Copper-requiring Enzymes Research Articles

The physiological and pathophysiological roles of copper in the nervous system.

Copper is a critical trace element in biological systems due the vast number of essential enzymes that require the metal as a cofactor, including cytochrome c oxidase, superoxide dismutase and dopamine-β-hydroxylase. Due its key role in oxidative metabolism, antioxidant defence and neurotransmitter synthesis, copper is particularly important for neuronal development and proper neuronal function. Moreover, increasing evidence suggests that copper also serves important functions in synaptic and network activity, the regulation of circadian rhythms, and arousal. However, it is important to note that because of copper's ability to redox cycle and generate reactive species, cellular levels of the metal must be tightly regulated to meet cellular needs while avoiding copper-induced oxidative stress. Therefore, it is essential that the intricate system of copper transporters, exporters, copper chaperones and copper trafficking proteins function properly and in coordinate fashion. Indeed, disorders of copper metabolism such as Menkes disease and Wilson disease, as well as diseases linked to dysfunction of copper-requiring enzymes, such as SOD1-linked amyotrophic lateral sclerosis, demonstrate the dramatic neurological consequences of altered copper homeostasis. In this review, we explore the physiological importance of copper in the nervous system as well as pathologies related to improper copper handling.

Retinoic Acid-Induced HL-60 Cell Differentiation Is Augmented by Copper Supplementation1–4

The objective of this study was to determine how copper influences the ability of HL-60 cells to differentiate into cells of the granulocytic lineage. We hypothesized that granulopoiesis requires copper because copper-deficient humans become neutropenic. Differentiation of HL-60 cells along the granulocytic lineage with retinoic acid was enhanced by copper. The results showed a greater number of cells were more differentiated when copper was added to the medium for 96 h. The respiratory burst activity of retinoic acid-induced cells was increased by copper supplementation, but intracellular superoxide anion generation was not affected. Supplementation with copper resulted in more cell-associated copper in both noninduced and induced cells; however, the induced cells accumulated three times more copper than the noninduced cells. Even though the amount of copper associated with retinoic acid-treated cells was greater than in untreated cells, the activity of a copper-requiring enzyme, copper/zinc superoxide dismutase, was significantly lower. Copper supplementation increased the activity of this enzyme in both retinoic acid-treated and untreated cells. Cytochrome c oxidase activity was not affected by retinoic acid treatment or by copper supplementation. Copper seems to play a specific role during the early stages of granulocyte differentiation.

Explosive seed dispersal depends on SPL7 to ensure sufficient copper for localized lignin deposition via laccases

Exploding seed pods evolved in the Arabidopsis relative Cardamine hirsuta via morphomechanical innovations that allow the storage and rapid release of elastic energy. Asymmetric lignin deposition within endocarpb cell walls is one such innovation that is required for explosive seed dispersal and evolved in association with the trait. However, the genetic control of this novel lignin pattern is unknown. Here, we identify three lignin-polymerizing laccases, LAC4, 11, and 17, that precisely colocalize with, and are redundantly required for, asymmetric lignification of endocarpb cells. By screening for C. hirsuta mutants with less lignified fruit valves, we found that loss of function of the transcription factor gene SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 7 (SPL7) caused a reduction in endocarpb cell-wall lignification and a consequent reduction in seed dispersal range. SPL7 is a conserved regulator of copper homeostasis and is both necessary and sufficient for copper to accumulate in the fruit. Laccases are copper-requiring enzymes. We discovered that laccase activity in endocarpb cell walls depends on the SPL7 pathway to acclimate to copper deficiency and provide sufficient copper for lignin polymerization. Hence, SPL7 links mineral nutrition to efficient dispersal of the next generation.

Abstract 1000: ATP7A mutation reduces resistance to chemotherapy agents in vitro and in vivo.

