Heme-a, the heme prosthetic group of cytochrome c oxidase, is increased in Alzheimer's disease.

Heme-a, is the heme prosthetic group of cytochrome c oxidase (COX), the terminal complex of the mitochondrial electron transport chain. We measured heme-a levels in postmortem brain tissue from nine patients diagnosed with dementia: Alzheimer's disease (AD) was the primary diagnosis in five, AD/diffuse Lewy body disease (DLBD) was diagnosed in two, DLBD was diagnosed in one, and DLBD (severe)/AD (mild) was diagnosed in one. Eight non-demented patients who died from non-neurological causes served as controls. When the primary diagnosis was AD (AD and AD/DLBD), levels of cerebral heme-a were increased almost two-fold on a protein basis compared to controls (p<0.001). Using perfused and non-perfused rats we showed that measured levels of cerebral heme-a were unaffected by the presence of blood in brain tissue. In mice we showed that levels of cerebral heme-a were unaffected by 24h of storage at 4 degrees C prior to freezing. These animal studies suggest that increased levels of cerebral heme-a in AD were not due to blood in postmortem brain or variation in postmortem interval.

Effect of an oral iron chelator or iron-deficient diets on uroporphyria in a murine model of porphyria cutanea tarda.

UNLABELLED: Porphyria cutanea tarda is a liver disease characterized by elevated hepatic iron and excessive production of uroporphyrin (URO). Phlebotomy is an effective treatment that probably acts by reducing hepatic iron. Here we used Hfe(-/-) mice to compare the effects on hepatic URO accumulation of two different methods of hepatic iron depletion: iron chelation using deferiprone (L1) versus iron-deficient diets. Hfe(-/-) mice in a 129S6/SvEvTac background were fed 5-aminolevulinic acid (ALA), which results in hepatic URO accumulation, and increasing doses of L1 in the drinking water. Hepatic URO accumulation was completely prevented at low L1 doses, which partially depleted hepatic nonheme iron. By histological assessment, the decrease in hepatic URO accumulation was associated with greater depletion of iron from hepatocytes than from Kupffer cells. The L1 treatment had no effect on levels of hepatic cytochrome P4501A2 (CYP1A2). L1 also effectively decreased hepatic URO accumulation in C57BL/6 Hfe(-/-) mice treated with ALA and a CYP1A2 inducer. ALA-treated mice maintained on defined iron-deficient diets, rather than chow diets, did not develop uroporphyria, even when the animals were iron-supplemented either directly in the diet or by iron dextran injection. CONCLUSION: The results suggest that dietary factors other than iron are involved in the development of uroporphyria and that a modest depletion of hepatocyte iron by L1 is sufficient to prevent URO accumulation.

Hexachlorobenzene stimulates uroporphyria in low affinity AHR mice without increasing CYP1A2.

Hexachlorobenzene (HCB), a weak ligand of the aryl hydrocarbon receptor (AHR), causes hepatic uroporphyrin (URO) accumulation (uroporphyria) in humans and animals. CYP1A2 has been shown to be necessary in the development of uroporphyria in mice. Using mice expressing the low affinity form of the AH receptor (AHRd), we investigated whether the enhancement of uroporphyria by HCB involves an obligatory increase in CYP1A2 as measured by specific enzyme assays and immunoblotting. We compared the ability of HCB, in combination with iron dextran and the porphyrin precursor, 5-aminolevulinate (ALA), to cause uroporphyria in a strain of mice (C57BL/6) which expresses the high affinity form of the receptor (AHRb(1)), with three strains of mice (SWR and two 129 sublines) expressing the low affinity AHRd. In C57BL/6 mice, HCB-enhanced uroporphyria was associated with a doubling of CYP1A2. HCB treatment produced uroporphyria in iron-loaded mice expressing AHRd, even though there was little or no increase in CYP1A2. Cyp1a2(-/-) mice in a 129 background were completely resistant to HCB-induced uroporphyria, and female Hfe(-/-) 129 mice, in which the levels of hepatic CYP1A2 were half of those of the male levels, responded poorly. The effect of exogenous iron, administered in the form of iron dextran, on HCB enhancement of uroporphryia could be replicated utilizing the endogenous hepatic iron accumulated in 129 Hfe(-/-) mice. In conclusion, some minimal basal expression of CYP1A2 is essential for HCB-mediated enhancement of uroporphyria, but increases in CYP1A2 above that level are not essential.

Role of CYP3A and CYP2E1 in alcohol-mediated increases in acetaminophen hepatotoxicity: comparison of wild-type and Cyp2e1(-/-) mice.

