Arthur A. Levin

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.

Distinct binding determinants for 9-cis retinoic acid are located within AF-2 of retinoic acid receptor alpha.

Retinoids exert their physiological action by interacting with two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which regulate gene expression by forming transcriptionally active heterodimeric RAR/RXR or homodimeric RXR/RXR complexes on DNA. Retinoid receptor activity resides in several regions, including the DNA and ligand binding domains, a dimerization interface, and both a ligand-independent (AF-1) and a ligand-dependent (AF-2) transactivation function. While 9-cis retinoic acid (RA) alone is the cognate ligand for the RXRs, both 9-cis RA and all-trans RA (t-RA) compete for binding with high affinity to the RARs. This latter observation suggested to us that the two isomers may interact with a common binding site. Here we report that RAR alpha has two distinct but overlapping binding sites for 9-cis RA and t-RA. Truncation of a human RAR alpha to 419 amino acids yields a receptor that binds both t-RA and 9-cis RA with high affinity, but truncation to amino acid 404 yields a mutant receptor that binds only t-RA with high affinity. Remarkably, this region also defines a C-terminal boundary for AF-2, as addition of amino acids 405 to 419 restores receptor-mediated gene activity to a truncated human RAR alpha lacking this region. It is interesting to speculate that binding of retinoid stereoisomers to unique sites within an RAR may function with AF-2 to cause differential activation of retinoid-responsive gene pathways.

The discovery of 9-cis retinoic acid. A hormone that binds the retinoid-X receptor

Two classes of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid-X receptors (RXRs), mediate the physiologic activity of retinoids. The RXRs can form biologically active heterodimers with the RARs and with other nuclear receptors, including the vitamin-D, thyroid hormone, and peroxisome proliferator-activated receptors. Thus, the RXRs may play a pivotal role in modulating the action of other hormones or ligands. The RXRs were originally classified as orphan receptors whose cognate ligand was unknown until recently, when 9-cis retinoic acid (9-cis RA) was discovered to bind directly and activate this family of receptors. Since 9-cis RA also binds and activates the RARs, it is interesting to speculate that this natural ligand may regulate a broad range of physiologic processes by mediating transcriptional activity through both RAR- and RXR-linked pathways.

Retinoids for the future: Investigational approaches for the identification of new compounds

One desired goal for future retinoid development is the dissociation of therapeutic benefit from teratogenic risk. It is not known if this result can be achieved, but increasing our understanding of the molecular mechanisms of retinoid action offers rational approaches to the attainment of this goal. The basis for these new approaches is the recent discovery of nuclear receptors for all-trans-retinoic acid and for the 9-cis form of retinoic acid. These receptors, like the steroid hormone receptors, are ligand-dependent transcription factors. Retinoids act by binding to these receptors and modulating specific gene pathways that ultimately control cell differentiation and development. Retinoic acid is required for normal embryonic development, and retinoic acid concentrations are regulated in the developing embryo. The malformations associated with in utero exposure to retinoids might be the result of inappropriate activation of specific receptors and inappropriate modulation of specific gene pathways. At least three nuclear receptors for all-trans-retinoic acid and three nuclear receptors for the 9-cis form of retinoic acid are known to exist, with the two receptor subfamilies interacting to form heterodimers. This multiplicity of receptors and receptor interactions suggests that differences may exist among receptors in the biologic effects they mediate. Although some retinoid ligands are not specific, others are highly selective for one particular receptor. Contrasting the effects of ligands with different specificities should increase understanding of the biologic responses associated with specific receptors and efforts to dissociate therapeutic activity from undesired effects.

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.

Receptors as tools for understanding the toxicity of retinoids

Retinoids are derivatives of vitamin A that have numerous biologic activities including induction of epithelial differentiation, pattern formation in embryos, and maintenance of spermatogenesis. Retinoids are used to treat various dermatologic maladies and specific forms of cancer but their use is limited by toxic liabilities: most notably teratogenesis. Retinoids interact with 2 families of receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). The RARs and RXRs bind and transactivate distinct response elements of numerous genes. This multiplicity of receptors and gene products provides us with multiple targets for developing novel receptor-selective agonists. We are exploiting our knowledge of ligand receptor interactions to design better, more selective drugs and to understand the toxicity of retinoids and their metabolic products.

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.