Effects of teratogenic exposures to Zn2+, Cd2+, Ni2+, Co2+, and Cu2+ on metallothionein and metallothionein-mRNA contents of Xenopus embryos.

Xenopus laevis embryos were analyzed for metallothionein by silver-saturation assay and metallothionein-mRNA by reverse transcriptase/polymerase chain reaction following exposures to the following metal chlorides at levels that caused > 95% malformations and < 7% mortality: Zn2+ (300 microM); Cd2+ (18 microM); Ni2+ (56 microM); Co2+ (1,800 microM); and Cu2+ (5.6 microM). At the beginning of the exposure (stages 8), metallothionein-mRNA and metallothionein levels averaged 2.0 x 10(6) copies/embryo and 19 pmol/embryo, respectively. In control embryos at stages 26, 36, 42, and 46, metallothionein-mRNA content averaged 9, 37, 104, and 97 copies x 10(6)/embryo, and metallothionein content averaged 6, 11, 15, and 18 pmol/embryo. In Zn(2+) -exposed embryos at the same stages, metallothionein-mRNA content averaged 116*, 11,400*, 3,210*, and 14 copies x 10(6)/embryo and metallothionein content averaged 10, 18*, 46*, and 90* pmol/embryo; in Cd(2+)-exposed embryos, metallothionein-mRNA content averaged 22, 7,170*, 1,783*, and 240 copies x 10(6)/embryo and metallothionein content averaged 8, 14, 33*, and 56* pmol/embryo, respectively (*P < 0.05 versus controls). Exposure-response curves (Cd2+, 1-18 microM; Zn2+, 3-300 microM) indicated that Cd2+ was 3- to 5-times more potent than Zn2+, based on metallothionein-mRNA response at stage 36 and metallothionein response at stage 46. In Ni(2+)-, Co(2+)-, or Cu(2+)-exposed embryos, metallothionein-mRNA and metallothionein contents did not differ significantly from controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Intraarticular carcinogenesis bioassays of CoCrMo and TiAlV alloys in rats.

Wear-debris powders of cobalt-chromium-molybdenum (CoCrMo) and titanium-aluminum-vanadium (TiAlV) alloys, which are widely used for orthopedic implants (eg, hip and knee prostheses), were tested for carcinogenic activity following intraarticular administration (20 mg/rat) to groups of 44 male Fischer-344 rats (Charles River Breeding Laboratories, North Wilmington, MA). Control groups received similar intraarticular injections of either a noncarcinogen (manganese powder, negative control rats) or a potent carcinogen (nickel subsulfide powder, positive control rats). The experimental groups of 8-12 rats were observed for 24 months after injection. No local tumors developed at the injection site in the negative control rats or in rats that received the CoCrMo or TiAlV powders; poorly differentiated or pleomorphic sarcomas developed at the injection site in 10 of the 12 positive control rats that were treated with nickel subsulfide. Incidences of primary tumors distant from the injection site did not differ significantly among the experimental groups. This study shows that, under experimental conditions, any carcinogenic activity of CoCrMo or TiAlV wear-debris powders is weak in comparison to nickel subsulfide. Based on this study and observations in other laboratories, intraarticular administration of test materials to rats provides a practical, reliable, and biologically relevant method for carcinogenesis testing of biomaterials used for orthopedic implants.

Lipovitellin 2 beta is the 31 kD Ni(2+)-binding protein (pNiXb) in Xenopus oocytes and embryos.

An Ni(2+)-binding protein (pNiXb, 31 kD) present in mature Xenopus laevis oocytes and in embryos from fertilization in N/F stage 42, was isolated and characterized. After oocytes or embryos were fractionated by PAGE, electroblotted onto nitrocellulose, and probed with 63Ni2+, pNiXb was detected by autoradiography. pNiXb, a yolk protein located in the embryonic gut, was purified from yolk platelets by ammonium sulfate precipitation, delipidation, gel filtration chromatography, and HPLC analysis. During these steps, pNiXb copurified with lipovitellin 2. The N-terminal sequence of purified pNiXb exactly matched that of Xenopus lipovitellin 2 beta, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Since pNiXb and lipovitellin 2 beta agree in N-terminal sequence, amino acid composition, and apparent molecular weight, they appear to be identical. Based on a metal-blot competition assay, the abilities of metal ions to compete with 63Ni2+ for binding to pNiXb were ranked: Zn2+ approximately Cu2+ approximately Co2+ > Cd2+ approximately Mn2+ > Sn2+. This study shows that Xenopus lipovitellin 2 beta is a metal-binding protein in vitro, and raises the possibility that it may function similarly in vivo.

