Relative embryotoxicity of two classes of chemicals in a modified zebrafish embryotoxicity test and comparison with their in vivo potencies

The zebrafish embryotoxicity test (ZET) is a fast and simple method to study chemical toxicity after exposure of the complete vertebrate embryo during embryogenesis in ovo. We developed a novel quantitative evaluation method to assess the development of the zebrafish embryo based on specific endpoints in time, the general morphology score (GMS) system. For teratogenic effects a separate scoring list was developed. The relative effects of eight glycol ethers and six 1,2,4-triazole anti-fungals were evaluated in this system and results were compared with in vivo developmental toxicity potencies.Methoxyacetic acid and ethoxyacetic acid appeared as the most potent glycol ether metabolites, inducing growth retardation and malformations. Other glycol ethers showed no developmental toxicity. Flusilazole appeared the most potent triazole, followed by hexaconazole, cyproconazole, triadimefon, myclobutanil and triticonazole, respectively.In general, the potency ranking of the compounds within their class in the ZET was comparable to their in vivo ranking.In conclusion, the ZET with the GMS system appears an efficient and useful test system for screening embryotoxic properties of chemicals within the classes of compounds tested. This alternative test method may also be useful for the detection of embryotoxic properties of other classes of chemicals.     

Comparison of the mouse Embryonic Stem cell Test, the rat Whole Embryo Culture and the Zebrafish Embryotoxicity Test as alternative methods for developmental toxicity testing of six 1,2,4-triazoles

The relatively high experimental animal use in developmental toxicity testing has stimulated the search for alternatives that are less animal intensive. Three widely studied alternative assays are the mouse Embryonic Stem cell Test (EST), the Zebrafish Embryotoxicity Test (ZET) and the rat postimplantation Whole Embryo Culture (WEC). The goal of this study was to determine their efficacy in assessing the relative developmental toxicity of six 1,2,4-triazole compounds,¹ flusilazole, hexaconazole, cyproconazole, triadimefon, myclobutanil and triticonazole. For this purpose, we analyzed effects and relative potencies of the compounds in and among the alternative assays and compared the findings to their known in vivo developmental toxicity. Triazoles are antifungal agents used in agriculture and medicine, some of which are known to induce craniofacial and limb abnormalities in rodents. The WEC showed a general pattern of teratogenic effects, typical of exposure to triazoles, mainly consisting of reduction and fusion of the first and second branchial arches, which are in accordance with the craniofacial malformations reported after in vivo exposure. In the EST all triazole compounds inhibited cardiomyocyte differentiation concentration-dependently. Overall, the ZET gave the best correlation with the relative in vivo developmental toxicities of the tested compounds, closely followed by the EST. The relative potencies observed in the WEC showed the lowest correlation with the in vivo developmental toxicity data. These differences in the efficacy between the test systems might be due to differences in compound kinetics, in developmental stages represented and in the relative complexity of the alternative assays.     

Assessment of the in vitro developmental toxicity of diethylstilbestrol and estradiol in the zebrafish embryotoxicity test

The present study investigated the developmental toxicity of diethylstilbestrol (DES) in the zebrafish embryotoxicity test (ZET). This was done to investigate whether the ZET would better capture the developmental toxicity of DES than the embryonic stem cells test (EST) that was previously shown to underpredict the DES-induced developmental toxicity as compared to in vivo data, potentially because the EST does not capture late events in the developmental process. The ZET results showed DES-induced growth retardation, cumulative mortality and dysmorphisms (i.e. induction of pericardial edema) in zebrafish embryos while the endogenous ERα agonist 17β-estradiol (E2) showed only growth retardation and cumulative mortality with lower potency compared to DES. Furthermore, the DES-induced pericardial edema formation in zebrafish embryos could be counteracted by co-exposure with ERα antagonist fulvestrant, indicating that the ZET captures the role of ERα in the mode of action underlying the developmental toxicity of DES. Altogether, it is concluded that the ZET differentiates DES from E2 with respect to their developmental toxicity effects, while confirming the role of ERα in mediating the developmental toxicity of DES. Furthermore, comparison to in vivo data revealed that, like the EST, in a quantitative way also the ZET did not capture the relatively high in vivo potency of DES as a developmental toxicant.     

