepatitis C virus is an RNA virus that is a major cause of acute and chronic hepatitis. It is contracted chiefly through parenteral exposure to infected material such as blood transfusions or injections with dirty needles. Those at highest risk for development of hepatitis C are injection-drug users, people who snort cocaine with shared straws, and health care workers who are at risk for needle-stick and other exposures. Although the incidence of acute hepatitis C infection has fallen dramatically in the United States during the past decade, the prevalence of infection remains high (approximately 2.7 million Americans) because chronic hepatitis C develops in about 75% of those infected. Both acute and chronic hepatitis C are asymptomatic in most patients. However, chronic hepatitis C is a slowly progressive disease and results in severe morbidity in 20% to 30% of infected persons. Chronic hepatitis C is associated with a host of extrahepatic manifestations, many of which may be seen by dermatologists. The most frequent of these are mixed cryoglobulinemia with leukocytoclastic vasculitis and porphyria cutanea tarda. (J Am Acad Dermatol 2001;44:159-79.) LEARNING OBJECTIVE: At the conclusion of this learning activity, participants should be familiar with the essentials of the virology of the hepatitis C virus and the major features of the human diseases caused by hepatitis C viral infection; the extrahepatic manifestations of hepatitis C viral infection, with particular emphasis upon dermatologic manifestations, including leukocytoclastic vasculitis, porphyria cutanea tarda, and lichen planus; and the current methods of management of hepatitis C and its extrahepatic manifestations.

Induction of heme oxygenase-1 by phenylarsine oxide. Studies in cultured primary liver cells.

Heme oxygenase-1, the major inducible isoform of heme oxygenase (HO), can be induced by heme and numerous other physical and chemical factors, many of which cause cellular 'stress'. This has led to the realization that HO-1 is a major highly conserved stress or heat shock protein. Recent work has implicated activation of mitogen-activated protein kinases and other kinases in the mechanism of induction of HO-1, and suggested that signal transduction pathways through tyrosine kinases are involved in induction of HO-1 gene expression by stress inducers. We hypothesized that phenylarsine oxide (PAO), an inhibitor of protein tyrosine phosphatases (PTPs), might up-regulate the HO-1 gene. Here, we show that a remarkably brief (1-15 min) exposure of normal hepatocytes to low concentrations (0.5-3 microM) of PAO produces a marked increase in mRNA and protein of HO-1. This increase is comparable to the level obtained by addition of heme (20 microM), and occurs without producing changes in cellular glutathione levels or stabilization of HO-1 message. Preincubation of cells with inhibitors of protein synthesis decreased the ability of PAO to increase levels of HO-1 mRNA, suggesting that the inductive effect requires de novo protein synthesis. Addition of thiol donors abrogated the PAO-mediated induction of HO-1 in a dose dependent fashion. Addition of genistein, a tyrosine kinase inhibitor, blunted the induction produced by both PAO and heme. After brief incubations with PAO or heme, cell extracts showed comparable increases in levels of protein tyrosine phosphorylation in general, and specifically in ZAP70 kinase. Our results are consistent with the proposition that induction of HO-1 by PAO involves inhibition of specific PTP(s), and that the mechanisms of induction of HO-1 by PAO and by heme may share some common pathways.

Zebrafish dracula encodes ferrochelatase and its mutation provides a model for erythropoietic protoporphyria.

Exposure to light precipitates the symptoms of several genetic disorders that affect both skin and internal organs. It is presumed that damage to non-cutaneous organs is initiated indirectly by light, but this is difficult to study in mammals. Zebrafish have an essentially transparent periderm for the first days of development. In a previous large-scale genetic screen we isolated a mutation, dracula (drc), which manifested as a light-dependent lysis of red blood cells [1]. We report here that protoporphyrin IX accumulates in the mutant embryos, suggesting a deficiency in the activity of ferrochelatase, the terminal enzyme in the pathway for heme biosynthesis. We find that homozygous drc(m248) mutant embryos have a G-->T transversion at a splice donor site in the ferrochelatase gene, creating a premature stop codon. The mutant phenotype, which shows light-dependent hemolysis and liver disease, is similar to that seen in humans with erythropoietic protoporphyria, a disorder of ferrochelatase.

Management of recurrent hepatitis C after liver transplantation: a concise review.

Recurrent hepatitis C infection and subsequent graft failure are increasingly recognized problems after orthotopic liver transplantation. Although many prospective therapeutic, controlled trials in primary hepatitis C disease have been reported, large-scale studies are yet to be performed in patients with posttransplant recurrent hepatitis C after liver transplantation. In this review, we summarize the current literature on the therapeutic approaches for recurrent hepatitis C and discuss the results of published studies on therapy with ribavirin or interferon (IFN) alone and on combination therapy with IFN plus ribavirin. Further, we discuss results of prophylactic approaches to the problem of recurrent hepatitis C after transplant. Finally, we discuss additional aspects of anti-hepatitis C virus therapy after liver transplantation.