Abstract The Menkes protein (ATP7A), a copper-translocating P-type ATPase, is responsible for the loading of copper from the cytosol into secretory compartments of the cell for incorporation into newly synthesized copper-requiring enzymes. Previous studies using cultured cell lines have suggest that ATP7A is able to modulate cellular accumulation of cisplatin, a platinum-containing chemotherapy agent. In this study, we generate isogenic ATP7A-WT and ATP7A-knockout mouse embryonic fibroblast cells line pairs. We demonstrate that ATP7A deletion significantly decreases cellular resistance to cell cycle non-specific platinum agents, cisplatin & carboplatin and cell cycle specific alkaloid agent, vincristine sulfate. In addition, loss of ATP7A sensitizes xenograft tumor growth to cisplatin toxicity. These findings identify the ATP7A copper transporter as an active participant in multidrug resistance pathways in vivo, underscoring this transporter as a potential drug target to augment platinum-based chemotherapeutics. Citation Format: Sha Zhu, Michael J. Petris. ATP7A mutation reduces resistance to chemotherapy agents in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1000. doi:10.1158/1538-7445.AM2013-1000

Mfc1 Is a Novel Forespore Membrane Copper Transporter in Meiotic and Sporulating Cells

To gain insight in the molecular basis of copper homeostasis during meiosis, we have used DNA microarrays to analyze meiotic gene expression in the model yeast Schizosaccharomyces pombe. Profiling data identified a novel meiosis-specific gene, termed mfc1(+), that encodes a putative major facilitator superfamily-type transporter. Although Mfc1 does not exhibit any significant sequence homology with the copper permease Ctr4, it contains four putative copper-binding motifs that are typically found in members of the copper transporter family of copper transporters. Similarly to the ctr4(+) gene, the transcription of mfc1(+) was induced by low concentrations of copper. However, its temporal expression profile during meiosis was distinct to ctr4(+). Whereas Ctr4 was observed at the plasma membrane shortly after induction of meiosis, Mfc1 appeared later in precursor vesicles and, subsequently, at the forespore membrane of ascospores. Using the fluorescent copper-binding tracker Coppersensor-1 (CS1), labile cellular copper was primarily detected in the forespores in an mfc1(+)/mfc1(+) strain, whereas an mfc1Δ/mfc1Δ mutant exhibited an intracellular dispersed punctate distribution of labile copper ions. In addition, the copper amine oxidase Cao1, which localized primarily in the forespores of asci, was fully active in mfc1(+)/mfc1(+) cells, but its activity was drastically reduced in an mfc1Δ/mfc1Δ strain. Furthermore, our data showed that meiotic cells that express the mfc1(+) gene have a distinct developmental advantage over mfc1Δ/mfc1Δ mutant cells when copper is limiting. Taken together, the data reveal that Mfc1 serves to transport copper for accurate and timely meiotic differentiation under copper-limiting conditions.

CtpA, a Copper-translocating P-type ATPase Involved in the Biogenesis of Multiple Copper-requiring Enzymes

The ctpA (ccoI) gene product, a putative inner membrane copper-translocating P1B-type ATPase present in many bacteria, has been shown to be involved only in the cbb(3) assembly in Rhodobacter capsulatus and Bradyrhizobium japonicum. ctpA was disrupted in Rubrivivax gelatinosus, and the mutants showed a drastic decrease in both cbb(3) and caa(3) oxidase activities. Inactivation of ctpA results also in a decrease in the amount of the nitrous oxide reductase, NosZ. This pleiotropic phenotype could be partially rescued by excess copper in the medium, indicating that CtpA is likely a copper transporter that supplies copper-requiring proteins in the membrane with this metal. Although CtpA shares significant sequence homologies with the homeostasis copper efflux P1B-type ATPases including the bacterial CopA and the human ATP7A and ATP7B, disruption of ctpA did not result in any sensitivity to excess copper. This indicates that the CtpA is not crucial for copper tolerance but is involved in the assembly of membrane and periplasmic copper enzymes in this bacterium. The potential roles of CtpA in bacteria in comparison with CopA are discussed.