Development of a flow-through system for the fish embryo toxicity test (FET) with the zebrafish (Danio rerio)

The acute fish test is still a mandatory component in chemical hazard and risk assessment. However, one of the objectives of the new European chemicals policy (REACH – Registration, Evaluation, Authorization and Restriction of Chemicals) is to promote non-animal testing. For whole effluent testing in Germany, the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) has been an accepted and mandatory replacement of the fish test since January 2005. For chemical testing, however, further optimization of the FET is required to improve the correlation between the acute fish test and the alternative FET. Since adsorption of the test chemical to surfaces may reduce available exposure concentrations, a flow-through system for the FET using modified commercially available polystyrene 24-well microtiter plates was developed, thus combining the advantages of the standard FET with those of continuous delivery of test substances. The advantages of the design presented include: small test footprint, availability of adequate volumes of test solution for subsequent chemical analysis, and sufficient flow to compensate for effects of non-specific adsorption within 24 h. The flow-through test system can also be utilized to conduct longer-term embryo larval fish tests, thus offering the possibility for teratogenicity testing.     

Developmental toxicity testing of the fume condensate extracts of bitumen and oxidized asphalt in a series of in vitro alternative assays

The potential developmental toxicity and mode-of-action of fume condensate extracts of bitumen and oxidized asphalt were evaluated in the aryl hydrocarbon receptor (AhR) CALUX assay, the zebrafish embryotoxicity test (ZET), and the mouse embryonic stem cell test (mEST). In the AhR CALUX assay, both fume condensate extracts showed a concentration-dependent AhR induction following 6-h of exposure, but this activity was substantially reduced after 24-h, indicating a transient AhR activation. The main effect observed in the ZET was early embryonic lethality that occurred mostly in the 24 h-post-fertilization (hpf). This typically reflects non-specific toxicity rather than in vitro developmental toxicity of the fume condensate extracts tested since this effect was not seen as a result of the whole cumulative exposure period in the ZET (up to 96 hpf). No malformations were seen in any zebrafish embryo exposed to these fume condensate extracts, although some developed pericardial and/or yolk-sac edemas. Furthermore, both fume condensate extracts tested negative in the mEST. In conclusion, the results show that fume condensate extracts of bitumen and oxidized asphalt do not induce any in vitro developmental toxicity, which is in line with the results observed in the in vivo prenatal developmental toxicity studies performed with the same materials.     

A category approach to predicting the developmental (neuro) toxicity of organotin compounds: The value of the zebrafish (Danio rerio) embryotoxicity test (ZET)

Zebrafish embryos were exposed to different organotin compounds during very early development (<100 h post fertilization). Morphology, histopathology and swimming activity (in a motor activity test) were the endpoints analyzed. DBTC was, by far, the most embryotoxic compound at all time points and endpoints studied. In fact, we observed a clear concordance between the effects observed in our zebrafish embryo model, and those observed with these compounds in full rodent in vivo studies. All organotin compounds classified as developmental (neuro) toxicants in vivo, were correctly classified in the present assay. Together, our results support the ZET model as a valuable tool for providing biological verification for a grouping and a read-across approach to developmental (neuro) toxicity.     

Correlations of patch test reactivity and the repeated open application test (ROAT)/provocative use test (PUT).

The clinical relevance of patch test reactions is often difficult to determine. Use tests have been developed to further evaluate the significance of patch test results. We review studies that have defined correlations between the threshold concentrations at patch testing and the outcome of use tests for particular chemical allergens. Results of patch testing with serial dilutions of colophony, cinnamic aldehyde, and isoeugenol have shown concordance with the outcome of use tests. On the other hand, poor correlations between patch test reactivity and the use test were demonstrated in another study on isoeugenol and on studies on hydroxycitronellal, formaldehyde and chromium. These studies shed light on some factors that may influence the outcome of use tests. Individual factors such as patch test sensitivity, regional variations in reactivity and percutaneous penetration appear to play significant roles. Exposure dose, length of time of exposure, and other factors yet to be determined also affect degree of reactivity. Because patients with low thresholds in serial dilution patch testing are known to react to lower concentrations of products at use testing, results of these studies may be used to help identify subjects with a high risk of developing clinical disease. From the public health standpoint, data obtained may be used as a guide in limiting exposure concentrations in consumer products.     