CYP2E1 is widely accepted as the sole form of cytochrome P450 responsible for alcohol-mediated increases in acetaminophen (APAP) hepatotoxicity. However, we previously found that alcohol [ethanol and isopentanol (EIP)] causes increases in APAP hepatotoxicity in Cyp2e1(-/-) mice, indicating that CYP2E1 is not essential. Here, using wild-type and Cyp2e1(-/-) mice, we investigated the relative roles of CYP2E1 and CYP3A in EIP-mediated increases in APAP hepatotoxicity. We found that EIP-mediated increases in APAP hepatotoxicity occurred at lower APAP doses in wild-type mice (300 mg/kg) than in Cyp2e1(-/-) mice (600 mg/kg). Although this result suggests that CYP2E1 has a role in the different susceptibilities of these mouse lines, our findings that EIP-mediated increases in CYP3A activities were greater in wild-type mice compared with Cyp2e1(-/-) mice raises the possibility that differential increases in CYP3A may also contribute to the greater APAP sensitivity in EIP-pretreated wild-type mice. At the time of APAP administration, which followed an 11 h withdrawal from the alcohols, alcohol-induced levels of CYP3A were sustained in both mouse lines, whereas CYP2E1 was decreased to constitutive levels in wild-type mice. The CYP3A inhibitor triacetyloleandomycin (TAO) decreased APAP hepatotoxicity in EIP-pretreated wild-type and Cyp2e1(-/-) mice. TAO treatment in vivo resulted in inhibition of microsomal CYP3A-catalyzed activity, measured in vitro, with no inhibition of CYP1A2 and CYP2E1 activities. In conclusion, these findings suggest that both CYP3A and CYP2E1 contribute to APAP hepatotoxicity in alcohol-treated mice.

Effect of iron and ascorbate on uroporphyria in ascorbate-requiring mice as a model for porphyria cutanea tarda.

UNLABELLED: Excess hepatic iron is known to enhance both porphyria cutanea tarda (PCT) and experimental uroporphyria. Since previous studies have suggested a role for ascorbate (AA) in suppressing uroporphyria in AA-requiring rats (in the absence of excess iron), the present study investigated whether AA could suppress uroporphyria produced by excess hepatic iron. Hepatic URO accumulation was produced in AA-requiring Gulo(-/-) mice by treatment with 3,3',4,4',5-pentachlorbiphenyl, an inducer of CYP1A2, and 5-aminolevulinic acid. Mice were administered either sufficient AA (1000 ppm) in the drinking water to maintain near normal hepatic AA levels or a lower intake (75 ppm) that resulted in 70 % lower hepatic AA levels. The higher AA intake suppressed hepatic URO accumulation in the absence of administered iron, but not when iron dextran (300-500 mg Fe/kg) was administered. This effect of iron was not due to hepatic AA depletion since hepatic AA content was not decreased. The effect of iron to prevent AA suppression of hepatic URO accumulation was not observed until a high hepatic iron threshold was exceeded. At both low and high AA intakes, hepatic malondialdehyde (MDA), an indicator of oxidative stress, was increased three-fold by high doses of iron dextran. MDA was considerably increased even at low iron dextran doses, but without any increase in URO accumulation. The level of hepatic CYP1A2 was unaffected by either AA intake. CONCLUSION: In this mouse model of PCT, AA suppresses hepatic URO accumulation at low, but not high hepatic iron levels. These results may have implications for the management of PCT.

Effect of insulin and glucagon on accumulation of uroporphyrin and coproporphyrin from 5-aminolevulinate in hepatocyte cultures.

Primary cultures of chick embryo hepatocytes have been used to study the mechanisms by which various drugs and other chemicals cause accumulation of porphyrin intermediates of the heme pathway. When these cultures are incubated with the heme precursor, 5-aminolevulinic acid (ALA), there is a major accumulation of protoporphyrin. However, in the presence of ALA, addition of insulin caused a striking increase in accumulation of uroporphyrin I and coproporphyrin III, whereas addition of glucagon mainly caused an increase in uroporphyrin I. Treatment with both insulin and glucagon resulted in additive increases in uroporphyrin, but not coproporphyrin. Antioxidants abolished the uroporphyrin I accumulation and increased coproporphyrin III. Insulin caused an increase in uptake of ALA and an increase in porphobilinogen accumulation, suggesting that the accumulation of uroporphyrin I is due to increased flux through the heme pathway. Apparently, this increased flux could particularly affect the utilization of the intermediate hydroxymethylbilane, which would result in accumulation of uroporphyrin I.

Genetic factors influence ethanol-induced uroporphyria in Hfe(-/-) mice.

