Short-term effects and teratogenicity of heptanol on embryos of Xenopus laevis

This study examined teratogenic and short-term effects of heptanol on Xenopus embryos. Embryos were exposed for 5 h to 2.5 mM heptanol at different developmental stages. Teratogenic effects were found in embryos treated from cleavage to early neurula stages. Other heptanol concentrations, shorter exposure time, and the effect of temperature were also assayed. Short-term effects of hepatanol exposure were studied during cleavage and all treated blastulae showed cell separation with delaminated blastomeres inside the blastocel. Disruption of cell adhesion in addition to the uncoupling effect could account for heptanol teratogenicity.     

Atrazine and malathion shorten the maturation process of Xenopus laevis oocytes and have an adverse effect on early embryo development

The use of pesticides has a negative impact on the environment. Amphibians have long been regarded as indicator species to pollutants due to their permeable skin and sensitivity to the environment. Studies have shown that population declines of some amphibians are directly linked with exposure to agricultural contaminants. In the past, much of the studies have focused on the toxic effect of contaminants on larvae (tadpoles), juvenile and adult frogs. However, due to the nature of their life cycle, amphibian eggs and early embryos are especially susceptible to the contaminants, and any alteration during the early reproductive stages may have a profound effect on the health and population of amphibians. In this study, we analyzed the effect of atrazine and malathion, two commonly used pesticides, on Xenopus laevis oocyte maturation and early embryogenesis. We found that both atrazine and malathion shortened the frog oocyte maturation process and resulted in reduced Emi2 levels at cytostatic factor-mediated metaphase arrest, and a high level of Emi2 is critically important for oocyte maturation. Furthermore, frog embryos fertilized under the influence of atrazine and/or malathion displayed a higher rate of abnormal division that eventually led to embryo death during early embryogenesis.     

Paraquat induced embryotoxicity on Xenopus laevis development

Paraquat (PQ, 1-1'-dimethyl-4,4'-bipyridylium dichloride) is an effective herbicide widely used in agriculture with a rate of application for aquatic weed control ranging from 0.1 to 2 parts per million. Considering its wide-spread presence in Italian wetlands, we studied its embryotoxic effects with the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). The percentage of mortality as well as the percentage of malformed larvae was investigated by probit analysis. The results showed that PQ was highly embryolethal. From a LC(50) of 0.138 mg/l and TC(50) of 0.267 mg/l, a TI(50) of 0.52 was derived; indicating that PQ is to be considered a non-teratogenic compound. Remarkable was the presence of a specific malformation, classified as ventral tail flexure, in the 29% of living larvae exposed to 0.125 mg/l PQ concentration. Their histological examination showed several zones of abnormal somites containing severely affected myocytes. This confirmed the molecular mechanism of PQ toxicity in cell microfilaments. Even at the lowest concentration of 0.0625 mg/l, the difference between the mean head-tail length of control and exposed larvae was statistically significant, a sign of growth retardation. All our data emphasize that PQ must be consider highly embryotoxic on amphibian development, and suggest that this herbicide should be strictly regulated in weed control programs.     

Comparative sensitivity of Xenopus tropicalis and Xenopus laevis as test species for the FETAX model

