Richard W. Lambrecht

Role of Bach1 and Nrf2 in up-regulation of the heme oxygenase-1 gene by cobalt protoporphyrin

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO‐1 is highly inducible by a large number of physical and chemical factors. CoPP is known to be a potent and effective inducer of HO‐1 activity in many tissues. Here we report that CoPP up‐regulates HO‐1 via Bach1 and Nrf2 in human liver cells. CoPP did not influence hepatic Bach1 or Nrf2 mRNA levels, but markedly reduced Bach1 protein levels by increasing degradation of Bach1 protein (t1/2 from 19 h to 2.8 h), and increased Nrf2 by decreasing degradation of Nrf2 protein (t1/2 from 2.5 h to 9 h). Silencing Bach1 by Bach1‐siRNA significantly increased levels of HO‐1 mRNA and protein, and addition of CoPP up‐regulated HO‐1 mRNA and protein further. However, silencing Nrf2 mRNA by Nrf2‐siRNA did not significantly change baseline HO‐1 mRNA or protein levels, but significantly decreased 5–10 µM CoPP‐mediated up‐regulation of HO‐1 mRNA levels compared with CoPP alone. Transfection with equal amounts of non‐Bach1 or non‐Nrf2 related control siRNA did not reduce Bach1 or Nrf2 mRNA or protein, confirming the specificity of Bach1‐ and Nrf2‐siRNA in Huh‐7 cells. We conclude that the pathway of CoPP‐mediated induction of HO‐1 involves the repression of Bach1 and up‐regulation of the Nrf2 protein by posttranscriptional site(s) of action. Because CoPP, unlike heme, is neither a prooxidant nor a substrate for HO‐1, it might be considered as a potential therapeutic agent in situations where up‐regulation of HO‐1 is desired.—Shan, Y., Lambrecht, R. W., Donohue, S. E., Bonkovsky, H. L. Role of Bach1 and Nrf2 in up‐regulation of the heme oxygenase‐1 gene by cobalt protoporphyrin. FASEB J. 20, E2258–E2267 (2006)

Iron as a co-morbid factor in nonhemochromatotic liver disease

Heavy iron overload, in both primary and secondary hemochromatosis, may cause fibrosis of parenchymal organs, especially the liver. The toxicity of iron is believed to involve increased oxidative stress, with iron-catalyzed production of reactive oxygen species causing oxidative damage to lipids, proteins, and nucleic acids. Lesser degrees of hepatic iron deposition are also associated with, and seem to be risk factors for, certain nonhemochromatotic liver diseases. Porphyria cutanea tarda is associated with hepatic iron overload and responds to iron-reduction therapy. Results of recent studies have demonstrated high prevalences (about 60%-80%) of HFE gene mutations in patients with porphyria cutanea tarda. Chronic hepatitis C is another risk factor for porphyria cutanea tarda. Other recent evidence indicates that the prevalence of HFE gene mutations is increased in chronic viral hepatitis and that patients with chronic hepatitis C harboring especially the C282Y mutation are more likely to suffer from advanced hepatic fibrosis or cirrhosis and to do so at younger ages. A role for modest iron overload in increasing severity of alcohol-induced liver disease has been well established from results of experimental studies. However, it is currently unresolved whether mild-to-moderate hepatic iron deposition or heterozygosity for the C282Y mutation plays a role in human alcoholic liver disease or in nonalcoholic fatty liver disease or nonalcoholic steatohepatitis. There is persuasive evidence that iron reduction decreases insulin resistance, and it likely also decreases oxidative stress, two key pathogenic features of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Iron loading has also been described after portosystemic shunts and in end-stage liver disease.

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.

IRON-INDUCED LIVER INJURY

Iron, either in the form of heme or non-heme compounds, is essential to life, but it can also pose serious health risks. The liver is a principal target for iron toxicity because it is chiefly responsible for taking up and storing excessive amounts of iron. The major hepatic toxicities of iron overload include damage to multiple cell types (hepatocytes, Kupffer cells, hepatic stellate cells) and to multiple subcellular organelles (mitochondria, lysosomes, and smooth endoplasmic reticulum). Heavy iron overload, as occurs in primary (hereditary) or secondary forms of hemochromatosis, may cause cirrhosis, liver failure, and hepatocellular carcinoma. In addition, iron has been shown to be a contributory factor in the development or progression of alcoholic liver disease, nonalcoholic liver steatohepatitis, chronic viral hepatitis, prophyria cutanea tarda, and, perhaps, in alpha 1-antitrypsin deficiency and end-stage liver disease, regardless of cause.

