Structure-Activity Relationships for Osteolathyrism. I. Effects of Altering the Semicarbazide Structure

Abstract

The toxic and osteolathyrogenic effects of 15 chemicals reflecting modifications of semicarbazide [H2N-N(H)-C(=O)-NH2] were evaluated using the Frog Embryo Teratogenesis Assay: Xenopus (FETAX). Osteolathyrism, the failure of connective tissue fibers to cross-link correctly, is expressed as gross alterations in the long axis of the embryo and a sinusoidal configuration of the notochord. Alteration (addition or subtraction) at the hydrazino (H2NNH-) end of the molecule sharply reduces osteolathyrogenic activity. Alteration at the amino (H2N-) end of the molecule results in a graded response. The replacement of oxygen with sulfur causes no loss of osteolathyrogenic activity. Among the myriad events constituting vertebrate organogenesis is the formation of intercellular connective tissue elements such as collagen and elastin. Failure of this developing collagen and elastin to polymerize properly is termed osteolathyrism (Selye, 1957). An osteolathyrogenic agent, therefore, is any chemical that produces increased connective tissue fragility and collagen extractability (Levene, 1973). This is thought to result because of inhibition of the copper-requiring enzyme lysyl oxidase (Harris et al., 1974) necessary for extracellular cross-linking in both collagen (Bailey, 1968) and elastin (Barrow et al., 1974) maturation. Studies of experimentally-induced osteolathyrism have utilized a number of 1These studies were supported by National Institute of Environmental Health Sciences Grant R01 ES04209 to T.W.S. 2 Supported by the U.S. Environmental Protection Agency under Cooperative Agreement CR813158 with T.W.S. 3 Supported in part by U.S. Environmental Protection Agency under Cooperative Agreement CR-813158 with T.W.S. and The University of Tennessee, College of Veterinary Medicine, Institute of Agriculture, Center of Excellence in Livestock Diseases and Human Health. TRANS. AM. MICROSC. SOC., 107(2): 113-126. 1988. ? Copyright, 1988, by the American Microscopical Society, Inc. This content downloaded from 207.46.13.110 on Fri, 09 Dec 2016 05:44:01 UTC All use subject to http://about.jstor.org/terms TRANS. AM. MICROSC. SOC. natural and synthetic chemicals (Levene, 1961). One such compound is semicarbazide. Neuman et al. (1956), in an effort to produce liver damage in chick embryos similar to that observed following administration of aminoguanidine, injected semicarbazide into incubating eggs. Although semicarbazide had no effect on the developing liver, it did induce skeletal abnormalities similar to those described by Chang et al. (1954) following exposure to beta-aminopropionitrile. This initial documentation of the osteolathyrogenic potential of semicarbazide was followed by the studies of Dasler (1958) using Sprague-Dawley rats and by Levy (1959) using salamander and toad embryos. These latter investigations described axial malformations in treated individuals and suggested that semicarbazide is a strong direct-acting teratogen. Recent work in our laboratory has documented in-depth the embryotoxic and teratogenic effects of semicarbazide (Schultz et al., 1985) and benzoyl hydrazine (Riggin & Schultz, 1986) using a frog embryo assay (FETAX). An in vitro bioassay using early embryos of Xenopus laevis and referred to as FETAX (Frog Embryo Teratogenesis Assay: Xenopus) was developed as a screening system (Dumont et al., 1983). FETAX has several advantages as a teratogenic screening system, including cost and time effectiveness. In addition, the frog embryo as a classic experimental embryological system is developmentally relevant to mammals undergoing events (i.e., cleavage, gastrulation, organogenesis) comparable to those of other vertebrates. In this investigation, we used FETAX to explore structure-activity relationships by examining the relative toxicity and osteolathyrogenicity of a series of 15 chemicals that are structural modifications of semicarbazide. MATERIALS AND METHODS Embryos were obtained from adult pairs of Xenopus laevis injected with human chorionic gonadotropin to induce ovulation and amplexus. Healthyappearing early to mid-blastula embryos were selected individually using a dissecting microscope. Groups of 25 embryos were each placed in 60 x 15mm Pyrex dishes in 10-ml solutions of graduated concentrations of the test chemical and FETAX solution (625 mg NaCl, 96 mg NaHCO3, 30 mg KCl, 15 mg CaC12, 60 mg CaSO4 2H20, and 75 mg MgSO4 per liter of distilled water) (Dawson & Bantle, 1987). Control embryos were placed in 100% FETAX solution. The tested chemicals were structural modifications of semicarbazide (Table I). Each was purchased from a commercial source (Aldrich Chemical Company, Milwaukee, Wisconsin, U.S.A.) and not repurified prior to use. Stock solutions of each test chemical at a concentration equal to the highest concentration tested were made fresh for each assay using FETAX solution as the diluent. The stock solutions were adjusted to pH 7.2 and final test solutions were made by appropriate dilutions of stock solutions. For concentration-effect experiments, exposure was for 96 h at 23?C ? 0.5 in an incubator with a 12L/12D photoperiod. A minimum of three replicates was tested for each concentration series. A 96 h, surviving embryos were fixed in 10% neutral buffered formalin. The mortality, number and types of mal114 This content downloaded from 207.46.13.110 on Fri, 09 Dec 2016 05:44:01 UTC All use subject to http://about.jstor.org/terms VOL. 107, NO. 2, APRIL 1988