Upstream regulatory elements in chick heme oxygenase-1 promoter: a study in primary cultures of chick embryo liver cells.

Previously, chick heme oxygenase-1 (cHO-1) gene was cloned by us and two regions important for induction by sodium arsenite were identified. These two regions were found to contain consensus sequences of an AP-1 (-1580 to -1573) and a MRE/cMyc complex (-52 to -41). In the current study, the roles of these two elements in mediating the sodium arsenite or cobalt chloride dependent induction of cHO-1 were investigated further. DNA binding studies and site-directed mutagenesis studies indicated that both the AP-1 and MRE/cMyc elements are important for the sodium arsenite induction, while cobalt chloride induction involves only the AP-1 element. Electrophoretic mobility shift assays showed that nuclear protein binding to the AP-1 element was increased by both sodium arsenite or cobalt chloride treatment, whereas the binding of proteins to the MRE/cMyc element showed a high basal expression in untreated cells and the binding activity was only slightly increased by sodium arsenite treatment. Site-directed mutagenesis studies showed that, to completely abolish sodium arsenite induction, both the AP-1 and MRE/cMyc elements must be mutated; mutation of either element alone resulted in only a partial effect. In contrast, a single mutation at AP-1 element was sufficient to reduce the cobalt chloride induction almost completely. The MRE/cMyc complex plays a major role in the basal level expression, and shares some similarities to the upstream stimulatory factor element (USF) identified in the promoter regions of mammalian HO-1 genes and other stress regulated genes. Because sodium arsenite is known to cause oxidative stress and because activation of AP-1 proteins has been shown to be a key step in the oxidative stress response pathway, we also explored the possibility that the induction of the cHO-1 gene by sodium arsenite is mediated through oxidative stress pathway(s) by activation of AP-1 proteins. We found that pretreatment with antioxidants (N-acetyl cysteine or quercetin) reduced the induction of the endogenous cHO-1 message or cHO-1 reporter construct activities induced by sodium arsenite or cobalt chloride. These antioxidants also reduced the protein binding activities to the AP-1 element in the electrophoretic mobility shift assays. In summary, induction of the cHO-1 gene by sodium arsenite or cobalt chloride is mediated by activation of the AP-1 element located at -1,573 to -1,580 of the 5'UTR.

Outcomes research in chronic viral hepatitis C: effects of interferon therapy.

Although chronic hepatitis C (CHC) is often mild and asymptomatic, it may lead to decompensated hepatic cirrhosis and death. CHC is now the single most important indication for liver transplantation in North America. CHC is also an important cause of morbidity. Recent work is reviewed and shows that the health-related quality of life (HRQOL) of patients with CHC is markedly reduced compared with that of age- and sex-matched controls. For as yet unknown reasons, this reduction is more severe in CHC patients than in those with chronic hepatitis B. Successful therapy of CHC with type 1 interferons (IFNs) leads to substantial improvement - to nearly normal levels - in patients' HRQOL. In addition, IFN or IFN plus ribavirin therapy for CHC is highly cost effective, despite its limited long term efficacy; estimates of the costs to gain one year of quality-adjusted life range from no cost (that is, therapy is cost saving) to US$11,400 (for those most difficult to cure). Thus, despite the limited effectiveness of current therapies for CHC, they are fully justified based on their beneficial effects on patients' HRQOL and their cost effectiveness.

Induction of the heme oxygenase-1 gene by metalloporphyrins.

Induction of expression of heme oxygenase-1 (HO-1) has been studied in primary cultures of chick embryo liver cells and in the LMH line of avian hepatoma cells. Cells were transiently transfected with selected constructs containing portions of the 5'-untranslated (promoter) region of the HO-1 gene linked to luciferase as reporter gene. LMH cells that had been stably transfected with selected wild type or mutant constructs were also studied. Metalloporphyrins, especially Fe protoporphyrin (heme) and Co protoporphyrin strongly induced luciferase expression in both types of transfected cells. Low concentrations of Zn mesoporphyrin, an inhibitor of HO activity, exerted a synergistic effect on heme-, but not Co protoporphyrin-dependent induction. The antioxidant and &bond;SH donor N-acetyl cysteine had little effect on the metalloporphyrin-dependent inductions of HO-1, in contrast to its marked inhibitory effect on the sodium arsenite-dependent induction of the HO-1 gene. Deletional analysis showed that the key element(s) required for the metalloporphyrin-dependent induction of HO-1 is located between -3.6 and -5.6 kb upstream of the transcription starting point. Data from electrophoretic mobility shift and site-directed mutagenesis experiments excluded a role for consensus AP-1 binding elements at -1576, -3647, or -4578 in the inductions produced by heme or Co protoporphyrin.