9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha.

Vitamin A (retinol) and its natural derivatives are required for many physiological processes. The activity of retinoids is thought to be mediated by interactions with two subfamilies of nuclear retinoic acid receptors, RAR and RXR. The RARs bind all-trans retinoic acid (t-RA) with high affinity and alter gene expression as a consequence of this direct ligand interaction. RXR alpha is activated by t-RA, yet has little binding affinity for this ligand. t-RA may be converted to a more proximate ligand that directly binds and activates RXR alpha, and we have developed a method of nuclear receptor-dependent ligand trapping to test this hypothesis. Here we report the identification of a stereoisomer of retinoic acid, 9-cis retinoic acid, which directly binds and activates RXR alpha. These results suggest a new role for isomerization in the physiology of natural retinoids.

Time course of testicular degeneration in rats induced by a synthetic retinoid (Ro 23-2895) and evidence for induction of hypovitaminosis A in the testes.

Eight-week-old male Sprague--Dawley rats were dosed by gavage with 90 mg/kg of Ro 23-2895, (all-E)-9-[2-(nonyloxy)phenyl]-2,4,6,8 nonatetraenoic acid, dissolved in Tween 80. Treated animals (n = 3--4) were sacrificed after 3, 7, 11 and 21 days of dosing. Control rats (n = 3) received an equal volume of Tween 80 and were sacrificed after 3 or 21 days. Cross sections of formalin fixed testes were embedded in glycolmethacrylate, sectioned at 3 microns, and stained with periodic acid-Schiff and hematoxylin. No morphologic alterations were observed in the control rats or in treated rats after 3 days. After 7 days of treatment, there were occasional tubules in which there was a delayed release of mature sperm and occasionally the retained sperm were being resorbed. The frequency and severity of these morphologic changes was increased after 11 days of treatment, and round spermatids were occasionally observed with marginated chromatin in their nuclei. After 21 days of treatment, there was a significant reduction in testicular weight accompanied by marked degenerative changes and in some cases almost a complete desquamation of the germinal epithelium. Multinucleated giant cells and germ cells with marginated chromatin in their nuclei were commonly observed and there was moderate to severe oligospermia in the tubules. Sertoli cell nuclei were swollen and showed lucent, vesiculated nucleoplasm. In a parallel 21-day study, treated rats (n = 10) showed an 80% reduction in plasma retinol and a 56% decrease in testicular retinol compared to vehicle-treated rats (n = 10). A 53% decrease in plasma testosterone levels was also observed in treated rats. The testicular lesions produced by treatment with Ro 23-2895 were similar to vitamin A deficiency, which supports the hypothesis that high doses of synthetic retinoids may cause testicular degeneration through interference of normal retinol homeostasis.

The reversibility of nitrobenzene-induced testicular toxicity: continuous monitoring of sperm output from vasocystotomized rats.

Exposure of rats to nitrobenzene produces a degeneration of the seminiferous epithelium of the testes. Sperm production was continuously monitored in rats surgically prepared by anastomosing the vas deferentia with the urinary bladder to evaluate the reversibility of nitrobenzene toxicity. Rates of sperm production were monitored by collecting urine and counting sperm microscopically with a hemocytometer. Six weeks after surgery, rats were dosed p.o. with a single dose of 300 mg/kg of nitrobenzene in corn oil. Sperm were not detected in the urine of treated rats between 32 and 48 days after treatment. Despite the fact that degenerative changes in the seminiferous tubules were observed histologically as early as 3 days after dosing, there was a 32-day lag period between treatment and cessation of sperm output in treated rats. Histological examination showed that pachytene spermatocytes and step 1-2 spermatids were the most susceptible cell stages to nitrobenzene and were observed forming into giant cells as early as 3 days after treatment. However, repair was substantial by 3 weeks after treatment and by days 76-100, the rate of sperm output reached 78% of the control group. By 100 days after treatment, there was greater than 90% regeneration of the seminiferous epithelium. Thus, a single oral dose of nitrobenzene induced testicular degeneration and approximately a 17-day period of aspermia resulted. Back-dating of the aspermic period to the timing of the spermatogenic cycle closely corresponded with the same germ cell stages that were observed degenerating in histologic examinations. Thus, changes in sperm output from vasocystotomized rats correlated well with histopathologic changes, demonstrating the value of this technique for toxicity studies.

Studies on the testicular effects of vitamin A palmitate in the Sprague-Dawley rat.

The purpose of the research was to investigate the mechanism of reported vitamin A-induced testicular degeneration. Three studies of vitamin A toxicity were conducted in male Sprague-Dawley rats; a 10-day study with daily ip injections of retinol palmitate at doses of 0, 115,000 and 230,000 IU/kg/day in adult rats; a 10-day study with juvenile rats treated with 115,000 IU/kg/day, pair-fed controls and ad lib.-fed controls; a 13-wk dietary study in which retinol palmitate beadlets were mixed in the food of juvenile rats at doses of 0, 60,000, 120,000 and 200,000 IU/kg/day; a second untreated group was pair-fed to the high-dose group. Even at doses that produced overt signs of hypervitaminosis A and mortality, minimal or no changes were observed in the testes. In the 10-day ip studies, only a 20% incidence of treated juvenile rats (115,000 IU/kg) and adult rats (230,000 IU/kg) showed sloughing germ cells in some of the tubule lumens of the testes, but the structure and integrity of the seminiferous epithelium was completely intact. No change in testicular morphology or spermatid counts was observed in the 13-wk dietary study. In all studies, testicular weights of treated rats were not significantly reduced when corrected for body weight or compared with pair-fed controls. In the 10-day ip studies, serum testosterone levels of treated rats did not differ from the respective pair-fed control rats, but in the 13-wk study, a dose-related reduction in testosterone occurred that was considered to be a direct effect of chronic vitamin A treatment. Seminal vesicle weights were decreased, as would be expected with decreased testosterone levels. Adrenal weights were increased in all studies. These findings suggest that the testes of rat are resistant to orally administered vitamin A palmitate and only slightly affected by ip administration.

