The decrease in uroporphyrinogen decarboxylase activity induced by ethanol predisposes rats to the development of porphyria and accelerates xenobiotic-triggered porphyria, regardless of hepatic damage.

We evaluated the porphyrinogenic ability of ethanol (20% in drinking water) per se, its effect on the development of sporadic porphyria cutanea tarda induced by hexachlorobenzene in female Wistar rats (170-190 g, N = 8/group), and the relationship with hepatic damage. Twenty-five percent of the animals receiving ethanol increased up to 14-, 25-, and 4.5-fold the urinary excretion of delta-aminolevulinate, porphobilinogen, and porphyrins, respectively. Ethanol exacerbated the precursor excretions elicited by hexachlorobenzene. Hepatic porphyrin levels increased by hexachlorobenzene treatment, while this parameter only increased (up to 90-fold) in some of the animals that received ethanol alone. Ethanol reduced the activities of uroporphyrinogen decarboxylase, delta-aminolevulinate dehydrase and ferrochelatase. In the ethanol group, many of the animals showed a 30% decrease in uroporphyrinogen activity; in the ethanol + hexachlorobenzene group, this decrease occurred before the one caused by hexachlorobenzene alone. Ethanol exacerbated the effects of hexachlorobenzene, among others, on the rate-limiting enzyme delta-aminolevulinate synthetase. The plasma activities of enzymes that are markers of hepatic damage were similar in all drug-treated groups. These results indicate that 1) ethanol exacerbates the biochemical manifestation of sporadic hexachlorobenzene-induced porphyria cutanea tarda; 2) ethanol per se affects several enzymatic and excretion parameters of the heme metabolic pathway; 3) since not all the animals were affected to the same extent, ethanol seems to be a porphyrinogenic agent only when there is a predisposition, and 4) hepatic damage showed no correlation with the development of porphyria cutanea tarda.

Fractionated illumination in oesophageal ALA-PDT: effect on ferrochelatase activity.

BACKGROUND AND OBJECTIVE: Administration of 5-aminolevulinic acid (ALA) induces accumulation of the photosensitive compound protoporphyrin IX (PpIX) in certain tissues. PplX can be used as photosensitizer in photodynamic therapy (PDT). More selective or higher PpIX accumulation in the area to be treated could optimize the results of ALA-PDT. Porphobilinogen deaminase (PBGD) is rate-limiting in PpIX formation whereas ferrochelatase converts PpIX into haem by chelation of ferrous iron into PpIX. This results in a moment of close interaction (ferrochelatase binding to PpIX) during which ferrochelatase could selectively be destroyed resulting in an increased PpIX concentration. The aim of the present study was to investigate whether illumination before PDT can selectively destroy ferrochelatase. and whether this results in higher PpIX accumulation and thereby increases the PDT effect. Furthermore, the effect of a second ALA dose was tested. STUDY DESIGN/MATERIALS AND METHODS: Oesophageal tissue of 60 rats were allocated to 2 groups of 30 animals each. In one group, enzyme and PpIX measurements were performed after ALA administration (200 mg/kg orally, n=20), or a second dose of 200 mg/kg ALA at 4 h (n=10), half of each group with and without illumination at 1 h with 12.5 J/cm diffuser length. In the second group, PDT was performed. Ten animals were illuminated at 3 h after ALA administration with 20 (n=5) or 32.5 J/cm (n=5), 10 animals were illuminated at 1 h (12.5 J/cm) and received intra-oesophageal PDT treatment (20 J/cm) at 3 h (n=5) or 4 h (n=5) after ALA. Additionally, 10 animals received a second dose of 200 mg/kg ALA at 4 h and were illuminated (20 J/cm) at 7 h after the first dose of ALA with (n=5) or without (n=5) illumination at 4 h (12.5 J/cm). RESULTS: Illumination with 12.5 J/cm at 1 h after ALA administration caused inhibition of the activity of ferrochelatase at 3 and 4 h after ALA (P=0.02 and P<0.001, respectively), but not at 7 h (P=0.3). In animals sacrificed at 4 h the ratio PBGD:ferrochelatase was higher in animals illuminated at 1 h compared to non-illuminated animals (P<0.001). PpIX concentration was highest (42.7 +/- 3.2 pmol/mg protein) at 3 h after ALA administration and did not increase by illumination at 1 h. Administration of a second dose of ALA did not result in higher PpIX accumulation. After PDT, no difference in epithelial or muscular damage was found between the various groups. CONCLUSION: Illumination at 1 h after ALA administration can cause selective destruction of ferrochelatase, resulting in a higher ratio of PBGD:ferrochelatase. This does not result in accumulation of more porphyrins, even when a second dose of ALA is given. Therefore, under the conditions used in this study fractionated illumination does not enhance ALA-PDT-induced epithelial ablation of the rat oesophagus.

Evaluation of the porphyrinogenic risk of antineoplastics.