Deletion of hepatic Ctr1 reveals its function in copper acquisition and compensatory mechanisms for copper homeostasis

Copper is a vital trace element required for normal growth and development of many organisms. To determine the roles for copper transporter 1 (Ctr1) in hepatic copper metabolism and the contribution of the liver to systemic copper homeostasis, we have generated and characterized mice in which Ctr1 is deleted specifically in the liver. These mice express less than 10% residual Ctr1 protein in the liver and exhibit a small but significant growth retardation, which disappears with age. Hepatic copper concentrations and the activities of copper-requiring enzymes are reduced; however, mild copper deficiency relative to Ctr1 protein deficit indicates compensatory mechanisms for copper metabolism. Copper concentrations of other organs did not alter despite the defect in hepatic copper uptake. Whereas biliary copper excretion is reduced, urinary copper concentration in these mice is higher than that of control mice. Our data indicate that Ctr1 plays a critical role in copper acquisition in the liver, and, when Ctr1 expression is compromised, compensatory mechanisms facilitate copper uptake and/or retention in the liver and excretion of copper via urine.

Copper deficiency in potato dextrose agar causes reduced pigmentation in cultures of various fungi

Potato dextrose agar (PDA) is one of the most commonly used media for the isolation and cultivation of fungi, with morphological features and pigmentation in culture often being important for identification of cultures. Cultivation of various fungi on different brands and batches of powdered (commercial) potato dextrose media revealed deficient pigmentation in five of 10 media tested. Reduced pigmentation on these media was correlated with low levels of copper and colony colour was restored by the addition of copper. Deficient pigmentation was most pronounced when copper levels in the medium were below 50 ng mL(-1) (50 p.p.b.; 0.8 microM). Differences in pigmentation and laccase activity of spore and mycelial preparations were quantified for representative species belonging to the genera Aspergillus, Fusarium, Trichoderma, Cladosporium and Penicillium grown on PDA containing different amounts of copper. A strong positive correlation between laccase activity and copper levels was observed. Differences were also found between batches of raw potatoes, with organically cultivated tubers having higher copper levels than those grown by conventional methods, possibly because of the use of copper-based fungicides in the former case. Routine addition of 1000 ng mL(-1) copper (or standard trace element solutions) to PDA and other undefined media is advised to avoid atypical culture pigmentation and possibly other consequences of reduced activity of copper-requiring enzymes.

Trafficking of a Secretory Granule Membrane Protein Is Sensitive to Copper

We explored the effect of copper availability on the synthesis and trafficking of peptidylglycine alpha-amidating monooxygenase (PAM), an essential cuproenzyme whose catalytic domains function in the lumen of peptide-containing secretory granules. Corticotrope tumor cell lines expressing integral membrane and soluble forms of PAM were depleted of copper using bathocuproinedisulfonic acid or loaded with copper by incubation with CuCl(2). Depleting cellular copper stimulates basal secretion of soluble enzyme produced by endoproteolytic cleavage of PAM in secretory granules and transit of membrane PAM though the endocytic pathway and back into secretory granules. Unlike many cuproenzymes, lack of copper does not lead to instability of PAM. Copper loading decreases cleavage of PAM in secretory granules, secretion of soluble enzyme, and the return of internalized PAM to secretory granules. The trafficking and stability of the soluble, luminal domain of PAM and truncated membrane PAM lacking a cytosolic domain are not affected by copper availability. Taken together, our data demonstrate a role for copper-sensitive cytosolic machinery in directing endocytosed membrane PAM back to secretory granules or to a degradative pathway. The response of PAM to lack of copper suggests that it facilitates copper homeostasis.

Lead and Copper Levels in Tea Samples Marketed in Beijing, China

Next to water, tea is the most widely consumed beverage (Marcos et al., 1998) because of its taste aroma and health benefits. On the basis of extensive animal experiments and some epidemiologic data, tea is considered to have numerous beneficial effects on the prevention of many diseases, including skin cancer (Katharine, 2001), Parkinson’s disease (Richard, 2001), myocardial infarction (Cheng, 2003), and coronary artery disease (Hirano et al., 2003). Tea is produced from the leaves of the tropical evergreen Camellia sinensis, which is indigenous to China. In China, tea sipping is an integral part of the culture. In the world, China is a major tea producer, supplying 23.3% of the world’s tea. However, the rapid industrialization of China over the past two decades has caused increased contamination to the environment, which inevitably has brought contamination to tea. During the growth period of the tea plant and during tea processing, tea itself can be contaminated by heavy metals, such as lead and copper, which might increase the metal body burden in humans. Lead is a physiologic and neurologic toxin that can affect almost every organ and system in the human body. It can reduce cognitive development and intellectual performance in children and damage kidneys and the reproductive system. Copper is an essential element, with both deficiencies and excesses associated with impaired health. Copper deficiency is known to cause various physiologic disorders such as anemia and bone abnormalities (Uauy et al., 1998) resulting from decreased activity of the copper-requiring enzymes. Copper excess can cause hepatic and kidney damage, hemolytic anemia (Evans, 1973), and methaemoglobinemia (Chugh et al., 1975). Therefore, after the pesticide contamination issue, heavy metal contamination in teas has become another noteworthy issue because it is related directly to heath and disease. The current study aimed to determine the lead and copper concentrations in tea samples collected from the Beijing market, and to give an overview of the current safety situation of teas marketed in Beijing, host of the Olympic Games in the year 2008.

