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Evaluation of developmental toxicity in zebrafish embryos and antiproliferative potential against human tumor cell lines of new derivatives containing 4-nitrophenyl group
Abstract
The aim of the studies was to evaluate the antiproliferative potential against human tumor cell lines of newly synthetized derivatives containing 4-nitrophenyl group, as well as its impact on developmental toxicity in zebrafish model. We selected 1-(4-nitrobenzoyl)-4-ethylsemicarbazide (APS-1) and 1-[(4-nitrophenyl)acetyl]-4-hexyl-thiosemicarbazide (APS-18) for research. The antiproliferative properties of semi-carbazide derivatives were assessed against human cancer cell lines derived from hepatocellular adenocarcinoma (HepG2), renal cell carcinoma (769-P), non-small cell lung cancer (NCI-H1563) and glioblastoma multiforme (LN229) in comparison to the physiological human embryonic kidney (HEK-293) cell line. The influence of the tested substances on the cell cycle and apoptosis was also evaluated. An acute toxicity test was performed based on OECD Guidelines (Test No. 236), and was carried out for the first 5 days post-fertilization. The following concentrations of APS-1 and APS-18 were tested: 125–2000 μM and 0.125–1000 μM, respectively. The presented studies on the antiproliferative properties of the new semicarbazide derivatives showed that the compounds APS-1 and APS-18 reduce the viability of human tumor lines. Particularly noteworthy is the strong and selective antiproliferative activity of APS-18 against all neoplastic cell lines, in particular against glioblastoma. Against this tumor line, the compound APS-1 showed an effective inhibitory effect. In the FET we noted that the direct exposure of zebrafish embryos to APS-1 and APS-18 in used range of concentration did not cause morphological abnormalities, including cardiotoxicity. On basis of obtained outcomes it could be concluded that APS-1 and APS-18 may constitute models for further research, design and synthesis of new, safer drugs with more favorable anticancer properties.
... The fish embryo acute toxicity test (FET) was carried out based on OECD Guidelines for the Testing Chemicals (test No. 236) (OECD, 2013) as described before (Szopa et al., 2023). ...
... Compound 1b has a 4-nitrophenyl-grouping, which may account for its potential antiproliferative properties. Similar observations have been made in other studies, which also tested semicarbazide derivatives with a substituent in the same position (Szopa et al., 2023;Wos et al., 2017). In one of them, results similar to ours were obtained, i.e. the strongest cytotoxic effect was observed against cells of glioma lines. ...
... Embryos/larvae were considered dead when there when marked coagulation was noted and/or no heartbeat was observed (Deng et al., 2009;Huang et al., 2018). In our earlier studies, we assessed the toxicity of another thiosemicarbazide derivative, i.e. 1-[(4-nitrophenyl) acetyl]-4-hexylthiosemicarbazide (APS18) (Szopa et al., 2023). Similar to 1b no significant effect of the tested newly synthesized compound (APS18) on survival of Danio rerio embryos in the FET test was observed. ...
... One study compared a semicarbazide molecule and a thiosemicarbazide bearing a 4-nitrophenyl substituent. The compound with the thiosemicarbazide scaffold possessed stronger antiproliferative properties [30]. Importantly, it is also believed that it is not the structure of the thiosemicarbazide itself that is important in the anticancer activity, but its structure as a whole [31]. ...
A series of N-Substituted 2-(benzenosulfonyl)-1-carbotioamide derivatives (WZ1–WZ4) were synthesized and characterized using spectral methods. A comprehensive activity study was performed for each compound. All compounds were tested for antibacterial activity. Moreover, in silico studies were carried out to determine the anticancer potential of the designed WZ1–WZ4 ligands. Based on molecular docking, aldehyde dehydrogenase was selected as a molecular target. The obtained data were compared with experimental data in vitro tests. Novel hybrids of the thiosemicarbazide scaffold and sulfonyl groups may have promising anticancer activity via the aldehyde dehydrogenase pathway. The best candidate for further studies appears to be WZ2, due to its superior selectivity in comparison to the other tested compounds.
