Alcira M. Del C. Batlle

Bilirubin : Its role in cytoprotection against oxidative stress

Bilirubin, the end product of heme catabolism in mammals, is generally regarded as a potentially cytotoxic, lipid-soluble waste product that needs to be excreted. However, in the last 10 years, in vitro and in vivo studies, have demonstrated that bilirubin exhibits potent anti-oxidant properties preventing the oxidative damage triggered by a wide range of oxidant-related stimuli. Therefore, the idea of a beneficial and physiological role for bilirubin in cytoprotection against short and long-lasting oxidant-mediated cell injury is highlighted here.

In vivo protection by melatonin against δ-aminolevulinic acid-induced oxidative damage and its antioxidant effect on the activity of haem enzymes

Princ FG, Maxit AG, Cardalda C, Batlle A, Juknat AA. In vivo protection by melatonin against δ‐aminolevulinic acid‐induced oxidative damage and its antioxidant effect on the activity of haem enzymes. J. Pineal Res. 1998; 24:1–8.

A simple method for measuring erythrocyte porphobilinogenase, and its use in the diagnosis of acute intermittent porphyria

Abstract 1. l. A simple method for measuring erythrocyte porphobilinogenase activity has been devised. 2. 2. Red blood cells are hemolysed by three freeze-thaws, the hemolysate is diluted 15 times and only 0.5 ml of this preparation is used for determining porphobilinogenase. in an incubation system also containing porphobilinogen, NaCl. MgCl 2 . Tris-HCl buffer pH 8.2; which is incubated aerobically, in the dark. at 45°. for 1 hr with shaking. 3. 3. The enzymic activity is quenched by adding TCA. 4. 4. Porphyrinogens are oxidized, protein precipitated centrifuged off and uroporphyrins formed are determined spectrophotometrically in the supernatant. 5. 5. The enzyme is stable up to 1 month if stored at —20°.

Kinetics of porphyrin accumulation in cultured epithelial cells exposed to ALA

1. The kinetics of porphyrin accumulation in cultured mammalian epithelial cells (CNCM-I-221) during exposure to ALA was investigated. 2. The total porphyrin synthesized is a function of ALA concentration and the incubation time. The cellular porphyrin content exhibited a saturation pattern, reaching a plateau at about 0.04 fmol porphyrins/cell. A biphasic time-dependent increase in the total porphyrin synthesized was observed. 3. After 3 hr of exposure to ALA the rate of synthesis increased to almost twice the initial rate, reaching between 0.02 and 0.05 fmol porphyrins/cell/hr depending on serum concentration in the medium. 4. Two effects of FBS on ALA-stimulated porphyrin accumulation were observed. Greater total porphyrin synthesis was found when incubations were made in 10% FBS compared to those in 1% FBS. 5. The higher serum concentration also caused a greater release into the medium of the porphyrins generated in the cells with a calculated half-life of 24 min in 10% serum-supplemented medium compared with 62 min in 1% serum. 6. The results obtained from cell synchronization experiments suggest that there is little obvious cell cycle-dependent variation in the synthesis of porphyrins from ALA. 7. The small differences in the intracellular porphyrin content that were observed may be attributed to a slight reduction in the rate of loss of porphyrins in G2/M cells.

Porphyrin biosynthesis. VIII. Avian erythrocyte porphobilinogen deaminase-uroporphyrinogen III cosynthetase, its purification, properties and the separation of its components

Abstract 1. 1. A method for the isolation and purification of porphobilinogenase, porphobilinogen deaminase and uroporphyrinogen isomerase from avian erythrocytes is described. 2. 2. Some properties of the isolated enzymes were studied. The optimal pH for porphobilinogenase and deaminase was 7.4. Purified porphobilinogenase was resolved into three bands on starch gel electrophoresis. The molecular weight of the purified enzymes was determined by gel filtration. The presence of porphobilinogen or NH 4 + at certain concentrations afforded protection against heat inactivation of isomerase, the heat labile enzyme. Initial porphyrin formation by porphobilinogenase was linear with time. 3. 3. The action of various compounds added to the system was studied. Thiol reagents inhibited both porphobilinogenase and deaminase, indicating the presence of thiol groups essential for activity. NH 4 + , hydroxylamine, adenine, ADP, ATP, some dicarboxylic acids and 2-methoxy-5-nitrotropone inhibited deaminase.

δ-Aminolevulinic acid transport in murine mammary adenocarcinoma cells is mediated by beta transporters

δ-aminolevulinic acid, the precursor of porphyrin biosynthesis has been used to induce the endogenous synthesis of the photosensitiser protoporphyrin IX for photodynamic therapy in the treatment of various tumours. The aim of this work was to characterise the δ-aminolevulinic acid transport system in the murine mammary adenocarcinoma cell line LM3 using 14C-δ-aminolevulinic acid, to finally improve δ-aminolevulinic acid incorporation in mammalian cells. Our results showed that δ-aminolevulinic acid is incorporated into these cells by two different mechanisms, passive diffusion which is important at the beginning of the incubation, and active transport. Specificity assays suggested that the transporter involved in δ-aminolevulinic acid incorporation is a BETA transporter, probably GAT-2.