The effects of mitochondrial iron homeostasis on cofactor specificity of superoxide dismutase 2

Many metalloproteins have the capacity to bind diverse metals, but in living cells connect only with their cognate metal cofactor. In eukaryotes, this metal specificity can be achieved through metal-specific metallochaperone proteins. Herein, we describe a mechanism whereby Saccharomyces cerevisiae manganese superoxide dismutase (SOD2) preferentially binds manganese over iron based on the differential bioavailability of these ions within mitochondria. The bulk of mitochondrial iron is normally unavailable to SOD2, but when mitochondrial iron homeostasis is disrupted, for example, by mutations in S. cerevisiae mtm1, ssq1 and grx5, iron accumulates in a reactive form that potently competes with manganese for binding to SOD2, inactivating the enzyme. Studies in mtm1 mutants indicate that iron inactivation of SOD2 involves the Mrs3p/Mrs4p mitochondrial carriers and iron-binding frataxin (Yfh1p). A small pool of SOD2-reactive iron also exists under normal iron homeostasis conditions and binds SOD2 when mitochondrial manganese is low. The ability to control this reactive pool of iron is critical to maintaining SOD2 activity and has important potential implications for oxidative stress in disorders of iron overload.

Menkes protein contributes to the function of peptidylglycine alpha-amidating monooxygenase.

Menkes protein (ATP7A) is a P-type ATPase involved in copper uptake and homeostasis. Disturbed copper homeostasis occurs in patients with Menkes disease, an X-linked disorder characterized by mental retardation, neurodegeneration, connective tissue disorders, and early childhood death. Mutations in ATP7A result in malfunction of copper-requiring enzymes, such as tyrosinase and copper/zinc superoxide dismutase. The first step of the two-step amidation reaction carried out by peptidylglycine alpha-amidating monooxygenase (PAM) also requires copper. We used tissue from wild-type rats and mice and an ATP7A-specific antibody to determine that ATP7A is expressed at high levels in tissues expressing high levels of PAM. ATP7A is largely localized to the trans Golgi network in pituitary endocrine cells. The Atp7a mouse, bearing a mutation in the Atp7a gene, is an excellent model system for examining the consequences of ATP7A malfunction. Despite normal levels of PAM protein, levels of several amidated peptides were reduced in pituitary and brain extracts of Atp7a mice, demonstrating that PAM function is compromised when ATP7A is inactive. Based on these results, we conclude that a reduction in the ability of PAM to produce bioactive end-products involved in neuronal growth and development could contribute to many of the biological effects associated with Menkes disease.