The safety of cyclophosphamide (CP) in the early developmental stages is not studied yet; it is important to study the responses at these stages because they might have relevance to CP-administered humans. We studied the developmental toxicity of CP by analysing physiological, morphological, and oxidative stress, neurotransmission enzymes, gene expression and histological endpoints in zebrafish embryos/larvae. The study lasted for 120 hpf at environmentally relevant concentrations of CP. No visible alterations were noticed in the control group. Delayed hatching, slow heart rate, yolk sac oedema, pericardial oedema, morphological deformities, the incompetence of oxidative stress biomarkers, excessive generation of ROS, apoptosis, inhibition of neurotransmitters and histopathological anomalies were observed in CP-treated groups. These alterations were found to be concentration- and duration-dependent effects for physiological and morphological endpoints, whereas concentration-dependent effects were antioxidants, ROS, apoptosis and histological endpoints. Biomarkers and gene expression were standardised using the integrated biomarker response-IBRv2 index. The IBRv2 index showed a concentration-dependent behaviour. A non-lethal developmental and teratogenic effect was observed in CP-treated zebrafish embryos/larvae at the studied concentrations. The studied biomarkers are sensitive, and the responses are interrelated; thus, their responses are useful to assess veiled and unseen hazards of pharmaceuticals.
Our study briefly reviews the data sources and methods used in compiling the International Agency for Research on Cancer (IARC) GLOBOCAN cancer statistics for the year 2020 and summarises the main results. National estimates were calculated based on the best available data on cancer incidence from population‐based cancer registries (PBCR) and mortality from the World Health Organization mortality database. Cancer incidence and mortality rates for 2020 by sex and age groups were estimated for 38 cancer sites and 185 countries or territories worldwide. There were an estimated 19.3 million (95% uncertainty interval [UI]: 19.0‐19.6 million) new cases of cancer (18.1 million excluding non‐melanoma skin cancer) and almost 10.0 million (95% UI: 9.7‐10.2 million) deaths from cancer (9.9 million excluding non‐melanoma skin cancer) worldwide in 2020. The most commonly diagnosed cancers worldwide were female breast cancer (2.26 million cases), lung (2.21) and prostate cancers (1.41); the most common causes of cancer death were lung (1.79 million deaths), liver (830000) and stomach cancers (769000).
Background: The Fish Embryo Acute Toxicity (FET) test with the zebrafish (Danio rerio) embryo, the OECD test guideline (TG) 236, has been designed as an alternative for acute fish toxicity testing such as the OECD Acute Fish Toxicity Test (TG 203). To provide equivalent sensitivity to the acute fish test, the original FET test was designed to use only four morphological core endpoints: coagulation of the embryo, lack of somite formation, lack of heart beat, and non-detachment of the tail. These endpoints were selected due to (1) their association with mortality, directly or indirectly, (2) improve the practicality for screening by well-trained technical staff, and (3) the endpoints being relatively simple morphological alterations.
Results: With the growing need to understand the developmental toxicity of compounds found in the environment, the FET protocol has repeatedly been extended to a multitude of additional morphological endpoints that also allow the monitoring of teratogenicity. As the extensive use of the FET test has generated a multitude of observations in the scientific literature, a harmonisation of the terminology used for the description of the morphological effects seen after chemical exposure has become necessary.
Conclusion: For this end, the present communication provides an overview of both common and selected more specific morphological effects seen in zebrafish embryos after exposure to a wide variety of chemical substances together with suggestions for a harmonised nomenclature.
Parasitic infections caused by different species of intestinal helminths still poses a threat to public health. There is a need to search for new, effective anthelmintic drugs. A series of novel thiosemicarbazides were synthesized and evaluated for their in vitro anthelmintic activity. The preliminary results showed that the most of synthesized compounds were very active. 4-Phenyl-1-[(1-methyl-4-nitroimidazol-2-yl)carbonyl]thiosemicarbazide and 4-(3-chlorophenyl)-1-[(1-methyl-4-nitroimidazol-2-yl)carbonyl]thiosemicarbazide showed a 100% mortality of nematodes and a high anthelmintic activity in both tested concentrations.
