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.

Vitamin D3, lactose, and xylitol stimulate chromaffin cell proliferation in the rat adrenal medulla.

Chronic consumption by rats of diets rich in sugars or sugar alcohols leads to an increased incidence of pheochromocytomas. This relationship is hypothesized to be based on altered Ca2+ homeostasis due to increased intestinal Ca2+ absorption. Other agents associated with pheochromocytomas in rats in long-term toxicity studies have been shown to increase chromaffin cell proliferation, leading to the suggestion that the tumors occur secondarily to increased chromaffin cell turnover. We have demonstrated marked stimulation of chromaffin cell proliferation by vitamin D3, a potent stimulus to Ca2+ absorption not previously associated with adrenal medullary toxicity. This effect is detectable during the first week of dietary supplementation and persists throughout a 4-week time course. Lactose and xylitol, representative of sugars and sugar alcohols associated with pheochromocytomas, are also mitogenic but to a lesser extent, with their effects first detectable during Week 4 of dietary supplementation. Vitamin D3, its active metabolite calcitriol, lactose, and xylitol all fail to stimulate proliferation of rat chromaffin cells in vitro. The mitogenic effects of these agents may be mediated presynaptically in vivo. The data suggest that altered Ca2+ homeostasis may increase chromaffin cell proliferation and support the hypothesis that diets containing high concentrations of sugars and sugar alcohols cause pheochromocytomas in rats secondarily by this mechanism.

Cell proliferation and global methylation status changes in mouse liver after phenobarbital and/or choline-devoid, methionine-deficient diet administration.

Our laboratory is testing the hypothesis that hypomethylation of DNA [a decreased content of 5-methylcytosine (5MeC) compared with cytosine] facilitates aberrant oncogene expression involved in tumorigenesis, using a model system of mouse strains with differing susceptibilities to liver tumorigenesis. The B6C3F1 (C57BL/6 x C3H/He) mouse serves as the relatively susceptible strain and C57BL/6 serves as the relatively resistant strain. Phenobarbital (PB) and/or administration of a choline-devoid, methionine-deficient diet (CMD) were employed as non-genotoxic hepatocarcinogens. We have examined hepatocyte and nonhepatocyte proliferation in conjunction with an assessment of global methylation changes in liver DNA of B6C3F1 and C57BL/6 mice following these promoter treatments. Bromodeoxyuridine incorporation into DNA, used to measure cell proliferation indirectly, was visualized by immunohistochemistry and quantified by a Macintosh-based image analysis system. Increased hepatocyte proliferation was demonstrated following all three treatments. This increase was larger in C57BL/6 (the relatively resistant strain) as compared with B6C3F1. In contrast, global hypomethylation was evident to a larger extent in the B6C3F1 mouse, as compared with C57BL/6. PB led to hypomethylation (>20% decrease as compared with controls) at weeks 1, 2 and 4 in B6C3F1, but not in C57BL/6 at the same time points. CMD diet administration led to hypomethylation in both strains. At week 1, 21 and 9% decreases in global methylation status were observed in B6C3F1 and C57BL/6 respectively. Evaluation of these data suggests that the heightened sensitivity of the B6C3F1 mouse compared with the C57BL/6 is due, in part, to a decreased capacity for, or fidelity of, maintaining normal methylation status. The relatively resistant strain is better able to maintain the normal methylation status of DNA in the face of a higher level of cell proliferation.

Sustained stimulation of rat adrenal chromaffin cell proliferation by reserpine.

Chronic administration of reserpine is associated with the development of pheochromocytomas in rats. Short-term administration of reserpine to rats has been shown to stimulate chromaffin cell proliferation, leading to the hypothesis that reserpine causes pheochromocytomas indirectly by providing a proliferative backdrop on which genetic damage may occur. However, it is not known whether the proliferative effects of reserpine persist long enough for this model to be tenable. In the present investigation, the effects of reserpine on bromodeoxyuridine (BrdU) incorporation into epinephrine (E)- and norepinephrine (NE)-type chromaffin cells were studied after 1, 4, and 12 weeks of reserpine administration. Reserpine administered in the diet at 10 or 50 ppm was shown to result in a persistent mitogenic stimulation of the rat adrenal medulla. Cells that incorporated BrdU at all time points appeared to be typical E- and NE-type chromaffin cells, and the ratio of BrdU-labeled E cells to BrdU-labeled NE cells was not altered by reserpine. An additional observation was that the ratio of all E cells to all NE cells declined after Week 1 and that the decline could be accelerated by administration of reserpine. This finding suggests that neural stimulation of chromaffin cells might play a role in age-related functional changes of the adrenal medulla during early adult life. The present observations support the hypothesis that reserpine induces pheochromocytomas indirectly by increasing chromaffin cell proliferation. They also decrease the likelihood that rat pheochromocytomas arise from preferential stimulation of proliferation of a particular cell type.

The effect of phenobarbital on the metabolism and excretion of thyroxine in rats.