Ctr6, a Vacuolar Membrane Copper Transporter inSchizosaccharomyces pombe

Aerobic organisms possess efficient systems for the transport of copper. This involves transporters that mediate the passage of copper across biological membranes to reach essential intracellular copper-requiring enzymes. In this report, we identify a new copper transporter in Schizosaccharomyces pombe, encoded by the ctr6(+) gene. The transcription of ctr6(+) is induced under copper-limiting conditions. This regulation is mediated by the cis-acting promoter element CuSE (copper-signaling element) through the copper-sensing transcription factor Cuf1. An S. pombe strain bearing a disrupted ctr6Delta allele displays a strong reduction of copper,zinc superoxide dismutase activity. When the ctr6+ gene is overexpressed from the thiamine-inducible nmt1(+) promoter, the cells are unable to grow on medium containing exogenous copper. Surprisingly, this copper-sensitive growth phenotype is not due to an increase of copper uptake at the cell surface. Instead, copper delivery across the plasma membrane is reduced. Consistently, this results in repressing ctr4(+) gene expression. By using a functional ctr6(+) epitope-tagged allele expressed under the control of its own promoter, we localize the Ctr6 protein on the membrane of vacuoles. Furthermore, we demonstrate that Ctr6 is an integral membrane protein that can trimerize. Moreover, we show that Ctr6 harbors a putative copper-binding Met-X-His-Cys-X-Met-X-Met motif in the amino terminus, which is essential for its function. Our findings suggest that under conditions in which copper is scarce, Ctr6 is required as a means to mobilize stored copper from the vacuole to the cytosol.

Characterization of Mouse Embryonic Cells Deficient in the Ctr1 High Affinity Copper Transporter: IDENTIFICATION OF A Ctr1-INDEPENDENT COPPER TRANSPORT SYSTEM

The trace metal copper is an essential cofactor for a number of enzymes that have critical roles in biological processes, but it is highly toxic when allowed to accumulate in excess of cellular needs. Consequently, homeostatic copper metabolism is maintained by molecules involved in copper uptake, distribution, excretion, and incorporation into copper-requiring enzymes. Previously, we reported that overexpression of the human or mouse Ctr1 copper transporter stimulates copper uptake in mammalian cells, and deletion of one Ctr1 allele in mice gives rise to tissue-specific defects in copper accumulation and in the activities of copper-dependent enzymes. To investigate the physiological roles for mammalian Ctr1 protein in cellular copper metabolism, we characterized wild type, Ctr1 heterozygous, and Ctr1 homozygous knock-out cells isolated from embryos obtained by the inter-cross of Ctr1 heterozygous mice. Ctr1-deficient mouse embryonic cells are viable but exhibit significant defects in copper uptake and accumulation and in copper-dependent enzyme activities. Interestingly, Ctr1-deficient cells exhibit approximately 30% residual copper transport activity that is saturable, with a K(m) of approximately 10 microm, with biochemical features distinct from that of Ctr1. These observations demonstrate that, although Ctr1 is critical for both cellular copper uptake and embryonic development, mammals possess additional biochemically distinct functional copper transport activities.

Walking the copper trail

New work from a group of researchers at the Northwestern University, Evanston (IL, USA) provides the first image of the docked complex between a copper chaperone and a copper-requiring enzyme, namely superoxide dismutase (SOD). Found in a range of organisms, copper chaperones are soluble metal receptors that function to deliver copper ions to specific copper-dependent proteins within the cell, in a manner that prevents cytoplasmic exposure of these ions and therefore oxidative damage. The study, published in Nature Structural Biology [(2001) 8, 751–755], shows the 2.9 Å resolution structure of a SOD–metallochaperone complex comprising one monomer of each protein. The researchers observed striking conformational rearrangements in both the chaperone and the target enzyme upon complex formation. Besides the purely biochemical importance, the finding has potential medical relevance because mutant forms of SOD with aberrant copper chemistry are associated with severe neurodegenerative disorders, such as familial amyothrophic lateral sclerosis (also known as Lou Gehrig's disease). Scientists hope that further understanding of the biochemistry of copper delivery, and of the function of copper, in SOD mutants, will lead to an effective approach to intervention in the destructive effects of this disease. AR

Detection of genetic defects in Menkes disease by direct mutation analysis and its implications in carrier diagnosis.

