Vitamin D3-induced proliferative lesions in the rat adrenal medulla

Adrenal medullary hyperplasia and pheochromocytomas are induced in rats by a variety of non-genotoxic agents, and we have hypothesized that these agents induce lesions indirectly by stimulating chromaffin cell proliferation. Vitamin D3, which has not been previously associated with adrenal medullary proliferative lesions, is the most potent in vivo stimulus to chromaffin cell proliferation yet identified. The present investigation utilized the vitamin D3 model to prospectively test the relationship between mitogenicity and focal proliferative lesions in the adrenal medulla and to determine early events in the pathogenesis of these lesions. Charles River Crl:CD BR rats were treated with 0; 5000; 10,000; or 20,000 IU/kg/day of vitamin D3 in corn oil (5 ml/kg) by oral intubation. Rats were killed after 4, 8, 12, or 26 weeks of treatment, following a final week of labeling with bromodeoxyuridine (BrdU) using a mini-pump. Adrenal sections were double-stained for BrdU and phenylethanolamine-N-methyl transferase (PNMT) to discriminate epinephrine (E) from norepinephrine (NE) cells or for vesicular acetylcholine transporter (VAchT) to identify cholinergic nerve endings. Vitamin D3 caused a 4-5-fold increase in BrdU labeling at week 4, diminishing to a 2-fold increase by week 26. An initial preponderance of labeled E cells gave way to a preponderance of labeled NE cells. By week 26, 17/19 (89%) animals receiving the 2 highest doses of vitamin D3 had focal adrenal medullary proliferative lesions, in contrast to an absence of lesions in control rats. The lesions encompassed a spectrum including BrdU-labeled "hot spots" not readily visible on H and E sections, hyperplastic nodules, and pheochromocytomas. Lesions were usually multicentric, bilateral, and peripheral in location, and almost all were PNMT-negative. The lesions were not cholinergically innervated, suggesting autonomous proliferation. Hot spots, hyperplastic nodules, and pheochromocytomas appear to represent a continuum rather than separate entities. Their development might involve selective responses of chromaffin cell subsets to mitogenic signals, influenced by both innervation and corticomedullary interactions. A number of non-genotoxic compounds that induce pheochromocytomas in rats are known to affect calcium homeostasis. The results of this study provide further evidence to support the hypothesis that altered calcium homeostasis is indirectly involved in the pathogenesis of pheochromocytomas, via effects on chromaffin cell proliferation.