Iron Levels in Hepatocytes and Portal Tract Cells Predict Progression and Outcomes of Patients With Advanced Chronic Hepatitis C

BACKGROUND & AIMS Iron may influence severity and progression of non-hemochromatotic liver diseases. Our aim was to assess the relationship of iron and HFE genetic variations to progression and outcomes in the HALT-C Trial and whether PegIFN therapy influenced iron variables. METHODS Participants were randomized to receive long-term PegIFN [n = 400] or no therapy [n = 413] for 3.5 y, with follow-up for up to 8.7 y [median 6.0 y]. Associations of patient characteristics with iron variables at baseline and over time were carried out using Kaplan-Meier analyses, Cox regression models, and repeated measures analysis of covariance. RESULTS Participants who developed clinical outcomes [CTP > 7, ascites, encephalopathy, variceal bleeding, SBP, HCC, death] had significantly higher baseline scores for stainable iron in hepatocytes and in portal tract cells than those without. There were significant direct correlations between stainable iron in portal triads and lobular and total Ishak inflammatory and fibrosis scores [P < 0.0001]. Iron in triads at baseline increased risk of outcomes (HR = 1.35, P = 0.02). Stainable iron in hepatocytes decreased, whereas that in portal stromal cells increased significantly [P < 0.0001] over time. Serum iron and TIBC fell significantly over time [P < 0.0001], as did serum ferritin [P = 0.0003]. Chronic PegIFN treatment did not affect stainable iron. HFE genetic variations did not correlate with outcomes, including development of hepatocellular carcinoma. CONCLUSIONS Stainable iron in hepatocytes and portal tract cells is a predictor of progression and clinical and histological outcomes in advanced chronic hepatitis C. Chronic low-dose PegIFN therapy did not improve outcomes, nor iron variables.

Effects of new anticonvulsant medications on porphyrin synthesis in cultured liver cells: Potential implications for patients with acute porphyria

Some patients with acute hereditary porphyrias have seizures and require anticonvulsant therapy, but many anticonvulsants induce exacerbations of the hepatic porphyrias. Recently, several new anticonvulsants have become available. Among these are gabapentin, vigabatrin, felbamate, lamotrigine, and tiagabine. Little is known about their potential for induction of porphyric attacks. We used a cell culture model of primary chicken embryo liver cells, which maintain intact heme synthesis and regulation, to study the effects of these new anticonvulsants on porphyrin accumulation. Treatment of the cells with deferoxamine (250 µM) led to a partial block in heme synthesis, simulating the conditions encountered in human beings with porphyria. Concomitant exposure of these cells to phenobarbital (2 mM) strongly induced accumulation of porphyrins, serving as a positive control in this model. Cells were treated for 20 hours with increasing doses (3.2 to 1,000 µM) of the newer anticonvulsants, with or without deferoxamine. For most of these anticonvulsants 5 to 100 µM is representative of the concentrations achieved in humans with therapeutic doses. Porphyrins were measured spectrofluorometrically as uro-, copro-, and protoporphyrins. Results were confirmed by high-pressure liquid chromatography. Neither vigabatrin nor gabapentin treatment, with or without deferoxamine, led to any increase in porphyrin accumulation. Similar doses of felbamate (with deferoxamine) led to a marked increase in (mainly proto-) porphyrin levels, qualitatively and quantitatively almost identical to the accumulation produced by phenobarbital. Lamotrigine or tiagabine (with deferoxamine) caused similar porphyrin accumulation. Tiagabine treatment up to 100 µM(with deferoxamine) also resulted in very high levels of predominantly proto-porphyrin. In contrast to the other anticonvulsants tested, tiagabine without deferoxamine led to mild porphyrin accumulation. In the presence of deferoxamine, phenobarbital, felbamate, lamotrigine, or tiagabine, but not gabapentin or vigabatrin, increased levels of the mRNA of ALA synthase, the first and rate-controlling enzyme of porphyrin synthesis. Such enzyme induction is a sine qua non for acute porphyric attacks. We conclude that neither vigabatrin nor gabapentin is porphyrogenic, whereas felbamate, lamotrigine, and, especially, tiagabine lead to much accumulation of porphyrins. The latter three anticonvulsants, therefore, may precipitate or exacerbate acute porphyric attacks in humans. We recommend use of vigabatrin or gabapentin, but not felbamate, lamotrigine, or tiagabine, in patients with acute porphyria and seizures.

DNA polymorphisms and response to treatment in patients with chronic hepatitis C: results from the HALT-C trial.