Studies on the mode of action for thyroid gland tumor promotion in rats by phenobarbital.

A study was conducted to determine the mode of action for phenobarbital promotion of thyroid follicular cell neoplasia in rats using an initiation-promotion model established by Hiasa et al. (Y. Hiasa, Y. Kitahori, M. Ohshima, T. Fujita, T. Yuasa, N. Konishi, and A. Miyashiro, 1982a, Carcinogenesis 3, 1187-1190). Seven groups of Charles River Crl: CD(SD)BR rats (20/sex/group) were treated with either saline or 700 mg/kg DHPN [N-bis(2-hydroxypropyl)nitrosamine] administered subcutaneously once a week for 5 weeks (Initiation Phase) followed by 15 weeks of treatment with control diet or diets containing 500 ppm of phenobarbital (Promotion Phase). Groups of rats were also treated with L-thyroxine (50 micrograms/kg/day) in the diet to determine its effect on thyroid gland tumor promotion by phenobarbital. The incidence of thyroid follicular adenomas in DHPN male rats treated with phenobarbital was markedly increased [83% (15/18 rats)] as compared to rats receiving DHPN alone [37% (6/16 rats)]. Thyroxine treatment completely blocked the tumor promoting effect of phenobarbital in that the tumor incidence [25% (5/20 rats)] was reduced back to or somewhat less than that observed with DHPN alone. In female rats no tumors were observed with DHPN nor was a promoting effect of phenobarbital observed. These results demonstrate the potential for a microsomal enzyme inducer such as phenobarbital to alter the incidence of thyroid gland neoplasia in the male rat. The inhibitory effect of L-thyroxine on tumor promotion by phenobarbital supports the hypothesis that the promoting effect of phenobarbital is mediated via increased pituitary secretion of thyroid stimulating hormone as a compensatory response to the known effects of phenobarbital on peripheral thyroxine metabolism and excretion.

Comparative acute toxicity of chlorocitrate and fluorocitrate in dogs.

The high-dose effects of chlorocitrate [(-)-threo-chlorocitric acid] were compared in vivo to another halogenated citrate analog, and a well-known inhibitor of the tricarboxylic acid (TCA) cycle, fluorocitrate. The compounds were given iv to two dogs per sex per group, and a control group received an equimolar amount of citric acid. Chlorocitrate (100 mg/kg) showed TCA cycle inhibition as did fluorocitrate (8 mg/kg) in that both caused depletion of ATP and accumulation of citrate in the liver. Chlorocitrate was a significantly weaker inhibitor of citrate metabolism than fluorocitrate as evidenced by a substantially lower accumulation of serum citrate despite a much higher dose. Both halocitrates produced a similar diabetes-like syndrome (hyperglycemia, glycosuria) mediated by a significant hyperglucagonemia and slight hypoinsulinemia. Chlorocitrate was more potent in this effect and a much greater buildup of plasma lactate ensued (18- versus 3.7-fold increase), enough to explain lethality observed in earlier studies. In contrast, fluorocitrate produced a severe life-threatening hypocalcemia (-30%), and hypercalcuria was observed. This effect on calcium distribution was only minimal with chlorocitrate. Both halocitrates had a similar depressive effect on circulation as evidenced by hypothermia, bradycardia, and elongation of the QT-interval. These changes were considered to be the result of lactic acidosis and the ongoing ion imbalance since heart ATP levels were not depleted.

Serum citrate as a peripheral indicator of fluoroacetate and fluorocitrate toxicity in rats and dogs.

The utility of serum citrate as a peripheral indicator of toxicity was tested as a possible investigational probe for compounds which inhibit citrate metabolism. Fluoroacetate (FA) and its putative toxic metabolite, fluorocitrate (FC), were given to rats and dogs in a series of studies. In rats, 3 mg/kg FA (po) caused a 46% depletion in heart ATP concentrations and a 15-fold increase in heart citrate concentrations. Both of these changes were significantly correlated with a fivefold elevation in serum citrate. In dogs, citrate accumulation was less pronounced (two-to threefold) in the heart and serum, and heart ATP concentrations were not significantly reduced. However, the time course of serum citrate elevations corresponded with the appearance of serious clinical signs and death. In range-finding studies with rats or dogs, serum citrate elevations were always observed in a dose-related pattern according to the dose of FA or FC administered. In contrast to FA, toxic doses of FC did not reduce heart ATP in either rats or dogs, and heart citrate accumulation was less marked than with FA. Both FA and FC produced significant hyperglycemia (twofold increase) in both rats and dogs and high correlations were established between serum glucose and serum citrate in both species. Serum total calcium was reduced (-18%) in dogs treated with FC (8 mg/kg, iv) and a strong inverse correlation to serum citrate was shown. This correlation is biologically meaningful in light of the known chelating effect of citrate on calcium. Clinical manifestations of tremors, tetany, and convulsions in FC-treated dogs were consistent with known symptoms of hypocalcemia. No decrease in total calcium was observed in rats treated with either FA or FC. Despite certain species differences in response to the two fluoro inhibitors, serum citrate levels were always reflective of nontoxic, toxic, or lethal doses.