Multiple factors contribute to the toxicity of the aromatic retinoid TTNPB (Ro 13-7410): interactions with the retinoic acid receptors.

The aromatic retinoid, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1 -propenyl] benzoic acid (TTNPB) is 1000-fold more teratogenic than all trans-retinoic acid (tRA) in several species. Factors that partially explain the potency of this retinoid include binding affinities to retinoid nuclear receptors (RARs) in the nanomolar range, reduced affinities for the cytosolic binding proteins (CRABPs), and slow rate of metabolism (M. A. Pignatello, F. C. Kauffman, and A. A. Levin, Toxicol. Appl. Pharmacol. 142, 319-327, 1997). The present work investigates the possible involvement of longer receptor occupancy and increased transcriptional activity of the ligand receptor complex in the greater toxicity of TTNPB. Ligand off-rates from nuclear receptors were determined in nucleosol fractions prepared from COS-1 cells transfected with cDNA encoding the appropriate RAR subtype. When assayed at 10 degrees C, [3H]TTNPB was displaced from the RARs at a significantly faster rate than that of [3H]tRA. The difference in displacement was reduced at 4 degrees C. These observations are consistent with the 10-fold lower affinity of TTNPB vs tRA for RARs and, therefore, do not explain the greater potency of TTNPB. The ability of TTNPB and tRA to activate the RARs was determined using a luciferase reporter gene transfected into JEG-3 cells with the appropriate RAR subtype. The expression of the reporter was driven by a retinoic acid response element (RARE) from the RAR beta gene, which was incorporated into the reporter plasmid. Dose-response for gene activation indicated that the potency of TTNPB and tRA in activating mRAR alpha, beta, and gamma was similar after 24 h with comparable EC50s in the nanomolar range. However, after 72 h, activation by TTNPB was greater than that of tRA as indicated by EC50s and threshold for activation. This study indicates that the higher potency of TTNPB in activating the RARs may be due to slower disappearance of the retinoid and, therefore, is a contributing factor to its greater toxicity.

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.

Multiple factors contribute to the toxicity of the aromatic retinoid, TTNPB (Ro 13-7410): binding affinities and disposition.

The aromatic retinoid (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1 -propenyl] benzoic acid (TTNPB) is 1000-fold more potent as a teratogen than all trans-retinoic acid (tRA) in several species and in the inhibition of chondrogenesis in the mouse limb bud cell culture. Factors responsible for the potency of TTNPB were investigated including binding to nuclear retinoic acid receptors (RARs and RXRs), cytosolic binding proteins (CRABPs), and metabolic disposition of TTNPB. For competitive binding assays and saturation kinetics, nucleosol or cytosol fractions were obtained from COS-1 cells transfected with cDNAs encoding the appropriate nuclear receptor or binding protein. TTNPB binds to RAR alpha, beta, and gamma with Kds in the nanomolar range; however, these binding affinities are 10-fold less than those of tRA. Although the affinities are high for TTNPB, it is unlikely that the binding affinities to nuclear receptors alone account for the potency of TTNPB. The binding affinities of TTNPB for the CRABPs are significantly lower than those of tRA. TTNPB did not compete with [3H]9-cis RA for binding to RXR alpha, beta, or gamma. Mouse limb bud cell cultures, a well characterized model for retinoid teratogenesis, were used to compare the metabolic disposition of TTNPB and tRA. In the media of limb bud cell cultures treated with either retinoid, the disappearance of TTNPB was significantly slower than that of tRA over 72 hr. Both retinoids reached approximately equal concentrations in cell uptake experiments; however, TTNPB disappeared from the limb bud cell at a significantly slower rate than did tRA. Collectively, these results indicate that high affinity binding to RARs, lower affinity to CRABPs, and resistance to metabolism contribute to the potency of TTNPB.

Automated and semiautomated analysis of rat alkaline phosphatase isoenzymes.

A semiautomated quantitative assay for rat serum alkaline phosphatase (ALP) isoenzyme determination was developed, incorporating selective precipitation of bone alkaline phosphatase (BALP) with wheat germ lectin and differential inhibition with levamisole for determination of intestinal alkaline phosphatase (IALP). The assays for each isoenzyme were linear over a broad range of activities. The within-run and between-run coefficients of variation were less than 11% for all 3 isoenzymes. Dilution of serum with saline results in an artifactual overestimation of BALP activity. Comparison of ALP and ALP isoenzyme activity in rats of various ages showed that BALP activity drops dramatically with increasing age of rats. IALP activity is greater in immature rats compared to that in mature rats. While there was no difference between male and female rats at 4 wk of age with regard to total ALP activity and activity of any of the isoenzymes, total ALP activity and activity of the individual isoenzymes were higher in males than in females at most ages over 4 wk. Gavage with corn oil resulted in increased serum IALP activity, and bile duct ligation resulted in increased liver alkaline phosphatase activity. This combined assay for the 3 ALP isoenzymes in rat serum is an efficient means of analysis of large numbers of samples and should increase markedly the specificity of serum ALP activity in identifying the target organ in toxicologic studies when serum ALP activity is increased.

Evaluation of an interleukin-1 receptor antagonist in the rat acetic acid-induced colitis model.

The anti-inflammatory activity of the IL-1 receptor antagonist, IL-1ra, was evaluated in the acetic acid (HOAc)-induced model of colitis in rats. Animals treated with 10 mg/kg IL-1ra or vehicle were evaluated for general health, acute phase response, and colonic in flammation 24 hours after the initiation of inflammation. A significant decrease in the accumulation of neutrophils in the colonic mucosa as measured by myeloperoxidase activity was seen in animals with HOAc induced colitis that were treated intraperitoneally with IL-1ra when compared to animals with colitis that had been treated with vehicle. IL-1ra also reduced colonic necrosis measured grossly, although there was no effect on the histology IL-1ra had a modest effect on the HOAc-induced acute phase response, as indicated by changes in the serum iron, albumin and transferrin, but the results were not statistically significant. The number of circulating erythrocytes and neutrophils was significantly increased in animals with HOAc-induced colitis and treated with IL-1ra, suggesting that IL-1ra under these experimental conditions inhibited the migration of neutrophils to the injured colon and also the overall intestinal necrosis in the colon as assessed by gross pathology. IL-1ra may be useful as an intestinal anti-inflammatory agent.