BACKGROUND/AIMS Certain host genetic polymorphisms reportedly affect the likelihood of a sustained virological response (SVR) to interferon treatment in subjects infected with hepatitis C virus (HCV). As part of the HALT-C trial we evaluated genetic associations among patients infected with HCV genotype 1 who had failed previous interferon treatment. METHODS SVR was determined 24 weeks after completing treatment with pegylated interferon alfa-2a and ribavirin. Eight single nucleotide polymorphisms (SNPs) were selected on the basis of previously reported associations with treatment response. Genotypes were assessed by polymerase chain reaction-based assays. The percentage of patients who achieved SVR was determined for each genotype and for an IL10 promoter diplotype. RESULTS Among 637 non-Hispanic Caucasian patients there were no significant associations between genotype for any individual SNP (IL10-1082, IL10-592, TNF-308, TNF-238, TGFB1 codon 25, CCL2-2518, EPHX1 codon 113 and AGT-6) and SVR, but SVR was more common among the patients who were homozygous for the ACC IL10 promoter diplotype (adjusted odds ratio, 3.24; 95% confidence interval, 1.33-7.78; p=0.001). CONCLUSIONS Among non-Hispanic Caucasian patients treated with peginterferon and ribavirin after failing previous treatment with interferon, homozygosity for the ACC IL10 promoter diplotype was associated with SVR.

Association of Hepatitis C Virus Infection with Serum Iron Status: Analysis of Data from the Third National Health and Nutrition Examination Survey

BACKGROUND There is growing evidence that mildly increased amounts of iron in the liver can increase hepatic injury, particularly if combined with other hepatotoxic factors, such as alcohol use, use of porphyrogenic drugs, or chronic viral hepatitis. In the present study, the association of hepatitis C virus (HCV) infection with serum measurements of iron status was assessed in the US population. METHODS We analyzed data from a total of 14,462 participants in the Third National Health and Nutrition Examination Survey. We excluded subjects who were aged <12 years, subjects for whom measurements of serum levels of iron or ferritin or the results of liver function tests were missing, and subjects who had a serum transferrin saturation of > or =50% (to help exclude subjects with hemochromatosis). RESULTS Mean serum levels of ferritin and iron (+/- standard error) were significantly higher among subjects with HCV infection (100+/-3 ng/mL and 229+/-17 microg/dL, respectively) than among subjects without liver disease (83+/-0.3 ng/mL and 101+/-2.1 microg/dL, respectively) (P<.0001). Serum levels of ferritin were directly and significantly correlated with serum levels of alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyl transpeptidase (r=0.25, r=0.24, and r=0.28, respectively; P<.0001), whereas platelet counts were inversely correlated with serum levels of ferritin (r=-0.12; P<.0001). CONCLUSION HCV infection is significantly associated with higher serum levels of ferritin and iron in the US population.

Altered Folate Availability Modifies the Molecular Environment of the Human Colorectum: Implications for Colorectal Carcinogenesis

Low folate status increases colorectal cancer risk. Paradoxically, overly abundant folate supplementation, which is not uncommon in the United States, may increase risk. The mechanisms of these effects are unknown. We conducted two translational studies to define molecular pathways in the human colon altered either by folate supplementation or by dietary folate depletion (followed by repletion). In the first study, 10 healthy, at-risk volunteers (with documented stable/normal folate intake) received supplemental folic acid (1 mg/d) for 8 weeks. In the second study, 10 similar subjects were admitted to a hospital as inpatients for 12 weeks to study folate depletion induced by a low folate diet. A repletion regimen of folic acid (1 mg/d) was provided for the last 4 of these weeks. Both studies included an 8-week run-in period to ensure stabilized folate levels prior to intervention. We obtained 12 rectosigmoid biopsies (from 4 quadrants of normal-appearing mucosa 10–15 cm from the anal verge) at baseline and at measured intervals in both studies for assessing the primary endpoints: genome-wide gene expression, genomic DNA methylation, promoter methylation (depletion/repletion study only), and p53 DNA strand breaks. Serum and rectosigmoid folate concentrations accurately tracked all changes in folate delivery (P < 0.05). In the first study, gene array analysis revealed that supplementation upregulated multiple inflammation- and immune-related pathways in addition to altering several 1-carbon–related enzymes (P < 0.001). In the second study, folate depletion downregulated genes involved in immune response, inflammation, the cell cycle, and mitochondrial/energy pathways; repletion reversed most of these changes. However, changes in gene expression after repletion in the second study (involving immune response and inflammation) did not reach the levels seen after supplementation in the first study. Neither genomic nor promoter-specific DNA methylation changed during the course of the depletion/repletion protocol, and genomic methylation did not change with supplementation in the first study. p53 DNA strand breaks increased with depletion after 12 weeks. In sum, depletion downregulates, whereas repletion or supplementation upregulates pathways related to inflammation and immune response. These findings provide novel support to the concept that excessive folate supplementation might promote colorectal carcinogenesis by enhancing proinflammatory and immune response pathways. These results indicate that modest changes in folate delivery create substantial changes in the molecular milieu of the human colon. Cancer Prev Res; 4(4); 530–43. ©2011 AACR.

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.