Two major risk factors for porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). We recently described an animal model for alcohol-induced uroporphyria, using Hfe(-/-) mice. In the present study we show that this effect is dependent on genetic background and ethanol dose. In the 129S6/SvEvTac (129) strain, treatment with 15% ethanol in the drinking water for 6.5 months produced an accumulation of hepatic uroporphyrin (URO) 4-fold higher than that observed with 10% ethanol, a 90% decrease in uroporphyrinogen decarboxylase activity (UROD), and further increased the activities of hepatic 5-aminolevulinate synthase (ALAS) and CYP1A2. Hepatic nonheme iron (NHFe) and hepatocyte iron staining were not further increased by 15% compared to 10% ethanol. Treatment of C57BL/6 Hfe(-/-) mice with 15% ethanol for 6.5 months did not increase hepatic URO. Although NHFe was increased by ethanol, the resulting level was only half that of ethanol-treated 129 Hfe(-/-) mice. ALAS induction was similar in both Hfe(-/-) strains. In wild-type 129 mice treated with ethanol for 6 to 7 months, administration of iron dextran increased hepatic URO accumulation and decreased UROD activity. In conclusion, this study demonstrates a strong effect of genetic background on ethanol-induced uroporphyria, which is probably due to a greater effect of ethanol on iron metabolism in the susceptible strain.

High-throughput non-heme iron assay for animal tissues.

Iron has been widely studied in nearly every realm of biology. However, current methodologies, such as genetic mapping or mutation screening, have been difficult to apply due to the lack of robust high-throughput methods for quantifying iron levels from cells or tissues. The measurement of total iron levels in tissues, usually done with atomic absorption spectroscopy, is impractical for large numbers of samples and includes the contribution of heme iron from hemoglobin contained in red blood cells. The measurement of non-heme iron by reaction with a bathophenanthroline reagent, a commonly used assay reported more than 30 years ago, is also not feasible for large-scale analyses because it is cuvette-based. We therefore have modified this method to a microplate format that will facilitate large-scale analysis. The microplate assay is highly sensitive and specific, and is a simple and effective method for the measurement of non-heme iron for animal tissues that will enable the application of high-throughput of genetic methodologies.

Cyp1a1(-/-) male mice: protection against high-dose TCDD-induced lethality and wasting syndrome, and resistance to intrahepatocyte lipid accumulation and uroporphyria.

To study liver toxicity and uroporphyrin (URO) accumulation and urinary excretion, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent ligand for the aryl hydrocarbon receptor (AHR), is often used as the prototype. In this study, we asked the question how important is the role of CYP1A1 in causing TCDD toxicity. Using a single large intraperitoneal dose of TCDD (200 microg/kg) and following the response over an 8-week period, we found this dose: (a) was lethal in less than 4 weeks to Cyp1a1(+/+) males but not to Cyp1a1(-/-) males or to females of either genotype; (b) caused a wasting syndrome in Cyp1a1(+/+) but not Cyp1a1(-/-) mice; (c) resulted in thymic atrophy, regardless of gender or genotype; (d) decreased spleen size and caused leukocytopenia in males but not females of either genotype; (e) caused hepatocyte hypertrophy in Cyp1a1(+/+) more so than in Cyp1a1(-/-) mice; (f) increased intrahepatocyte lipids and total liver fat content in Cyp1a1(+/+) more than Cyp1a1(-/-) males and females; and (g) caused uroporphyria in Cyp1a1(+/+) males much more than Cyp1a1(+/+) females, or in Cyp1a1(-/-) mice. Contrary to Cyp1a2(-/-) knockout mice that exhibited 15 times less accumulation of TCDD in liver than Cyp1a1/1a2(+/+) wild-type mice, Cyp1a1(-/-) mice did not show this altered TCDD distribution-indicating that CYP1A2 but not CYP1A1 is the major hepatic TCDD-binding "sink". Our data demonstrate that CYP1A1 contributes to high-dose TCDD-induced toxicity, uroporphyria, and lethality.

A mechanism of oxygen sensing in yeast. Multiple oxygen-responsive steps in the heme biosynthetic pathway affect Hap1 activity.

Heme plays central roles in oxygen sensing and utilization in many living organisms. In yeast, heme mediates the effect of oxygen on the expression of many genes involved in using or detoxifying oxygen. However, a direct link between intracellular heme level and oxygen concentration has not been vigorously established. In this report, we have examined the relationships among oxygen levels, heme levels, Hap1 activity, and HAP1 expression. We found that Hap1 activity is controlled in vivo by heme and not by its precursors and that heme activates Hap1 even in anoxic cells. We also found that Hap1 activity exhibits the same oxygen dose-response curves as Hap1-dependent aerobic genes and that these dose-response curves have a sharp break at approximately 1 microM O2. The results show that the intracellular signaling heme level, reflected as Hap1 activity, is closely correlated with oxygen concentration. Furthermore, we found that bypass of all heme synthetic steps but ferrochelatase by deuteroporphyrin IX does not circumvent the need for oxygen in Hap1 full activation by heme, suggesting that the last step of heme synthesis, catalyzed by ferrochelatase, is also subjected to oxygen control. Our results show that multiple heme synthetic steps can sense oxygen concentration and provide significant insights into the mechanism of oxygen sensing in yeast.