The use of Xenopus tropicalis as an alternative test species for the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) model was evaluated. Five test substances with varying developmental toxicity potential were evaluated using the traditional FETAX (X. laevis) and a modified assay to accommodate the use of X. tropicalis. Two separate definitive concentration-response tests were performed with ethanol, semicarbazide, copper, 6-aminonicotinamide (6-AN) and atrazine. In order to evaluate the impact of culture temperature on species sensitivity, tests with X. tropicalis were performed concurrently at 27 degrees C (optimum temperature) and 23 degrees C (traditional FETAX temperature). Tests with X. laevis were performed only at 23 degrees C (optimal for X. laevis). Regardless of culture temperature, tests with X. laevis and X. tropicalis indicated that each of the compounds possessed teratogenic potential: semicarbazide>6-AN>atrazine approximately copper>ethanol. Results from these studies indicated that these two species responded similarly to the test compounds. Xenopus tropicalis was somewhat less sensitive to 6-AN, semicarbizide and atrazine when tested at 27 degrees C than at 23 degrees C. Ethanol, copper and atrazine were reasonably equipotent in X. tropicalis and X. laevis in terms of teratogenic response (EC50 for malformation), whereas 6-AN and semicarbizide were less potent in X. tropicalis than in X. laevis. No substantial differences (order of magnitude) in potency were observed between X. laevis and X. tropicalis with any of the test materials evaluated. Malformation syndromes induced in both species were similar in X. tropicalis and X. laevis. These results suggested that X. tropicalis could be used effectively as a test organism for the FETAX model.     

Effects of perfluorooctanesulfonate and perfluorobutanesulfonate on the growth and sexual development of Xenopus laevis

Perfluorobutanesulfonate (PFBS), as a substitute for perfluorooctanesulfonate (PFOS), is widespread in the environment and biotic samples as well as PFOS. To investigate effects of PFOS and PFBS on the growth and sexual development of amphibians, we exposed Xenopus laevis tadpoles at a series of concentrations of PFOS and PFBS (0.1; 1; 100; 1,000 μg/l) as well as 17-beta-estradiol (E2, 100 ng/l) and 5 alpha-androstan-17-beta-ol-3-one (DHT, 100 ng/l) from stage 46/47 to 2 months postmetamorphosis. We found that neither PFOS nor PFBS had a significant effect on the survival and growth. However, they caused hepatohistological impairment at higher concentrations (100; 1,000 μg/l). Unlike E2, PFOS at all concentrations did not alter the sex ratio and induce intersex, but caused degeneration of spermatogonia in testes except for the lowest concentration. PFBS had no effect on the sex ratio and gonadal histology. PFOS and PFBS promoted expression of estrogen receptor (ER) and androgen receptor (AR), but not affected aromatase expression in the brain. The increase in expression of ER and AR suggests an increase in the responsiveness to the corresponding sex hormone and potential effects on sexual development. Our results show that PFBS as well as PFOS have adverse effects on hepato-histology and sexual development on X. laevis. Also, PFOS- and PFBS-induced increase in ER and AR expression highlights the need to further study effects of PFOS and PFBS on subsequently gonadal development, sexual dimorphism, and secondary sex characteristics in X. laevis. It is debatable that PFBS is widely used as a substitute of PFOS.     

Physical effects of chlorpyrifos on two stages of xenopus laevis

The effects of a 96-h static exposure to chlorpyrifos were examined in two developmental stages of Xenopus laevis (premetamorph and metamorph). Measures of effect included body length, mass, and swimming ability. Most parameters indicate that metamorphs were more sensitive than premetamorphs. For larvae exposed as premetamorphs, body length and mass were significantly lower after exposure to 0.001 mg/L and 0.1 mg/L, respectively. Premetamorph swimming activity was increased by a concentration of 0.001 mg/L. Body length and swimming activity were the most sensitive endpoints in metamorphs, showing effects at 0.0001 mg/L. The corresponding body mass was not affected until exposure to 0.1 mg/L. Based on recent environmental data, chlorpyrifos has a 0.1-32.8% probability of exceeding a 96-h time-weighted average of 0.0001 mg/L in U.S. surface waters.     