Malformations persist after metamorphosis of Xenopus laevis tadpoles exposed to Ni2+, Co2+, or Cd2+ in FETAX assays.

This study was performed to determine whether malformations induced in Xenopus laevis embryos by exposures to divalent nickel, cobalt, or cadmium chlorides in FETAX assays persist after the tadpoles undergo metamorphosis to juvenile frogs. Embryos were exposed for four days to EC50 concentrations of Ni2+, Co2+, or Cd2+ under the standard conditions of FETAX assays; thereafter, the exposures were discontinued and the tadpoles were kept in aquaria through metamorphosis. Controls were treated similarly, without exposure to metals. At 13 weeks of age, surviving frogs were killed and examined for malformations. Control and metal-exposed groups of Xenopus did not differ significantly in their median ages at metamorphosis, mean body weights, or survival at 13 weeks. Overall incidences of malformations found in Ni(2+)-, Co(2+)-, or Cd(2+)-exposed frogs at 13 weeks of age were 55, 40, and 51%, respectively (P < 0.01 vs. 3% in controls). The malformations of metal-exposed frogs included retinal depigmentation, diastematomyelia, scoliosis, kyphosis, phocomelia, sacro-pelvic and hind-limb deformities, and dysplasia of the heart, kidney, ovary and gut.

Ocular malformations of Xenopus laevis exposed to nickel during embryogenesis.

The pathogenesis of eye anomalies induced by exposure to Ni2+ (as nickel chloride) during embryogenesis was studied in the frog, Xenopus laevis. Eyes of control and Ni(2+)-exposed tadpoles were examined without staining using a dissecting microscope, by light microscopy of histological sections, and by electron microscopy. The ocular abnormalities of Ni(2+)-exposed tadpoles included (a) microphthalmia, (b) hypopigmentation, (c) hernias and cysts of the choroid and retina, and (d) iris coloboma; cataracts were uncommon. The pathogenesis of the ocular lesions appears to involve diffuse or focal dysplasia and loss of the retinal pigment epithelium, with dystrophy of photoreceptor outer segments and protrusion of neuroretina through gaps in the pigment epithelium. This study confirms that Ni2+ is a potent ocular teratogen for Xenopus embryos and points to the retinal pigment epithelium as a primary cellular target for Ni(2+)-induced embryotoxicity.

Mg(2+)-deprivation enhances and Mg(2+)-supplementation diminishes the embryotoxic and teratogenic effects of Ni2+, Co2+, Zn2+, and Cd2+ for frog embryos in the FETAX assay.

The influence of Mg2+ on the embryotoxicity and teratogenicity of Ni2+, Co2+, Zn2+, and Cd2+ for Xenopus embryos was studied by an adaptation of the FETAX protocol. In seven assays, 25 groups of embryos were grown from 5 to 101 hours post-fertilization in FETAX media that contained five graded MgCl2 concentrations (0, 6.2, 62, 620, or 6,200 mumol per L), with or without added NiCl2 (56 mumol per L), CoCl2 (1,800 mumol per L), ZnCl2 (300 mumol per L), or CdCl2 (18 mumol per L). In FETAX assays performed with the standard Mg2+ concentration (620 mumol per L), the incidence of malformations in control embryos averaged 5.4 (SD +/- 1.3) percent; the incidence of malformations in the controls was increased at low Mg2+ concentrations (32 +/- 7 percent at 62 mumol per L; 100 percent at greater than or equal to 6.2 mumol per L). The specified additions of Ni2+, Co2+, Zn2+, or Cd2+ caused death in < 10 under standard conditions (620 mumol Mg2+ per L). Mg(2+)-deprivation greatly enhanced and Mg(2+)-supplementation significantly reduced the incidence and severity of the teratogenic and embryotoxic effects of Ni2+, Co2+, Zn2+, and Cd2+ (p < 0.0001 by analysis of variance [ANOVA]). To explain these findings, the authors speculate that Mg2+ competes with the other divalent metal ions for a carrier mechanism involved in metal absorption or cellular uptake, or for binding to critical molecular targets.