The use of antineoplastics is common in cancer therapy, and some of them have been associated with the development of porphyria in patients with cancer. However, knowledge of their effects on the haeme metabolic pathway is at present scarce and unclear. So, the present study evaluates the porphyrinogenic ability of nine antineoplastics (both alkylating and non-alkylating). These were tested either alone or in conjunction with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (latent porphyria model) in chick embryos and in mice. The results obtained suggest that the use of cyclophosphamide, azathioprine, 5-fluorouracil, busulphan, procarbazine and hexamethylmelamine be avoided in the treatment of porphyric patients. On the other hand, dacarbazine, chlorambucil and melphalan are non-porphyrinogenic. We also provide evidence showing that neither the presence of the mustard group in the structure of the antineoplastic nor alterations in ferrochelatase or protoporphyrinogen oxidase activities are responsible for the porphyrinogenic ability of cyclophosphamide.

Heme availability affects corticosterone and aldosterone biosynthesis in rat adrenal.

In this paper, we studied the effect of heme availability on corticosterone and aldosterone synthesis in rat adrenal. We found that hemin stimulated corticosterone and aldosterone production in adrenal homogenates in a dose-dependent fashion. Hemin administration to rats also provoked an increase in both corticosterone and aldosterone content in adrenal. 3,5-Diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC), an inhibitor of liver ferrochelatase activity, was able to inhibit this enzyme in rat adrenal. This resulted in an impairment of heme concentration and consequently adrenal ALA-synthase and porphyrin content were increased. Thus, it was proven that DDC inhibits heme biosynthesis in adrenal as it does in liver. In vivo experiments with rats showed that DDC was able to partially blocked ACTH-mediated corticosterone and aldosterone production while hemin administration was able to partially restore it. These data indicate that heme availability affects steroid biosynthesis in rat adrenal.

The effect of L-cysteine and N-acetylcysteine on porphyrin/heme biosynthetic pathway in cells treated with 5-aminolevulinic acid and exposed to radiation.

The effects of L-cysteine (LC) and N-acetylcysteine (NAC) on porphyrin accumulation in a human dermal microvascular endothelial cell line (HMEC-1) and a human epidermoid carcinoma cell line (A431) loaded with 5-aminolevulinic acid (ALA) and exposed to ultraviolet A (UVA) and blue light radiation were determined. Porphyrin accumulation was decreased in the presence of 0.1-7.5 mM LC (24.8%-31.4% suppression in HMEC-1 cell; 35.8%-48.9% suppression in A431 cells), and in the presence of 0.1-10.0 mM NAC (30.9%-58.0% suppression in HMEC-1 cells; 8.5%-45.3% in A431 cells). The suppression occurred in a LC or NAC dose-dependent fashion. The above was associated with partial reversal of suppression of ferrochelatase (FeC) activity in HMEC-1 cells and in A431 cells. As compared to FeC activity in cells treated with ALA and irradiation, enzyme activity was higher (by 31.9%-62.1%) in the presence of LC (1.0 mM or 5.0 mM) and in the presence of NAC (1.0 mM or 5.0 mM). These data indicate that LC and NAC have protective effects on porphyrin- and irradiation-induced diminution of FeC activity in HMEC-1 cells and A341 cells in vitro.

Formation of N-methyl protoporphyrin in chemically-induced protoporphyria. Studies with a novel porphyrogenic agent.

1-[4-(3-Acetyl-2,4,6-trimethylphenyl)-2,6-cyclohexanedionyl]-O-eth yl propionaldehyde oxime (for short ATMP) is a novel porphyrogenic agent causing hepatic protoporphyria in the mouse. Mice given a single dose of the drug showed 24 h later a 70% inhibition of liver ferrochelatase and marked accumulation of protoporphyrin. These changes were not seen in similarly treated rats, guinea pigs, hamsters or chick embryos. A green pigment was isolated from the liver of mice treated with ATMP and identified by its electronic absorption spectrum and chromatographic properties on HPLC as N-methyl protoporphyrin. The ATMP pigment markedly inhibited the enzyme ferrochelatase in vitro, thus supporting its identification as N-methyl protoporphyrin. Two inhibitors of liver cytochrome P450, compound SKF 525-A and piperonyl butoxide, when given before ATMP, afforded protection against ATMP-induced porphyria and production of N-methyl protoporphyrin, suggesting a role of cytochrome P450 in the induction of the metabolic disorder. The most likely interpretation for these findings is therefore that ATMP is metabolized in the mouse to a reactive species, which in turn alkylates the haem moiety of liver cytochrome P450, thus producing N-methyl protoporphyrin. This inhibits ferrochelatase and, as a secondary response, protoporphyrin accumulates. This pathway of metabolism to the postulated reactive metabolite presumably does not occur to a significant extent in the other species examined and hence is the likely basis for the species difference in protoporphyria.