Feminizing/demasculinizing effects of polychlorinated biphenyls on the secondary sexual development of Xenopus laevis

We have previously demonstrated that polychlorinated biphenyls (PCBs) have caused phenotypic feminization/demasculinization of gonadal development in Xenopus laevis. Whether PCBs affect secondary sexual development has remained unknown. In this study, X. laevis tadpoles were exposed to Aroclor1254 and PCB(3) from stage 46/47 (system of Nieuwkoop and Faber) for up to 1 month postmetamorphosis. After 24 months postmetamorphosis, the degree of secondary sexual development was examined. Male oviducts were observed in some of the PCB-exposed male frogs, but not in control males. These male oviducts had not completely developed in histological structure when compared with mature female oviducts. Larynx weight and width of PCB-exposed males were significantly less than those of control males. Laryngeal histology showed that PCBs inhibited cartilaginous and muscular development of male frogs, i.e. elastic cartilages had not completely developed and laryngeal muscle fibers were smaller. In a further study on adult male frogs, a decrease in serum testosterone level was found in PCB-exposed frogs compared with controls, but serum estradiol level was not significantly affected. Our study suggests that PCBs can cause phenotypic feminization/demasculinization of male genital ducts and larynges, and these effects may, in part, result from the decrease in serum testosterone level in X. laevis.     

Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis

There is a growing international concern that commonly used environmental contaminants have the potential to disrupt the development and functioning of the reproductive system in amphibians. One such chemical of interests is the herbicide atrazine. Effects of atrazine on sex differentiation were studied using wild-type Xenopus laevis tadpoles and all-ZZ male cohorts of X. laevis tadpoles, produced by mating wild-type ZZ male to sex-reversed ZZ male (female phenotype). Stage 49 tadpoles were exposed to 0.1-100 ppb atrazine or 0.27 ppb (1 nM) 17beta-estradiol (E(2)) until all larvae completed metamorphosis (stage 66). Metamorphosis, gonadal morphology and histology, CYP19 (P450 aromatase) mRNA induction, and hepatic vitellogenin (VTG) induction were investigated. Effects of atrazine on VTG-induction were also assessed in vitro in primary-cultured X. laevis hepatocytes. Atrazine had no effect on metamorphosis of developing wild-type or all-male X. laevis larvae. Statistical increase in female ratios was observed in 10 and 100 ppb atrazine groups in comparison with control group. While no hermaphroditic froglet was observed in all atrazine groups. In ZZ males, sex reversal was induced by 0.27 ppb E(2), but not by atrazine at concentrations of 0.1 and 1.0 ppb. In addition, neither P450 aromatase mRNA in the gonad nor hepatic VTG were induced by atrazine. Furthermore, VTG was not induced by 1000 ppb atrazine in primary-cultured hepatocytes. Our results indicate that female ratios in developing X. laevis tadpoles were increased by 10 and 100 ppb atrazine under the present experimental conditions. While the other endpoints showed no effect in the range of 0.1-100 ppb atrazine. These results suggest that effect of atrazine on sexual differentiation was not caused by estrogenic action and has no induction ability of P450 aromatase gene in gonad.     

Teratogenic effects of two antifungal triazoles, triadimefon and triadimenol, on Xenopus laevis development: craniofacial defects

Triadimefon and triadimenol, fungicides used in agriculture, are suspected of producing craniofacial malformations. As the results of FETAX analysis showed that Triadimefon was highly teratogenic, we studied the action of these triazoles on the development of the branchial apparatus in Xenopus, using early molecular markers and analysis of the cartilaginous-muscular elements of tadpoles. Teratogenic effects were observed, after exposure at the neurula stage, at the level of cartilages and muscles of the 1st and 2nd branchial arches. By in situ hybridization, we observed that in exposed specimens the territories of the branchial arches are perturbed. From these results, we considered the craniofacial malformations related to the influence of triazoles on the differentiation of branchial arches. Comparing the anomalies caused by triazoles and by retinoic acid (RA) led us to the conclusion that triazoles can affect endogenous RA content, as has been shown for mammals.     

