Differences in chromatin from normal and leukemic human cells as shown by digestion with restriction nucleases

Sequence homology was found by computer analysis between potato spindle tuber viroid (PSTV) RNA and U3B snRNA of Novikoff hepatoma cells. This homology is colinear in arrangement, extends in length to 81% of the entire U3B snRNA molecule and is involved in the PSTV molecule unique sites which, if depicted in terms of the secondary structure of the circular PSTV molecule, reveal a conspicuous regularity in their location. A strong relation in primary structure between PSTV and U3B snRNA is demonstrated by statistical analysis.     

Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. Lightinduced herbicidal damage is mediated by the photoactive porphyrin. Here we investigate whether diphenyl ether herbicides can affect porphyrin synthesis in rat and chick hepatocytes. In rat hepatocyte cultures, protoporphyrin, as well as coproporphyrin, accumulated after treatment with acifluorfen or acifluorfen methyl. Combination of acifluorfen methyl with an esterase inhibitor to prevent the conversion of acifluorfen methyl to acifluorfen resulted in a greater accumulation of porphyrins than caused by acifluorfen methyl or acifluorfen alone. In vitro enzyme studies of hepatic mitochondria isolated from rat and chick embryos demonstrated that protopor-phyrinogen oxidase, the penultimate enzyme of heme biosynthesis, was inhibited by low concentrations of acifluorfen, nitrofen, or acifluorfen methyl with the latter being the most potent inhibitor. These findings indicate that diphenyl ether treatment can cause protoporphyrin accumulation in rat hepatocyte cultures and suggest that this accumulation was associated with the inhibition of protoporphyrinogen oxidase. In cultured chick embryo hepatocytes, treatment with acifluorfen methyl plus an esterase inhibitor caused massive accumulation of uroporphyrin rather than protoporphyrin or coproporphyrin. Specific isozymes of cytochrome P450 were also induced in chick embryo hepatocytes. These effects were not observed in the absence of an esterase inhibitor. These results suggest that diphenyl ether herbicides can cause uroporphyrin accumulation similar to that induced by other cytochrome P450-inducing chemicals such as polyhalogenated aromatic hydrocarbons in the chick hepatocyte system.     

Antioxidant inhibition of oxygen radicals for measurement of total antioxidant capacity in biological samples

Few methods for assessing total antioxidant capacity (TAC) include both the percentage of inhibition and the length of inhibition in the measurement. Available methods require above ambient constant temperature incubation, reaction preheating, and/or separate assays for testing hydrophilic and hydrophobic samples. We describe a high-throughput method, antioxidant inhibition of oxygen radicals (AIOR), that overcomes these difficulties. AIOR uses peroxyl radicals to trigger a decrease in fluorescence of the indicator molecule, uroporphyrin I, which is delayed by the presence of antioxidants. The area under the curve is measured by a fluorescence spectrophotometer in a 96-well microplate format, and TAC results are expressed as millimole/liter Trolox equivalents. AIOR is performed at ambient temperature and is applicable to samples in either aqueous or common organic solvents. The reaction between uroporphyrin I and the peroxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) was found to be of first-order kinetics with a mean rate constant (k) of 0.0254. Applications to measure antioxidant capacity are demonstrated on individual chemicals and biological samples. The method has good linearity, within- and between-assay precision, and recovery.     

Absolute requirement for iron in the development of chemically induced uroporphyria in mice treated with 3-methylcholanthrene and 5-aminolevulinate

Accumulating evidence, including experiments using cytochrome P450 1a2 (Cyp1a2) gene knock-out mice (Cyp1a2(−/−)), indicates that the development of chemically induced porphyria requires the expression of CYP1A2. It has also been demonstrated that iron enhances and expedites the development of experimental uroporphyria, but that iron alone without CYP1A2 expression, as in Cyp1a2(−/−) mice, does not cause uroporphyria. The role of iron in the development of porphyria has not been elucidated. We examined the in vivo effect of iron deficiency on hepatic URO accumulation in experimental porphyria. Mice were fed diets containing low (iron-deficient diet (IDD), 8.5 mg iron/kg) or normal (normal diet (ND), 213.7 mg iron/kg) levels of iron. They were treated with 3-methylcholanthrene (MC), an archetypal inducer of CYP1A, and 5-aminolevulinate (ALA), precursors of porphyrin and heme. We found that uroporphyrin (URO) levels and uroporphyrinogen oxidation (UROX) activity were markedly increased in ND mice treated with MC and ALA, while the levels were not raised in IDD mice with the same treatments. CYP1A2 levels and methoxyresorufin O-demethylase (MROD) activities, the CYP1A2-mediated reaction, were markedly induced in the livers of both ND and IDD mice treated with MC and ALA. UROX activity, supposedly a CYP1A2-dependent activity, was not enhanced in iron-deficient mice in spite of the fact of induction of CYP1A2. We showed that a sufficient level of iron is essential for the development of porphyria and UROX activity.     

Leishmania spp.: delta-aminolevulinate-inducible neogenesis of porphyria by genetic complementation of incomplete heme biosynthesis pathway

To further develop the Leishmania model for porphyria based on their deficiencies in heme biosynthesis, three Old World species were doubly transfected as before for Leishmania amazonensis with cDNAs, encoding the 2nd and 3rd enzymes in the pathway. Expression of the transgenes was verified immunologically at the protein level and functionally by uroporphyrin neogenesis that occurs only after exposure of the double-transfectants to delta-aminolevulinate. All species examined were equally deficient in heme biosynthesis, as indicated by the accumulation of uroporphyrin as the sole porphyrin and the production of coproporphyrin upon further transfection of one representative species with the downstream gene. The results obtained thus demonstrate that at least the first five enzymes for heme biosynthesis are absent in all species examined, rendering their transfectants inducible with aminolevulinate to accumulate porphyrins and thus useful as cellular models for human porphyrias.     

Ferritin accumulation and uroporphyrin crystal formation in hepatocytes of C57BL/10 mice: a time-course study

To establish the time-sequence relationship between ferritin accumulation and uroporphyrin crystal formation in livers of C57BL/10 mice, a biochemical, morphological and morphometrical study was performed. Uroporphyria was induced by the intraperitoneal administration of hexachlorobenzene plus iron dextran and of iron dextran alone. Uroporphyrin crystal formation started in hepatocytes of mice treated with hexachlorobenzene plus iron dextran at 2 weeks and in mice treated with iron dextran alone at 9 weeks. In the course of time, uroporphyrin crystals gradually increased in size. Uroporphyrin crystals were initially formed in hepatocytes in the periportal areas of the liver, in which also ferric iron staining was first detected. The amount and the distribution of the main storage form of iron in hepatocytes, ferritin, did not differ between the two treatment groups. Ferritin accumulation preceded the formation of uroporphyrin crystals in hepatocytes in both treatment groups. Moreover, uroporphyrin crystals were nearly always found close to ferritin iron. We conclude that uroporphyrin crystals are only formed in hepatocytes in which also iron (ferritin) accumulates. Hexachlorobenzene accelerates the effects of iron in porphyrin metabolism, but does not influence the accumulation of iron into the liver.     

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.