Hexachlorobenzene treatment on hepatic mitochondrial function parameters and intracellular coproporphyrinogen oxidase location.

These studies try to elucidate why isocoproporphyrin appears in hexachlorobenzene-poisoned rats' feces. Chronic exposure of hexachlorobenzene to rats produces an experimental model for human porphyria cutanea tarda. After 8 weeks of treatment, rats showed high porphyrin excreta and 50% inhibition of liver uroporphyrinogen decarboxylase activity. Uroporphyrin plus heptacarboxylic porphyrin exceeded coproporphyrin in urine, whereas in feces, isocoproporphyrin, from abnormal pentacarboxylic porphyrinogen III oxidative decarboxylation by liver coproporphyrinogen oxidase, became the main porphyrin. Trypsin-treated mitochondria showed that the outer and inner membrane permeability barrier was highly conserved after hexachlorobenzene intoxication. In digitonin-treated hexachlorobenzene mitochondria, coproporphyrinogen oxidase was free in the mitochondrial intermembrane space, whereas in normal mitochondria, 30% to 50% remained anchored to the inner membrane. Hexachlorobenzene led to a decrease in respiratory control and ADP/O ratios (uncoupled mitochondria). Albumin restored oxidative phosphorylation, indicating no irreversible inner membrane damage. Normal and hexachlorobenzene mitochondria oscillatory studies exhibited similar damping factor values, showing that hexachlorobenzene had no significant effect on membrane fluidity and elasticity. Mitochondrial uncoupling could explain the free state of the enzyme within the intermembrane space. The free state of the enzyme makes it more flexible and would allow pentacarboxylic porphyrinogen III, whose levels are increased, to compete with coproporphyrinogen III and being transformed into dehydroisocoproporphyrinogen, the liver forerunner of fecal isocoproporphyrin.

Function and structure of rat hepatic coproporphyrinogen oxidase.

Rat hepatic coproporphyrinogen oxidase, the sixth enzyme in the heme biosynthetic pathway, was purified 1340-fold with a yield of 39.7%. To obtain the soluble enzyme, different methods were applied to disrupt mitochondria, with sonication giving the highest yield (85%). The minimum catalytic form of enzyme was a dimer with a molecular mass of 77 +/- 4 kDa. The existence of aggregated forms was possible since in fractions of gel filtration elution activity was observed with higher molecular mass. We determined a Stokes radius of 36.3 A, a sedimentation coefficient (S20,w) of 5.06 S, and frictional ratio of 1.29, suggesting a nearly globular shape of the protein. Regardless of the type of salt, high ionic strength inhibits the enzyme, probably modifying its native structure. Experiments with amino acid modifiers showed that histidine, arginine, and tryptophan are involved in the catalytic process. Non-ionic detergents and phospholipids activated the enzyme, probably because they reproduce its natural hydrophobic environment. The present study describes a simple method for the purification of rat liver coproporphyrinogen oxidase, introducing for the first time data on the structure and function of the protein in a tissue often used as a laboratory model in biological studies, and contributing to the study of human hereditary coproporphyria.

Simple and rapid method for the determination of coproporphyrinogen oxidase activity.

Coproporphyrinogen oxidase, the sixth enzyme in the biosynthetic heme pathway, catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX. A reversed-phase high pressure liquid chromatography method was developed to measure coproporphyrinogen oxidase enzymatic activity in rat liver. With this method, the separation, identification and quantification of coproporphyrin III (oxidized substrate) and protoporphyrin IX (oxidized product) present in the assays could be carried out with no need of derivatization and in less than 15 min. Rat and human liver coproporphyrinogen oxidase basal activities determined using this method were 0.41+/-0.05 nmol of protoporphyrin IX/h per mg of hepatic protein and 0.87+/-0.06 protoporphyrin IX/h per mg of hepatic protein, respectively. Kinetic studies showed that optimum pH for rat CPGox is 7.3, and that its activity is linear in the range of protein concentrations and incubation times assayed. The present paper describes a sensitive, specific and rapid fluorometric high performance liquid chromatography method to measure coproporphyrinogen oxidase, which could be applied to the diagnosis of human coproporphyria, and which is also suitable for the study of lead and other metal poisoning that produce alterations in this enzymatic activity.

Effect of acute lead treatment on coproporphyrinogen oxidase activity in HepG2 cells.

Acute toxic effects of lead were evaluated on porphyrin synthesis and coproporphyrinogen oxidase (CO) activity in an in vitro model, using HepG2 cells, a hepatoma cell line of human origin. Lead concentrations for exposure treatments were 0.5, 1.0, 2.5, 5.0 microM. No significant changes were found in treated cells with respect to uroporphyrin cellular or media concentrations. Cellular protoporphyrin increased in dose response shape, but no changes in extracellular content were found. Extracellular coproporphyrin concentration increased in a dose response manner without changes in cellular content. The CO activity was depressed in dose response shape, reaching 62% of control activity at 5.0 microM of lead treatment. The CO activity in Pb-treated cells was recovered after dithiothreitol (DTT) treatment, suggesting that sulphydryl groups play an essential role in the enzyme activity. The dose-response increase of coproporphyrin secretion accompanied by the depression of CO activity supports the suggestion that lead causes CO inhibition, as observed in this cellular model.