cDNA cloning and sequence analysis of Xenopus laevis preproendothelin-1

Endothelin (ET)-like immunoreactivity has been observed not only in mammals, but also in amphibians. The biological actions of ET are similar in amphibians and mammals, and amphibian ET-related receptors have been cloned and characterized. The cDNA sequences of mature and precursor forms of ET-related peptides, however, have not been reported in any amphibian until now. To identify the ET-related peptides, we screened the Xenopus laevis intestine cDNA library using the rapid amplification of cDNA ends method and cloned cDNAs encoding preproendothelin-1. The deduced amino acid sequence of X. laevis preproendothelin-1 comprises 223 amino acids, including a putative signal sequence of 19 amino acids, a mature ET-1 of 21 amino acids, as well as big ET-1 and ET-1-like sequences. X. laevis ET-1 is identical to mammalian ET-1 as well as ET-1 peptide, recently purified from the stomach of the European green frog, Rana ridibunda. This is the first report describing the cDNA encoding preproendothelin-1 in an amphibian species.     

Effect of methoxychlor on various life stages of Xenopus laevis.

The toxicological effects of the organochlorine pesticide methoxychlor were evaluated at various life stages of the South African clawed frog, Xenopus laevis, in an effort to determine stage-specific sensitivity. A battery of four separate assays, including a short-term (4-day) early embryo-larval assay (FETAX) (NF stages 8-46 [Nieuwkoop and Faber, 1994]), 30-day hind limb development assay (NF stages 8-54), 18-day metamorphic climax assay (NF stages 58-66), and 30-day adult reproduction assay were performed. Test concentrations for the FETAX, hind limb development, metamorphic climax, and reproductive assays ranged from 0.0001-1.0 mg/l, 0.0001-0.1 mg/l, 0.0001-0.1 mg/l, and 0.001-0.1 mg/l, respectively. Results from the short-term embryo-larval assay indicated that increased embryo-lethality, malformation, and growth inhibition were not induced at /=0.01 mg/l delayed hind limb digit differentiation. Follicular hyperplasia of the thyroid glands was noted in specimens exposed to 0.1 mg/l methoxychlor. Results from the 18-day metamorphic climax assay indicated that methoxychlor inhibited the rate of tail resorption in a concentration-dependent manner. Whole body tissue triiodothyronine (T(3)) profiles showed a reduced and delayed surge during climax compared to controls. For the reproductive assessment, adult female X. laevis were super-ovulated and both female and male were then exposed to varying concentrations of methoxychlor. A concentration-dependent reduction in ovary weight and the number of viable oocytes was observed. In exposed male specimens, a concentration-dependent reduction in testis weight and sperm count was found. Methoxychlor was found to accumulate in the ovary, and to a lesser extent in the testis. Based on breeding studies in which exposed females were bred with control males and exposed males bred with control females, the frequency of amplexus, fertilization, and embryo viability was also affected by adult female methoxychlor exposure, and to a lesser extent by male exposure. Overall, these results suggested that sensitivity to methoxychlor is most dramatic during the reproductive and metamorphic phases of the life cycle and least sensitive during early embryo-larval development.     

Exposure to low concentrations of di-n-butyl phthalate during embryogenesis reduces survivability and impairs development of Xenopus laevis frogs

Di-n-butyl phthalate (DBP) appears to be a ubiquitous environmental contaminant, as indicated by its presence in air, water, and soil worldwide (Giam et al., 1980; ATSDR, 2001; Peterson & Freeman, 1982) and the presence of its major metabolite, monobutyl phthalate (MBP), in random human urine samples (Blount et al., 2000). Studies indicate that exposure to a variety of endocrine-disrupting chemicals, such as DBP, may be partially responsible for reported global amphibian declines; if so, amphibians may serve as ecological harbingers for the future of human health. Therefore, this study was undertaken to investigate the effects of environmentally relevant concentrations of DBP on development in Xenopus laevis African clawed frogs. Developmental effects of DBP on Xenopus embryos were determined using the 96-h frog embryo teratogenesis assay-Xenopus (FETAX). Embryos (n = 300/group) were exposed from gastrulation (stage 8-11) through primary organogenesis (stage 46) to 0.1, 0.5, 1, 5, 10, or 15 ppm DBP dissolved in 0.01% dimethyl sulfoxide (DMSO), vehicle alone (0.01% DMSO; solvent control), or FETAX culture medium only (control; n = 600). At 96 h, mortalities for control, solvent control, and 0.1, 0.5, 1, 5, 10, and 15 ppm DBP were 5, 4, 6, 5, 5, 9, 18, and 52%, respectively; the incidence of developmental malformations in the surviving tadpoles was 7, 9, 15, 37, 51, 53, 90, and 100%. The average length of embryos was significantly lower in all DBP treatment groups. Thus, DBP significantly affected development of Xenopus embryos at low, environmentally relevant concentrations.     