Critical duration of exposure for developmental chlorpyrifos-induced neurobehavioral toxicity

Developmental exposure of rats to the pesticide chlorpyrifos (CPF) causes persistent neurobehavioral impairment. In a parallel series of studies with zebrafish, we have also found persisting behavioral dysfunction after developmental CPF exposure. We have developed a battery of measures of zebrafish behavior, which are reliable and sensitive to toxicant-induced damage. This study determined the critical duration of developmental CPF exposure for causing persisting neurobehavioral effects. Tests of sensorimotor response (tap startle response and habituation), stress response (novel tank diving test) and learning (3-chamber tank spatial discrimination) were conducted with adult zebrafish after early developmental CPF exposure. The CPF exposure level was 100 ng/ml with durations of 0-1, 0-2, 0-3, 0-4 and 0-5 days after fertilization. Developmental CPF exposure had persisting behavioral effects in zebrafish tested as adults. In the tactile startle test, CPF exposed fish showed decreased habituation to startle and a trend toward increased overall startle response. In the novel tank exploration test, exposed fish showed decreased escape diving response and increased swimming activity. In the 3-chamber learning test, the 0-5 day CPF exposure group had a significantly lower learning rate. There was evidence for persisting declines in brain dopamine and norepinepherine levels after developmental CPF exposure. In all of the measures the clearest persistent effects were seen in fish exposed for the full duration of five days after fertilization. In a follow-up experiment there were some indications for persisting behavioral effects after exposure during only the later phase of this developmental window. This study demonstrated the selective long-term neurobehavioral alterations caused by exposure to CPF in zebrafish. The zebrafish model can facilitate the determination of the molecular mechanisms underlying long-term neurobehavioral impairment after developmental toxicant exposure.     

The long-term study in rodents for identifying carcinogens: some controversies and suggestions for improvements

Despite increasing standardisation and international harmonization of the long-term carcinogenicity study in rodents there are still areas of controversy such as high-dose selection, duration of the test period, combining or not combining the chronic toxicity and the carcinogenicity study, the use of historical control data, conditions for redundancy of the study, and significance of studies of restricted design. It is discussed that the 'Maximum Tolerated Dose', or doses above and below the 'metabolic break-point' should be included. In general a test period of 24 months is adequate, but the protocol should be flexible enough to allow extension beyond 24 months. Major advantages of the combined chronic toxicity/carcinogenicity study are that the toxicity and carcinogenicity data are obtained with the same sample of the test compound using the same batch of animals kept on the same diet under the same environmental conditions. It is discussed that the use of historical control data will not lead to a final conclusion on carcinogenic or non-carcinogenic potential of a compound. A long-term carcinogenicity study is considered redundant when adequate (semi-chronic) toxicity studies including reproduction and mutagenicity tests and pre-screens for carcinogenicity, do not indicate mutagenic or carcinogenic activity, and when there is a wide margin (e.g. 1000) between the 'no-observed-adverse effect level' and the (presumed) exposure level in humans. Studies of restricted design and conduct may clearly demonstrate carcinogenicity but also may easily lead to inconclusive results.     

Persistent behavioral impairment caused by embryonic methylphenidate exposure in zebrafish

As more adults take the stimulant medication methylphenidate to treat attention deficit hyperactivity disorder (ADHD) residual type, the risk arises with regard to exposure during early development if people taking the medication become pregnant. We studied the neurobehavioral effects of methylphenidate in zebrafish. Zebrafish offer cellular reporter systems, continuous visual access and molecular interventions such as morpholinos to help determine critical mechanisms underlying neurobehavioral teratogenicity. Previously, we had seen that persisting neurobehavioral impairment in zebrafish with developmental chlorpyrifos exposure was associated with disturbed dopamine systems. Because methylphenidate is an indirect dopamine agonist, it was thought that it might also cause persistent behavioral impairment after developmental exposure. Zebrafish embryos were exposed to the ADHD stimulant medication methylphenidate 0-5 days post fertilization (12.5-50 mg/l). They were tested for long-term behavioral effects as adults. Methylphenidate exposure (50 mg/l) caused significant increases in dopamine, norepinepherine and serotonin on day 6 but not day 30 after fertilization. In the novel tank diving test of predatory avoidance developmental methylphenidate (50 mg/l) caused a significant reduction in the normal diving response. In the three-chamber spatial learning task early developmental methylphenidate (50 mg/l) caused a significant impairment in choice accuracy. These data show that early developmental exposure of zebrafish to methylphenidate causes a long-term impairment in neurobehavioral plasticity. The identification of these functional deficits in zebrafish enables further studies with this model to determine how molecular and cellular mechanisms are disturbed to arrive at this compromised state.     

