Toxoplasma gondii: mechanism of the parasitostatic action of 6-thioxanthine.

In contrast to the cytocidal effect of 6-thiopurines on mammalian cells, the action of 6-thioxanthine on Toxoplasma gondii was only parasitostatic. 6-Thioxanthine was a substrate of the parasite's hypoxanthine-guanine phosphoribosyltransferase. That enzyme converted 6-thioxanthine to 6-thioxanthosine 5'-phosphate which accumulated to near millimolar concentrations within parasites incubated intracellularly in medium containing the drug. 6-Thioxanthosine 5'-phosphate was the only detectable metabolite of 6-thioxanthine. The absence of 6-thioguanine nucleotides explains the lack of a parasitocidal effect because the incorporation of 6-thiodeoxyguanosine triphosphate into DNA is the mechanism of the lethal effect of 6-thiopurines on mammalian cells. Extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate incorporated more labeled hypoxanthine or xanthine into their nucleotide pools than did control parasites. The basis for this increased nucleobase salvage remains unexplained. It was not due to up-regulation of hypoxanthine-guanine phosphoribosyltransferase and could not be explained by reduced use of labeled nucleotides for nucleic acid synthesis. Extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate used labeled hypoxanthine almost entirely to make adenine nucleotides while control parasites made both adenine and guanine nucleotides. Both extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate and control parasites efficiently used labeled xanthine to make guanine nucleotides. These observations suggested that inosine 5'-phosphate-dehydrogenase was inhibited while guanosine 5'-phosphate synthase was not. Assay of inosine 5'-phosphate dehydrogenase in soluble extracts of T. gondii confirmed that 6-thioxanthosine 5'-phosphate was an inhibitor. We conclude that 6-thioxanthine blocks the growth of T. gondii by a depletion a guanine nucleotides.

Role of small differences in CYP1A2 in the development of uroporphyria produced by iron and 5-aminolevulinate in C57BL/6 and SWR strains of mice.

Previous work has implicated CYP1A2 in experimental uroporphyria caused by polyhalogenated aromatic compounds, and in uroporphyria caused by iron and 5-aminolevulinate (ALA) in the absence of inducers of CYP1A2. Here we examined whether the different susceptibilities of SWR and C57BL/6 strains of mice to uroporphyria in the absence of inducers of CYP1A2 are related to different levels of CYP1A2. Enzymological assays (ethoxy- and methoxyresorufin dealkylases, and uroporphyrinogen oxidation) and immunoblots indicated that there was about twice the amount of hepatic CYP1A2 in SWR mice compared with C57BL/6 mice. Immunohistochemistry revealed that CYP1A2 was located centrilobularly in the liver, and the staining was more intense in SWR mice than in C57BL/6 mice. Hepatic non-heme iron was about double in SWR compared with C57BL/6 mice. In SWR mice given iron dextran, hepatic iron was 1.7-fold that of C57BL/6 mice given iron dextran. SWR mice administered ALA in the drinking water accumulated much less hepatic protoporphyrin than did C57BL/6 mice. To confirm the importance of small increases in CYP1A2, C57BL/6 mice were given a low dose of 3-methylcholanthrene (MC) (15 mg/kg), as well as iron and ALA. There was about a 5- to 6-fold increase in hepatic uroporphyrin accumulation after 32 days on ALA compared with animals not given MC. In these animals, CYP1A2 was increased by 10-fold at 2 days, but returned to basal levels by 14 days. We conclude that small and transient differences in CYP1A2 may be important in the development of uroporphyria.

Ascorbic acid deficiency in porphyria cutanea tarda.

Porphyria cutanea tarda (PCT), the most common form of porphyria, is manifested as skin photosensitivity caused by excess hepatic production of uroporphyrin and heptacarboxylporphyrin. In experimental animal models, ascorbic acid modulates chemically induced uroporphyrin accumulation. The purpose of this study was to determine whether ascorbic acid is decreased in the plasma of patients with PCT. Plasma was obtained after an overnight fast from 21 PCT patients, 16 of whom were infected with hepatitis C virus (HCV), and from a separate group of 9 patients with HCV infection but not PCT. Thirteen PCT patients were studied when they had active disease and 8 after treatment-induced remission. Plasma ascorbic acid was low (<23 micromol/L) in 11 (85%) of the 13 untreated PCT patients and deficient (<11 micromol/L) in 8 (62%). Two patients with normal ascorbic acid levels (45 and 62 micrommol/L) had consumed multivitamins. In 2 patients with deficient ascorbic acid, plasma levels returned to normal after phlebotomy treatment. Of the 8 patients studied during remission, 4 had normal ascorbic acid values and 4 were deficient (5 to 8 micromol/L). Plasma ascorbic acid values were normal for all patients who had HCV but no PCT. These data suggest that plasma ascorbic acid concentrations are commonly low in PCT, but this decrease is unrelated to HCV infection. Ascorbic acid deficiency may be one of the factors that contributes to the pathogenesis of PCT.