Teratogenic hazard of BPEI-coated silver nanoparticles to Xenopus laevis

Silver nanoparticles (AgNPs) are among the most exploited antimicrobial agents and are used in many consumer products. Size and surface reactivity are critical physico-chemical properties responsible for NPs toxicity, and surface coatings, often used to functionalize or stabilize AgNPs, can influence their toxic profile and biocompatibility. In the current study the developmental toxicity of (1) negatively charged citrate-coated AgNPs (Cit-AgNPs), (2) positively charged branched polyethylenimine-coated AgNPs (BPEI-AgNPs), and (3) Ag⁺ (from 0.0625 to 0.75?mg Ag/L) was investigated by the standard Frog Embryo Teratogenesis Assay – Xenopus (FETAX). In order to identify the most sensitive developmental phase, embryos were also exposed during different embryonic stages. Morphological and bio-physical studies were performed to characterize tissue lesions and NP uptake. The results suggest that Ag⁺ was strongly embryo-lethal. Contrary to Cit-AgNPs, the positively charged BPEI-AgNPs exert a concentration-dependent effect on lethality and malformations of embryos. The BPEI-AgNPs showed the highest teratogenic index (TI?=?1.6), pointing out the role of functional coating in determining the developmental hazard. The highest susceptibility to BPEI-AgNPs was during early embryogenesis, when embryos are still enclosed in the fertilization envelope, and the post-stomodeum opening stages, when NPs ingestion occurs. In BPEI-AgNPs treated larvae, the histological examination revealed irregular intestinal diverticula coupled with edematous connective tissue. Small NPs aggregates are mapped throughout the intestinal mucosa and secondary target organs by two-photon excitation microscopy. We conclude that a teratogenic risk may be associated with BPEI-AgNPs exposure, but the modality of NP-tissue interactions and the teratogenic mechanism need further investigations to be better defined.     

Toxicity of selenium to developing Xenopus laevis embryos.

Se in the form of sodium selenite is toxic to Xenopus laevis embryos and tadpoles continuously exposed to concentrations above 1 ppm. Concentrations of 2 ppm and above result in severe developmental abnormalities and increased mortality. Uptake and loss of radioactive Se from water are rapid, but depuration is not complete indicating that some Se can remain bound by the organism. The facts that Se is toxic at low levels to Xenopus embryos and tadpoles, can cause developmental abnormalities, and accumulates in tissues suggest that increased release of Se compounds into the environment poses a potential threat to aquatic organisms.     

Effects of THA on ionic currents in myelinated axons of Xenopus laevis

In voltage-clamp experiments with the myelinated nerve fibre of Xenopus laevis, 9-amino-1,2,3,4-tetrahydroacridine (THA) decreased both Na+ and K+ currents and shifted the steady state inactivation potential curve in a negative direction. The effects may be described as (a) a decrease of the permeability constant PNa, (b) a modified potential dependence of the inactivating system and (c) a decrease of PK. The Na+ system was affected more than the K+ system.     