Characterization of socio-psychological stress-induced antinociception in the formalin test in mice

The antinociceptive effect induced by exposure to socio-psychological (PSY) stress using a communication box was assessed by the formalin test in mice, compared with those by exposure to footshock (FS) stress and forced swimming (SW) stress. After the termination of stress exposure, whereas exposure to FS- and SW-stress resulted in the attenuation of the formalin-induced biphasic pain response over 15 min, no appreciable antinociceptive effect was found in the case of PSY stress. When exposure to PSY stress was started during the period of early or late phase of pain after the formalin injection, the antinociceptive effect was maintained for 5-15 min; however, further exposure to PSY stress was not effective for producing antinociception. In the tail-pinch test, likewise, exposure to PSY stress longer than 5 min rather decreased the intensity of antinociception. We conclude that PSY stress in this tonic pain paradigm produces antinociception, but further continuous exposure to the emotional stress caused mice to become recuperative even in such a fear-inducing situation.     

Reconsidering sufficient and optimal test design in acute toxicity testing

In dose–response analysis, regression analysis and hypothesis testing are the main tools of choice. These methods, however, have specific requirements for the design of acute toxicity experiments. To produce meaningful results, both approaches require a constant exposure concentration over the duration of the test, and regression analysis makes an additional demand for at least two doses with partial mortality at the end of the test. These requirements, however, result from the limitations of the statistical techniques, which only use the observations at the end of the test. In practice, most standard protocols for acute testing prescribe that observations are made at several points in time (often daily). In this contribution, I demonstrate how dynamic modelling can make use of this information to produce robust estimates of LC50 as function of time, with confidence intervals, from data sets that violate the requirements for standard dose–response analysis. This form of modelling invites an entirely different, more flexible, view on experimental design, which could lead to a more efficient use of test animals and, at the same time, a stronger support for environmental risk assessment as well as the science of ecotoxicology.     

Ethanol in goldfish: Effect of prior exposure in a test procedure

Goldfish acquire tolerance to ethanol (Greizerstein and Smith, 1973). The present studies were designed to determine whether prior exposure to the test procedure contributed to tolerance development.The actions of ethanol were measured by the time required for the appearance of overturn (loss of righting reflex) and the brain ethanol content at the instant of overturn following immersion of goldfish in 3.1% (w/v) ethanol. Neither the time required for onset of action nor ethanol brain levels at overturn were significantly affected by 1–5 exposures to the 3.1% solution over an interval of 2–5 days.These findings further demonstrate the use of the overturn test in goldfish as a behavioral measure that is relatively uninfluenced by repeated exposure to the test situation.     

Quantitative relationships between patch test reactivity and use test reactivity: an overview

Use tests such as the provocative use test (PUT) or repeated open application test (ROAT) have been created to better understand the clinical significance of patch test results. It has been suggested that since these tests typically utilize only one substance at a time and avoid occlusion, they minimize the occurrence of irritation and false positives and, thus, are more reflective of real-life exposure to an allergen. In this analysis, we compare and analyze different studies comparing patch test and use test reactivity. With regard to colophony, cinnamic aldehyde, methyldibromo glutaronitrile, 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, and isoeugenol, increased patch test sensitivity resulted in increased use test sensitivity. However, this was not true for formaldehyde or chromium. The reason for the latter allergens' divergence from the trend is not yet understood. Additionally, we note the presence of an increasing slope in the relationship between use test reactivity and minimum eliciting concentration on patch testing for methyldibromo glutaronitrile. Expansion of databases relating serial dilution patch test reactivity and use test data should aid dermatologic management, public health policy, and an understanding of the complexity of allergic contact dermatitis in humans.     