The effect of phenobarbital on thyroid function and the metabolism and biliary excretion of thyroxine in rats was determined. Phenobarbital, administered for 2 weeks at a dose of 100 mg/kg/day, resulted in an increase in hepatic and thyroid gland weights, decreased circulating levels of T4, T3 and rT3, and increased TSH levels in male and female rats. After 3 months of treatment liver and thyroid weights were still increased; however, hormone values were not as markedly affected indicating that the rats had partially compensated for the effect on thyroid function. In thyroidectomized rats the plasma clearance of thyroxine was increased with phenobarbital. In bile duct cannulated phenobarbital-treated male rats the hepatic uptake at 4 hr was markedly increased. Bile flow was increased and the 4-hr cumulative biliary excretion of administered radioactivity was increased by 42%. Most of the increase in the excretion (76%) was accounted for by an increase in the excretion of thyroxine-glucuronide in phenobarbital-treated rats. Hepatic thyroxine-glucuronyltransferase activity in phenobarbital-treated rats expressed as picomoles per milligram of protein was increased by 40%; enzyme activity per gram of liver was increased by about twofold which, coupled with increased hepatic weight, resulted in about a threefold increase in total hepatic thyroxine-glucuronyltransferase activity in phenobarbital-treated rats as compared to that of controls. Qualitatively similar effects on metabolism, excretion, and enzyme induction were noted in female rats; however, the magnitude of increase was less than that observed in male rats. It is concluded that the effect of phenobarbital on thyroid function in rats is primarily a result of its effects on the hepatic disposition of thyroid hormone. The induction of thyroxine-glucuronyltransferase appears to play an important role in the increased metabolism and excretion of thyroxine in phenobarbital-treated rats.

Effect of metronidazole on fertility and testicular function in male rats.

The reproductive toxicology of metronidazole was studied in rats. Male Charles River Crl:CD(SD)BR rats (10/group) were treated with metronidazole as a dietary admixture at doses of 0 (control), 25, 100, and 400 mg/kg/day for 8 weeks. The reversibility of effects after a 3 1/2-month recovery period was determined in separate groups of 10 control and 10 rats treated with 400 mg/kg/day of metronidazole. After 2 and 4 weeks of metronidazole treatment, mating performance and fertility in treated and control animals were comparable. After 6 weeks of treatment, all high-dose rats were infertile; however, fertility in low- and middose rats was not affected. High-dose male rats killed after 8 weeks of treatment showed markedly decreased testicular and epididymal weights, and markedly decreased testicular spermatid counts and epididymal sperm counts. Most of the few epididymal sperm present in high-dose rats were viable, but morphologically abnormal. Histologically, severe degeneration of the seminiferous epithelium was observed in the testes of high-dose rats; the tubules were generally devoid of primary or secondary spermatocytes and spermatids. Rats treated with the low and middle doses of metronidazole exhibited normal testicular and epididymal weights, and normal testicular spermatid counts and epididymal sperm reserves. Epididymal sperm viability and morphology of treated and control animals were comparable. Minimal histologic changes were observed in the testes of middose rats, including degenerative fragmentation of spermatozoa and spermatids. In high-dose recovery rats, fertility was restored in most rats by 8 weeks after the cessation of treatment; however, the testicular and epididymal weights and sperm counts of rats killed after 3 1/2 months of recovery had increased but were still significantly decreased in treated rats as compared with controls. Histologically, spermatogenesis was observed in most tubules; however, a portion of atrophic tubules persisted. It is concluded that high doses of metronidazole produce infertility in the male rat through inhibition of spermatogenesis as early as the stage of the primary spermatocyte. This effect is partially reversible.

Reproduction studies in rats treated with ornidazole.

Reproduction studies were performed with ornidazole, a compound with trichomonacidal activity. Male rats were treated for 61 days prior to mating and female rats were treated for 2 weeks prior to mating and throughout gestation and lactation at doses of 0 (control), 25, 100, and 400 mg of ornidazole/kg/day. A decrease in the pregnancy rate was observed in high-dose rats without altered mating performance. Crossover matings between high-dose treated and control male and female rats showed that male but not female fertility was affected and that the effect on fertility was reversible within several days after the cessation of treatment. Testicular and epididymal weights were not altered in treated male rats. Histopathological examination revealed that spermatogenesis and the testes were normal and that the epididymides of treated male rats contained normal appearing sperm. It is concluded that ornidazole, at high dosages, produces infertility in the male rat; however, unlike many other 5-nitroimidazole compounds which are reported to inhibit spermatogenesis, no effect on spermatogenesis was observed under the conditions of these studies. This in conjunction with the rapid reversibility of infertility suggests that the mode of action of ornidazole involves a rapidly reversible effect on epididymal sperm function.

The effect of ornidazole on fertility and epididymal sperm function in rats.

A comprehensive study of male fertility and sperm production and function was performed in 20 control and 20 rats treated with ornidazole, a compound with trichomonacidal activity. Rats were treated for 4 weeks at dosages of 0 (control) and 400 mg/kg/day of ornidazole during which fertility was assessed by weekly matings. Testicular sperm production and epididymal sperm function were assessed in one-half of the rats while the reversibility of effects after a 2-week recovery period was assessed in the remaining half. Male rats treated with ornidazole were infertile during the second week of treatment. After 4 weeks of treatment, testicular and epididymal weights, testicular spermatid counts, epididymal sperm reserves, sperm morphology, and sperm viability were similar in treated and control rats. A quantitative assessment of epididymal sperm motility using a dark-field photomicroscope with a stroboscopic light source revealed that ornidazole markedly inhibited sperm motility. Although the percentage of nonmotile sperm was not substantially increased in treated rats, the vigor of tail movement was markedly decreased which resulted in decreased sperm velocity. Restoration of fertility and normal sperm motility and velocity were observed in the group of recovery rats assessed 2 weeks after the cessation of ornidazole treatment. It is concluded that ornidazole, at a high dosage of 400 mg/kg/day, produces infertility in male rats by inhibiting epididymal sperm motility in terms of decreased sperm velocity. These effects are rapidly reversible after the cessation of treatment.