Menkes disease (McKusick 309400) is an X-linked recessive disturbance of copper metabolism leading to death in early childhood. The main phenotypic features are progressive psychomotor retardation, peculiar hair, hypopigmentation and connective tissue disturbances. Most of the clinical symptoms can be explained by malfunctioning of one or more of the known copper-requiring enzymes (Horn et al 1992). Copper accumulation in cultured cells and in tissues provides the ultimate biochemical test for both prenatal and postnatal diagnosis. The Menkes locus has been mapped to Xq13.3 (Tumer et al 1992) and the gene defective in Menkes disease has recently been isolated by three different groups including ours (Chelly et al 1993; Mercer et al 1993; Vulpe et al 1993)

Extracellular matrix 4: The elastic fiber

The elastic properties of many tissues such as the lung, dermis, and large blood vessels are due to the presence of elastic fibers in the extracellular space. These fibers have been shown by biochemical and ultrastructural analysis to be composed of two distinct components, a more abundant amorphous component and a 10-12 nm microfibrillar component, which is located primarily around the periphery of the amorphous component. The protein elastin makes up the highly insoluble amorphous component and is responsible for the elastic properties. Elastin is found throughout the vertebrate kingdom and possesses an unusual chemical composition rich in glycine, proline, and hydrophobic amino acids, consonant with its characteristic physical properties. The 72-kDa biosynthetic precursor, tropoelastin, is secreted into the extracellular space where it becomes highly cross-linked into a rubber-like network through the activity of the copper-requiring enzyme lysyl oxidase. Analysis of the elastin gene has demonstrated that hydrophobic and cross-linking domains are encoded in separate exons and that there is significant alternative splicing, resulting in multiple isoforms of tropoelastin. The elastin gene promoter contains many potential binding sites for various modulating factors indicative of a complex pattern of transcriptional regulation. The microfibrils contain several proteins, including fibrillin, and probably act as an organizing scaffold in the formation of the elastin network. There appears to be a fibrillin gene family in which each protein contains multiple repeats of a motif previously found in epidermal growth factor and a second motif observed in transforming growth factor beta 1-binding protein. Mutations in the fibrillin gene located on human chromosome 15 have been strongly implicated as the cause of the Marfan syndrome.

Cu/Zn superoxide dismutase activity does not parallel copper levels in copper supplemented HL-60 cells.

The objective of this research was to develop a method for measuring Cu/Zn-superoxide dismutase (Cu/Zn-SOD) (E.C. 1.15.1.1) in HL-60 cells and subsequently examine the relationship between cellular copper levels and the activity of this copper-requiring enzyme. In cells such as the neutrophil or HL-60 promyelocyte cell line, the activity of Cu/Zn-SOD cannot be measured because of an increase in the oxidation rate of the substrate by some unknown compound in the cells. Others have utilized heat treatment to inactivate the responsible compounds, however, we found that heat treatment of HL-60 cells resulted in a loss of over half of the activity of the enzyme. The method described here utilizes sodium azide to inhibit the substance(s) that are responsible for the enhanced rate of pyrogallol's oxidation. Gel filtration data confirmed that the compound responsible for the enhanced rate of pyrogallol oxidation was sensitive to azide and did not affect Cu/Zn-SOD activity. When HL-60 cells were incubated with various levels of copper, Cu/Zn-SOD activity did not reflect the cellular copper levels.

HL-60 Cells Can Be Made Copper Deficient by Incubating with Tetraethylenepentamine , ,

A system for studying copper deficiency was developed in a cell culture model. HL-60 cells were incubated with three chelators known to bind copper. One chelator, tetraethylenepentamine (TEPA), reduced cellular copper levels and the activities of two copper-requiring enzymes, Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and cytochrome c oxidase. The specificity of the chelator was assessed by incubating cells with both copper and TEPA and, in other experiments, with zinc and TEPA. Copper levels, Cu/Zn-SOD activity and cytochrome c oxidase activity were restored to control values when copper and TEPA were added to cultures simultaneously, indicating the TEPA was responsible for reducing these aspects of copper metabolism. Incubating with both zinc and TEPA reduced copper levels relative to the control, but did not reduce Cu/Zn-SOD activity to the same extent as TEPA alone. The chelation of copper was a time-dependent process that was stable for at least 4 d. Cell growth and viability were not affected by TEPA. Respiratory burst activity, an indicator of differentiation, was not affected by TEPA, demonstrating that the reduction of Cu/Zn-SOD activity was due to copper chelation and not due to changes in Cu/Zn-SOD protein levels that occur during differentiation. Loss of copper, as well as a reduction of the activity of two copper-requiring enzymes, provides evidence that TEPA is a useful compound for creating a functional copper deficiency in cell culture.

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

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