Prolactin-releasing effect of zetidoline, a new neuroleptic agent, in the rat

The effect of a new neuroleptic agent, zetidoline, 1-(3-chlorophenyl)-3-[2-(3,3-dimethyl-l-azetidinyl)ethyl]imidaz olidin-2-one (ZET), on prolactin release was studied in both male and female rats and compared to that of the classic antipsychotic drugs chlorpromazine and haloperidol. Time-course and dose-effect studies showed that ZET induces a rapid and short lasting increase in plasma prolactin levels, with a significant increase after a dose as low as 0.33 mg/kg, i.p.. The overall patterns of prolactin release appeared to be similar in both sexes but the response was markedly greater in females than in males. The prolactin-releasing effect of ZET was counteracted by apomorphine and L-dopa, which indicates that blockade of dopamine receptors is the basis of its neuroendocrine action. As a prolactin-releaser, ZET was found to be about 5 times as potent as chlorpromazine and about one-ninth as potent as haloperidol.     

Effect of combined exposure to silica nanoparticles and cadmium chloride on female zebrafish ovaries

Silica nanoparticles (SiNPs) and cadmium chloride (CdCl₂) are two important environmental pollutants. In previous research, found that SiNPs in zebrafish larvae can amplify the cardiovascular damage caused by cadmium. Whether SiNPs in the ovaries can amplify the adverse effects of cadmium on the zebrafish ovaries is worth studying problem. In this study, sexually mature female zebrafish were used as model organisms and exposed to 1 μmol/L CdCl₂ and/or 25 μg/mL SiNPs for 30 days. The results showed that the structure and function of ovaries in the sole and combined exposure groups changed significantly, resulting in reduced ovarian quality, decreased number of mature oocytes, and the development of malformed offspring. A deep-sequencing analysis showed that organisms’ lipid metabolism and transportation, estrogen metabolism, and response to the maturation, meiosis, and vitellogenin synthesis of oocytes were significantly affected by single exposure or combined exposure. These findings provide further insights into the harm of cooperation of CdCl2 and/or SiNPs to the aquatic ecosystems.     

Developmental chlorpyrifos effects on hatchling zebrafish swimming behavior

Chlorpyrifos (CPF), a widely used organophosphate insecticide and potent acetylcholinesterase inhibitor, interferes with neurobehavioral development. Rat models have been key in demonstrating that developmental CPF exposure causes learning deficits and locomotor activity alterations, which persist into adulthood. Complementary nonmammalian models can be useful in determining the neurodevelopmental mechanisms underlying these persisting behavioral effects. Zebrafish (Danio rerio) with their clear chorion and extensive developmental information base provide an excellent model for assessment of molecular processes of toxicant-impacted neurodevelopment. We have developed methods for assessing spatial discrimination learning in adult zebrafish and have documented persisting effects of developmental CPF exposure on swimming activity and learning after low and high doses of CPF (10 and 100 ng/ml) administered to zebrafish embryos on Days 1–5 postfertilization (pf). In the current study, we developed methods for behavioral assessment of CPF exposure on swimming activity in newly hatched zebrafish. An equal area segmented annular grid (concentric circles divided into quadrants through the diameter) was made in a 16-mm diameter cylinder. The test area was placed on a heating device secured to an Olympus SZH10 dissecting scope stage. Zebrafish embryos were exposed to 10 ng/ml CPF, 100 ng/ml CPF, or vehicle control (25 μl/ml DMSO) (n=8–10/treatment group). Each treatment group was kept in a total volume of 25 ml of egg water (60 mg/ml Instant Ocean) including DMSO with or without CPF mixed to above dilutions in an incubator set at 28.5°C. CPF dilutions or vehicle were changed daily with exposure ending on Day 5 pf. Testing of larval zebrafish was performed on Days 6 and 9 pf. The fish were placed in the test cylinder with 1.5 ml of egg H₂O (28.5°C). After a 2-min acclimation period, the swimming activity of the fish was measured for a 3-min testing session. The 100 ng/ml CPF dose caused significant slowing of swimming activity on Days 6 and 9 pf and had persisting effects of impairing spatial discrimination and decreasing response latency in adulthood. Developmental exposure to 10 ng/ml of CPF did not cause a significant change in locomotor activity during the period soon after hatching. CPF exposure during early development caused clear behavioral impairments detectable during the posthatching period. In a previous study, we found that early developmental CPF exposure caused behavioral alterations in zebrafish, which lasted throughout adulthood. The molecular mechanisms by which early developmental CPF exposure produces these behavioral impairments expressed in adulthood can now be studied in the zebrafish model.