Effect of 1,10-phenanthroline and desferrioxamine in vivo on prolyl hydroxylase and hydroxylation of collagen in various tissues of rats

Abstract

We studied prolyl hydroxylase (PH) activity in various tissues of rats after systemic and local administrations of 1,10-phenanthroline and Desferrioxamine. We assayed PH in tissue extracts without adding iron to the incubation medium; we ascertained hydroxylation of collagen by measuring 3H release from biosynthetically 3,4- 3H-proline-labeled collagen substrate. We studied the effect of both Fe 2+ and Fe 3+ chelating agents in tissues with a relatively high rate of collagen synthesis such as fetal skin and skin of newborn rats, 17-β-estradiol-stimulated uterus of immature rats, and carrageenan granuloma tissue. In addition, in some organs we studied collagen hydroxylation by measuring the ratio of proline to hydroxyproline either in highly purified samples or in the digest from samples treated with protease-free collagenase. Finally, the extent of underhydroxylation of collagen from some tissues of rats treated with either one or both chelating agents was determined using a 3,4,- 3H-proline-labeled collagen as a substrate to partially purified PH. Neither systemic nor local injections of 1,10-phenanthroline or Desferrioxamine alone inhibited PH in any of the models. But simultaneous administration of both agents inhibited PH and hydroxylation of collagen in some models. Although local injections of 1,10-phenanthroline into granuloma tissue did not inhibit PH activity, hydroxylation of collagen synthesized in the granuloma from 4 to 16 hr after injection was reduced significantly. This seeming discrepancy is explained by the finding that PH is active even without the addition of Fe 2+ in the assay medium; a strong reducing environment (α-ketoglutarate, ascorbic acid) reduces Fe 3+, thus providing ferrous ions essential for PH activity. Both forms of iron, therefore, must be chelated simultaneously to affect PH activity in the assay system. In some experiments, within 8 hr after systemic administration of 1,10-phenanthroline, the activity of PH in skin almost doubled and remained significantly elevated for 40 hr. The mechanism responsible for this phenomenon is not known. The results of this study indicate that previously reported inhibitory effects of some Fe 2+ chelating agents on collagen synthesis in various models of fibrosis may not be related to inhibition of PH or to collagen hydroxylation.