Uroporphyrin accumulation in hepatoma cells expressing human or mouse CYP1A2: relation to the role of CYP1A2 in human porphyria cutanea tarda.

In experimental animals, CYP1A2 is absolutely required for the development of uroporphyria induced by treatment with polyhalogenated aromatic compounds or other compounds. Although the role of this CYP in clinical uroporphyria, porphyria cutanea tarda (PCT), is not clear, Cyp1a2(-/-) mice are resistant to the development of uroporphyria. Here, we compared the abilities of human and mouse CYP1A2 expressed in mouse hepatoma Hepa-1 cells to: (i) catalyze CYP1A2-dependent methoxyresorufin demethylase (MROD), and (ii) support uroporphyrin (URO) accumulation. Both CYP1A2 orthologs were expressed at similar levels as indicated by immunodetectable CYP1A2 proteins and MROD activities. URO accumulation was increased in cultures expressing either ortholog when supplemented with 5-aminolevulinic acid, the porphyrin precursor. Cells expressing mouse CYP1A2 produced more URO than cells expressing human CYP1A2. The results indicate that human CYP1A2 can support URO accumulation in hepatoma cells and thus may play a role in human PCT.

Uroporphyria caused by ethanol in Hfe(-/-) mice as a model for porphyria cutanea tarda.

Two major risk factors for the development of porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). To develop an animal model, Hfe knockout mice were treated continuously with 10% ethanol in drinking water. By 4 months, uroporphyrin (URO) was detected in the urine. At 6 to 7 months, hepatic URO was increased and hepatic uroporphyrinogen decarboxylase (UROD) activity was decreased. Untreated Hfe(-/-) mice or wild-type mice treated with or without ethanol did not show any of these biochemical changes. Treatment with ethanol increased hepatic nonheme iron and hepatic 5-aminolevulinate synthase activity in Hfe(-/-) but not wild-type mice. The increases in nonheme iron in Hfe(-/-) mice were associated with diffuse increases in iron staining of parenchymal cells but without evidence of significant liver injury. In conclusion, the results of this study suggest that the uroporphyrinogenic effect of ethanol is mediated by its effects on hepatic iron metabolism. Ethanol-treated Hfe(-/-) mice seem to be an excellent model for studies of alcohol-mediated PCT.

Uroporphyria in mice: thresholds for hepatic CYP1A2 and iron.

In mice treated with 5-aminolevulinic acid (ALA) and polyhalogenated aromatic compounds, the levels of both hepatic cytochrome P450 (CYP)1A2 and iron-which can be quite different among inbred strains-are critical in causing experimental uroporphyria. Here we investigate the development of uroporphyria as a function of CYP1A2 and iron levels in the liver of mice having a common C57BL/6 genetic background. We compared Cyp1a2(-/-) knockout mice, Cyp1a2(+/-) heterozygotes, Cyp1a2(+/+) wild type, and Cyp1a2(+/+) mice pretreated with a low dose of 3,3',4,4',5-pentachlorobiphenyl (PCB126) (4 microg/kg). Cyp1a2(+/-) mice contain about 60% of the hepatic CYP1A2 content of Cyp1a2(+/+) mice, and the PCB126-pretreated Cyp1a2(+/+) mice have about twice the wild-type levels of CYP1A2. ALA- and iron-treated Cyp1a2(+/+) mice are known to accumulate hepatic uroporphyrin; this accumulation was increased 7-fold by pretreatment with the low dose of PCB126. ALA- and iron-treated Cyp1a2(+/-) heterozygote mice accumulated no uroporphyrin in 4 weeks, but by 8 weeks accumulated significant amounts of uroporphyrin. As previously reported, the ALA- and iron-treated Cyp1a2(-/-) knockout mouse has no CYP1A2 and exhibits no detectable uroporphyrin accumulation. Iron dose-response curves in ALA- and PCB126-treated Cyp1a2(+/+) mice showed that hepatic iron levels greater than 850 microg/g liver were required to produce significant uroporphyrin accumulation in the liver. Other measures of hepatic effects of iron (iron-response element-binding protein [IRP]-iron response element [IRE] binding activity and accumulation of protoporphyrin from ALA) decreased when the level of iron was considerably lower than 850 microg/g liver. At low iron doses, accumulation of iron was principally in Kupffer cells, whereas at the higher doses (required to stimulate uroporphyrin accumulation), more iron was found in parenchymal cells. We conclude that small changes in hepatic CYP1A2 levels can dramatically affect uroporphyria in C57BL/6 mice, providing the animals have been sufficiently loaded with iron; these data might be clinically relevant to acquired (sporadic) porphyria cutanea tarda, because humans show greater than 60-fold genetic differences in hepatic basal CYP1A2.