Embryotoxicity and teratogenicity of Cu2+ and Zn2+ for Xenopus laevis, assayed by the FETAX procedure.

Cupric chloride (CuCl2) and zinc chloride (ZnCl2) were tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure in the South African frog, Xenopus laevis. The median teratogenic concentrations (EC50) of Cu2+ and Zn2+ were 2.5 and 40 mumol per L. The median embryolethal concentrations (LC50) of Cu2+ and Zn2+ were 22 and 850 mumol per L. The teratogenic indices (TI = LC50/EC50) were 8.8 for Cu2+ and 21 for Zn2+. Both metal ions were shown to be potent teratogens for Xenopus, causing concentration-related increases of eye, gut, facial, notochord, and cardiac anomalies.

Teratogenicity of cadmium chloride in the South African frog, Xenopus laevis.

The teratogenicity of cadmium chloride was tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure. In five assays, groups of Xenopus embryos were grown in media containing concentrations of 0.75-56 mumol/l; controls were incubated in medium without cadmium chloride. Exposures began 5 h post-fertilization and ended 101 h post-fertilization. In control groups, > 95% of embryos survived at 101 h and the incidence of malformations was < 7%. In Cd(2+)-exposed groups, concentration-dependent mortality and numerous malformations were observed, including gut malrotation, ocular anomalies, bent notochord, misshapen fin, facial dysplasia, cardiac deformities and dermal blisters. Other abnormalities included stunted growth and hypopigmentation. The minimum concentration of cadmium chloride that inhibited growth was 18 mumol/l. The median embryolethal concentration (LC50) was 32 (SE +/- 4) mumol/l; the median teratogenic concentration (EC50) was 3.7 (SE +/- 1) mumol/l; the teratogenic index (TI = LC50/EC50) was 8.6. This study demonstrates that cadmium chloride is teratogenic for Xenopus laevis and provides a standardized experimental model for studying the molecular mechanisms of cadmium teratogenesis.

Embryotoxicity and teratogenicity of cadmium chloride in Xenopus laevis, assayed by the FETAX procedure.

The embryotoxicity and teratogenicity of cadmium chloride (CdCl2) were tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure in the South African frog, Xenopus laevis. In five assays, groups of Xenopus embryos were grown in media that contained CdCl2 at concentrations ranging from 0.75 to 56 mumol per L; control groups were incubated in the same medium without added CdCl2. The exposures to CdCl2 were begun at the blastula stage (five hours post-fertilization) and were terminated 96 hours later (101 hours post-fertilization). The embryos were counted, fixed in formalin, and examined by microscopy to score malformations and measure head-to-tail lengths. In control groups, greater than or equal to 95 percent of the embryos survived at 101 hours post-fertilization, and the incidence of malformations was less than or equal to 7 percent. In Cd(2+)-exposed groups, there was concentration-dependent mortality, and the embryos showed a concentration-related pattern of malformations, including gut malrotation, ocular anomalies, bent notochord, misshapen dorsal fin, facial dysplasia, cardiac deformities, and dermal blisters. Other abnormalities, not categorized as malformations, included stunted growth and hypopigmentation. The minimum concentration of CdCl2 that inhibited growth (MCIG) was 18 mumol per L. The median embryolethal concentration (LC50) of CdCl2 was 32 (SE +/- 4) mumol per L; the median teratogenic concentration (EC50) was 3.7 (SE +/- 1) mumol per L; the teratogenic index (TI = LC50/EC50) was 8.6. This study demonstrates that CdCl2 is teratogenic for Xenopus laevis and provides a standardized experimental model for studies of the molecular mechanisms of cadmium teratogenesis.