Genotoxic and stress inductive potential of cadmium in Xenopus laevis larvae

The present investigation evaluates the toxic potential of Cd in larvae of the frog Xenopus laevis after 12 days of exposure to environmentally relevant contamination levels, close to those measured in the river Lot (France). Several genotoxic and detoxification mechanisms were analyzed in the larvae: clastogenic and/or aneugenic effects in the circulating blood by micronucleus (MN) induction, metallothionein (MT) production in whole larvae, gene analyses and Cd content in the liver and also in the whole larvae. The results show: (i) micronucleus induction at environmental levels of Cd contamination (2, 10, 30 microgL(-1)); (ii) an increased and concentration-dependent quantity of MT in the whole organism after contamination with 10 and 30 microgCdL(-1) (a three- and six-fold increase, respectively) although no significant difference was observed after contamination with 2 microgCdL(-1); (iii) Cd uptake by the whole organism and by the liver as a response to Cd exposure conditions; (4) up-regulation of the genes involved in detoxification processes and response to oxidative stress, while genes involved in DNA repair and apoptosis were repressed. The results confirm the relevance of the amphibian model and highlight the complementarity between a marker of genotoxicity, MT production, bioaccumulation and genetic analysis in the evaluation of the ecotoxicological impact.     

Developmental toxicity of methoprene and several degradation products in Xenopus laevis

Methoprene is an insect juvenile growth hormone mimic, which inhibits pupation and is used for the control of emergent insect pests such as mosquitoes. Researchers have hypothesized that methoprene use in US may be a contributing factor to the recent increase in malformed amphibians. However, little is known concerning the developmental toxicity of methoprene and its degradation products in amphibians. In these studies, the aqueous stability and developmental toxicity of methoprene and several degradation products (methoprene acid, methoprene epoxide, 7-methoxycitronellal, and 7-methoxycitronellic acid) were examined. Xenopus laevis embryos (stage 8) were exposed to the test chemicals for 96 h. Assays were conducted under static renewal (24 h) conditions and chemical concentrations in water were measured at the beginning and end of the renewal periods. Methoprene exposure did not result in developmental toxicity at concentrations up to 2 mg/l, which is slightly higher than its water solubility. Methoprene acid, a relatively minor degradation product, produced developmental toxicity when concentrations exceeded 1.25 mg/l. Methoprene epoxide and 7-methoxycitronellal caused developmental toxicity at concentrations of 2.5 mg/l and higher. 7-Methoxycitronellic acid was not developmentally toxic at a test concentration as high as 30 mg/l. The five test chemicals had differential stability in aqueous solution that was in some instances affected by the presence of test organisms. These data indicate that methoprene and its degradation products are not potent development toxicants in X. laevis. This, in combination with the fact that field applications of sustained-release formulations of methoprene result in methoprene concentrations that do not typically exceed 0.01 mg/l, suggests that concerns for methoprene-mediated developmental toxicity to amphibians may be unwarranted.     

Mitochondrial dysfunction from malathion and chlorpyrifos exposure is associated with degeneration of GABAergic neurons in Caenorhabditis elegans

Toxicity resulting from off-target effects, beyond acetylcholine esterase inhibition, for the commonly used organophosphate (OP) insecticides chlorpyrifos (CPS) and malathion (MA) was investigated using Saccharomyces cerevisiae and Caenorhabditis elegans model systems. Mitochondrial damage and dysfunction were observed in yeast following exposure to CPS and MA, suggesting this organelle is a major target. In the C. elegans model, the mitochondrial unfolded protein response pathway showed the most robust induction from CPS and MA treatment among stress responses examined. GABAergic neurodegeneration was observed with CPS and MA exposure. Impaired movement observed in C. elegans exposed to CPS and MA may be the result of motor neuron damage. Our analysis suggests that stress from CPS and MA results in mitochondrial dysfunction, with GABAergic neurons sensitized to these effects. These findings may aid in the understanding of toxicity from CPS and MA from high concentration exposure leading to insecticide poisoning.