δ-aminolaevulinate synthetase in extracts of cultured soybean cells

1. 1. δ-Aminolaevulinate synthetase has been detected in extracts of soybean callus tissues and the enzyme activity reached its maximum when callus were 11 days old. 2. 2. The presence of a compound which seems to control δ-aminolaevulinate synthetase activity was demonstrated. The enzyme was present in the soluble fraction and was very labile. 3. 3. When crude extracts or 500 × g supernatant were stored at 4−6°, the apparent activity of δ-aminolaevulinate synthetase increased by as much as 3–6 times, while the activities of δ-aminolaevulinate dehydratase and succinyl-CoA synthetase did not significantly change during the storage. Activation was dependent on concentrations of cells suspensions during disruption and aging. 4. 4. Gel filtration with Sephadex G-25 of 2000 × g supernatants produced an enzyme fraction 30% more active. An increase in enzyme activity was observed when dark-grown callus were exposed to light. 5. 5. The addition of ATP, gibberellic acid and δ-aminolaevulinate to the culture media diminished activity; iron deficiency also produced an δ-aminolaevulinate synthetase less active.

Effect of acetaminophen on heme metabolism in rat liver

BACKGROUND AND AIMS Acetaminophen (APAP) or paracetamol is a hepatotoxic drug through mechanisms involving oxidative stress. To know whether mammalian cells possess inducible pathways for antioxidant defense, we have to study the relationship between heme metabolism and oxidative stress. METHODS fasted female Wistar rats received a single injection of APAP (3.3 mmol kg(-1) body weight) and then were killed at different times. Heme oxygenase-1 (HO), delta-aminolevulinic acid (ALA) synthase, ALA dehydratase, and porphobilinogenase activities, lipid peroxidation, GSH, catalase and glutathione peroxidase, were measured in liver homogenates. The antioxidant properties of bilirubin and S-adenosyl-L-methionine were also evaluated. RESULTS APAP increased lipid peroxidation (115% +/- 6; S.E.M., n=12 over control values) 1 h after treatment. GSH reached a minimum at 3 h (38% +/- 5) increasing thereafter. At the same time antioxidant enzymes reached minimum values (catalase, 5. 6 +/- 0.4 pmol mg(-1) protein, glutathione peroxidase, 0.101 +/- 0.006 U mg(-1) protein). HO induction was observed 6 h after treatment reaching a maximum value of 2.56 +/- 0.12 U mg(-1) protein 15 after injection. ALA synthase (ALA-S) induction occurred after enhancement of HO, reaching a maximum at 18 h (three-fold the control). ALA dehydratase activity was first inhibited (31 +/- 3%) showing a profile similar to that of GSH, while porphobilinogenase activity was not modified along the whole period of the assay. Administration of bilirubin (5 micromol kg(-1) body weight) or S-adenosyl L-methionine (46 micromol kg(-1) body weight) 2 h before APAP treatment entirely prevented the increase in malondialdehyde (MDA) content, the decrease in GSH levels as well as HO and ALA-S induction. CONCLUSION This study shows that oxidative stress produced by APAP leads to increase in ALA-S and HO activities, indicating that toxic doses of APAP affect both heme biosynthesis and degradation.

Effect of reactive oxygen species promoted by δ-aminolevulinic acid on porphyrin biosynthesis and glucose uptake in rat cerebellum

1. delta-Aminolevulinic acid (ALA) has been reported to promote reactive oxygen species (ROS). Overproduction and accumulation of ALA, as it occurs in acute intermittent porphyria (AIP), can be the origin of an endogenous source of ROS, which can then exert their oxidative damage to cell structures. 2. To investigate the induction of lipid peroxidation by ALA, thiobarbituric acid reactive substances and conjugated diene formation were measured by using minimal tissue units (MTUs) obtained from rat cerebellum. Malondialdehyde levels increased with ALA concentration and incubation time (72% at 1.0 mM ALA and 127% at 4.0 mM ALA for 4 hr), and conjugated diene formation was enhanced 50% in incubations with 1.0 mM ALA for 4 hr. 3. ALA-promoted ROS by exposure of cerebellum MTUs to 1.0 mM ALA during different intervals (1-4 hr) was partly reduced by the addition of antioxidants such as superoxide dismutase (SOD; 50 U/ml), catalase (4.5 microM) and dimethylsulfoxide (150 mM), demonstrating the involvement of O2-., H2O2 and OH. in ALA autooxidation. 4. Porphobilinogen biosynthesis was 170% increased when cerebellum MTUs were incubated with 1.0 mM ALA for 4 hr in the presence of SOD, suggesting that protein damage was promoted by ALA autooxidation. 5. These findings provide the first experimental evidence of the involvement of ALA-promoted ROS in the damage of proteins related to porphyrin biosynthesis, specially ALA-D. Oxidation of this enzyme would lead to further accumulation of ALA in AIP patients, which may be the origin of the well-known neuropsychiatric manifestations.

Paraquat-generated Oxidative Stress in Rat Liver Induces Heme Oxygenase-1 and Aminolevulinic Acid Synthase

The in vivo effect of the known herbicide, paraquat, on both hepatic oxidative stress and heme metabolism was studied. A marked increase in lipid peroxidation and a decrease in reduced glutathione (GSH) content were observed 1 h after paraquat administration. The activity of liver antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase was decreased 3 h after paraquat injection. Heme oxygenase-1 induction started 9 h after treatment, peaking at 15 h. i -aminolevulinic acid synthase induction occurred once heme oxygenase had been enhanced, reaching its maximum (1.5-fold of control) at 16 h. i -aminolevulinic acid dehydratase activity was 40% inhibited at 3 h showing a profile similar to that of GSH, while porphobilinogenase activity was not modified along the whole period of the assay. Administration of f -tocopherol (35 mmol/kg body weight) 2 h before paraquat treatment entirely prevented the increase in thiobarbituric acid reactive substances (TBARS) content, the decrease in GSH levels as well as heme oxygenase-1 and i -aminolevulinic acid synthase induction. This study shows that oxidative stress produced by paraquat leads to an increase in i -aminolevulinic acid synthase and heme oxygenase-1 activities, indicating that the herbicide affects both heme biosynthesis and degradation.