Teratogenicity of Ni2+ in Xenopus laevis, assayed by the FETAX procedure.

The teratogenicity of Ni2+ was tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure in the South African frog, Xenopus laevis. In seven assays, beginning at 5 h postfertilization, groups of Xenopus embryos were incubated for 96 h in media that contained Ni2+ (added as NiCl2) at concentrations ranging from 1 x 10(-7) to 3 x 10(-3) mol/L; control groups were incubated in the same medium without added NiCl2. At 101 h postfertilization, surviving embryos were counted, fixed in formalin, and examined by microscopy to determine their developmental stages, malformations, and head-to-tail lengths. In control embryos, survival was greater than or equal to 95% and malformations were less than or equal to 7%. Malformations were found in greater than 95% of embryos exposed to Ni2+ concentrations greater than or equal to 5.6 mumol/L. The most frequent malformations in Ni(2+)-exposed embryos were ocular, skeletal, and intestinal deformities; less common malformations included facial, cardiac, and integumentary deformities. Other abnormalities, not categorized as malformations, included stunted growth, dermal hypopigmentation, and coelomic effusions or hemorrhages. The median embryolethal concentration (LC50) of Ni2+ was 365 (SE +/- 9) mumol/L; the median teratogenic concentration (EC50) was 2.5 (SE +/- 0.1) mumol/L; the Teratogenic Index (TI = LC50/EC50) was 147 (SE +/- 5), indicating that Ni2+ is a potent teratogen for Xenopus laevis. Experiments in which Ni(2+)-exposures were limited to specific 24 h periods showed that Xenopus embryos were most susceptible to Ni(2+)-induced malformations on the second and third days of life, during the most active period of organogenesis.

Teratogenicity of cobalt chloride in Xenopus laevis, assayed by the FETAX procedure.

The teratogenicity of cobalt chloride (CoCl2) was tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure in the South African frog, Xenopus laevis. In five assays, beginning at 5 h post-fertilization, groups of Xenopus embryos were incubated for 96 h in media that contained CoCl2 at concentrations ranging from 1.8 x 10(-6) to 1.8 x 10(-2) mol/L; control groups were incubated in the same medium without added CoCl2. At 101 h post-fertilization, surviving embryos were counted, fixed in formalin, and examined by microscopy to score malformations and measure head-to-tail lengths. In control embryos, survival was greater than or equal to 95% and malformations were less than or equal to 5%. Malformations were found in greater than 99% of embryos exposed to Co2+ levels greater than or equal to 56 mumol/L. Co2+)-exposed embryos showed a concentration-related pattern of malformations, comprising gut malrotation, ocular anomalies, kinked tail, craniofacial dysplasia, cardiac deformities, and dermal blisters. Other concentration-dependent abnormalities, not categorized as malformations, included stunted growth, edema, ventral distention, and hypopigmentation. The median embryolethal concentration (LC50) of CoCl2 was 10.4 (SE +/- 0.4) mmol/L; the median teratogenic concentration (EC50) was 25 (SE +/- 2) mumol/L; the teratogenic index (TI = LC50/EC50) was 416 (SE +/- 13), indicating that CoCl2 is a potent teratogen for Xenopus laevis.

Carcinogenesis bioassays of nickel oxides and nickel-copper oxides by intramuscular administration to Fischer-344 rats.

Five nickel oxides and nickel-copper oxides, with chemical compositions, physicochemical properties, and biological characteristics that were previously reported, were tested for carcinogenicity by administration to groups of male Fischer-344 rats as a single im injection (20 mg Ni/rat). Two additional groups of rats received injections of the glycerol vehicle (Negative Controls) or nickel subsulfide (alpha Ni3S2, 20 mg Ni/rat, Positive Controls). Within the observation period of 2 yr post-injection, the following numbers of sarcomas developed at the injection site: Negative Controls, 0/15; Positive Controls, 15/15; Compound A (INCO black NiO, prepared at less than 650 degrees C), 6/15; Compound B (grey NiO, calcined at 735 degrees C), 0/15; Compound F (green NiO, calcined at 1,045 degrees C), 0/15; Compound H (oxidized Ni-Cu matte, Ni/Cu = 2.5:1, calcined at 850 degrees C), 13/15; Compound I (oxidized Ni-Cu matte, Ni/Cu = 5:1, calcined at 850 degrees C), 15/15. The Ni- and Ni/Cu-oxides that induced sarcomas (Compounds A, H, and I) had measurable dissolution rates in body fluids and were strongly positive in an erythrocytosis stimulation assay, demonstrating Ni bioavailability. Compound A contained detectable Ni[III] and Compounds H and I contained Cu, plus traces of Fe, Co and S, which may all promote oxygen free-radical reactions. In contrast, the compounds that did not induce sarcomas (Compounds B and F) were essentially insoluble in body fluids, did not stimulate erythrocytosis, and were practically devoid of Ni[III], Cu, Fe, Co, or S. Thus, the bioavailability of nickel and the presence of constituents that promote oxygen free-radical reactions evidently influence the carcinogenicity of nickel oxides and related compounds.

Uptake and release of 63Ni2+ by Xenopus embryos during early cleavage stages.

Uptake and release of 63Ni was studied in dejellied Xenopus laevis embryos exposed to 63Ni2+ (0.3-30 mumol/l) for 0.5-h intervals during the period 1-4.5 h post-fertilization (i.e. from first cleavage to early blastula stage). At first cleavage, the mean uptake of 63Ni by embryos was 12-17 times that by non-fertilized eggs, suggesting that conversion of the vitelline envelope to the fertilization envelope enhanced integumental permeability to 63Ni2+. 63Ni uptake by embryos at the 1-2-cell stage averaged 1.8-2.5 times that at the early blastula stage. An average of 5% of total 63Ni in washed embryos was recovered in isolated fertilization envelopes, indicating that 63Ni2+ passed through the envelope into internal compartments. Progressive increases of 63Ni uptake were seen with increasing exposure levels; after exposure during 1-1.5 h post-fertilization to the highest concentration of 63Ni2+ (30 mumol/l), 63Ni uptake averaged 11.4 (SD +/- 5.1) pmol/embryo. Rapid efflux of 63Ni was noted after 63Ni2(+)-exposed embryos were transferred to nickel-free medium; mean 63Ni contents at 0.25 h and 2 h post-exposure diminished to 50% and 15% of the initial values, regardless of the exposure level. The finding that Xenopus embryos are permeable to 63Ni2+ during early cleavage stages provides a convenient experimental system to investigate the embryotoxicity and teratogenicity of nickel.

Toxicity to alveolar macrophages in rats following parenteral injection of nickel chloride.

Alveolar macrophages collected by pulmonary lavage from male Fischer-344 rats at intervals (1-72 hr) after NiCl2 injection (62-500 mumol/kg, sc) were tested by several techniques. Within 1 to 4 hr, the macrophages showed morphological and biochemical signs of activation (hypertrophy, ruffled plasma membrane, increased cyclic AMP concentration, and markedly diminished 5'-nucleotidase activity, assayed by concanavalin A inhibition). Functional impairment (reduced phagocytic activity) was first seen at 24 hr; lipid peroxidation (increased malondialdehyde concentration) was not detected until 48 hr. Dose- and time-related effects of NiCl2 on 5'-nucleotidase activity, phagocytic activity, malondialdehyde concentration, and nickel content of alveolar macrophages were observed 24 to 72 hr postinjection. Diminished cell viability occurred only at 72 hr after the highest dosage of NiCl2. In alveolar macrophages from 63NiCl2-treated rats, 63Ni was located primarily in the cytoplasm, based upon liquid scintillation counting and autoradiography; fractionations of macrophage cytosol by gel filtration chromatography showed that 63Ni was bound to several high- and low-molecular-weight constituents. This study demonstrates that sc administration of NiCl2 to rats caused nickel uptake into and activation of alveolar macrophages, followed by reduced phagocytic capacity. The alveolar macrophage was a cellular target for nickel toxicity following parenteral exposure to NiCl2.