Thiosemicarbazide is a useful structural moiety that has the biological potential. Optimization of this structure can result in groundbreaking discovery of a new class of therapeutic agents. In the light of this, 1-(2,4-dichlorophenoxy)acetyl-4-(1-naphthyl)thiosemicarbazide (1) and 1,4-bis[(2,4-dichlorophenoxy)acetylthiosemicarbazide]phenyl (2) were synthesized and characterized by spectroscopic method. Cytotoxicity of obtained compounds was evaluated on MKN74 gastric cancer cell line and human skin fibroblast BJ based on methylthiazolyldiphenyl-tetrazolium bromide (MTT) test. The apoptosis/necrosis and cell cycle analysis were conducted using image cytometry. Additionally, in DNA of treated cells, abasic sites (AP) and double strands breaks (DSB) presence were measured. Intercalating properties of active compounds were evaluated using the UV-spectroscopic method. Among newly synthesized derivatives, compound 2 showed toxic effects on gastric cancer cells with simultaneous lack of toxicity to normal fibroblasts. Cell cycle analysis revealed that both compounds influence cell division mainly at the stage of replication. Simultaneously with DNA synthesis disorders, DNA damages like AP-sites and DSBs were observed. Spectroscopic studies revealed possible DNA intercalating properties of tested compounds. Obtained results indicate that the newly synthesized thiosemicarbazide derivatives are a promising group of compounds with potential anticancer activity resulted from interactions with DNA and cell cycle interrupt.
Background:
Infectious pathogens are strong and modifiable causes of cancer. The aim of this study was to improve estimates of the global and regional burden of infection-attributable cancers to inform research priorities and facilitate prevention efforts.
Methods:
We used the GLOBOCAN 2018 database of cancer incidence and mortality rates and estimated the attributable fractions and global incidence for specific anatomical cancer sites, subsites, or histological subtypes known to be associated with ten infectious pathogens classified as human carcinogens. We calculated absolute numbers and age-standardised incidence rates (ASIR) of infection-attributable cancers at the country level. Estimates were stratified for sex, age group, and country, and were aggregated according to geographical regions and World Bank income groups.
Findings:
We found that, for 2018, an estimated 2·2 million infection-attributable cancer cases were diagnosed worldwide, corresponding to an infection-attributable ASIR of 25·0 cases per 100 000 person-years. Primary causes were Helicobacter pylori (810 000 cases, ASIR 8·7 cases per 100 000 person-years), human papillomavirus (690 000, 8·0), hepatitis B virus (360 000, 4·1) and hepatitis C virus (160 000, 1·7). Infection-attributable ASIR was highest in eastern Asia (37·9 cases per 100 000 person-years) and sub-Saharan Africa (33·1), and lowest in northern Europe (13·6) and western Asia (13·8). China accounted for a third of worldwide cancer cases attributable to infection, driven by high ASIR of H pylori (15·6) and hepatitis B virus (11·7) infection. The cancer burden attributed to human papillomavirus showed the clearest relationship with country income level (from ASIR of 6·9 cases per 100 000 person-years in high-income countries to 16·1 in low-income countries).
Interpretation:
Infection-attributable cancer incidence, in addition to the absolute number of cases, allows for refined geographic analyses and identification of populations with a high infection-associated cancer burden. When cancer prevention is largely considered in a non-communicable disease context, there is a crucial need for resources directed towards cancer prevention programmes that target infection, particularly in high-risk populations. Such interventions can markedly reduce the increasing cancer burden and associated mortality.
Funding:
International Agency for Research on Cancer.
Thiosemicarbazones (TSCs) are a class of Schiff bases usually obtained by the condensation of thiosemicarbazide with a suitable aldehyde or ketone. TSCs have been focus of chemists and biologists due to their wide range of pharmacological effects. One of the promising areas in which these excellent metal chelators are being developed is their use against cancer. TSCs have a wide clinical antitumor spectrum with efficacy in various tumor types such as leukemia, pancreatic cancer, breast cancer, non-small cell lung cancer, cervical cancer, prostate cancer and bladder cancer. To obtain better activity different series of TSCs have been developed by modifying the heteroaromatic system in their molecules. These compounds possessed significant antineoplastic activity when the carbonyl attachment of the side chain was located at a position α to the ring nitrogen atom, whereas attachment of the side chain β or γ to the heterocyclic N atom resulted in inactive antitumor agents. In addition, replacement of the heterocyclic ring N with C also resulted in a biologically inactive compound suggesting that a conjugated N,N,S-tridentate donor set is essential for the biological activities of thiosemicarbazones. Several possible mechanisms have been implemented for the anticancer activity of thiosemicarbazones.
Cell lines are in vitro model systems that are widely used in different fields of medical research, especially basic cancer research and drug discovery. Their usefulness is primarily linked to their ability to provide an indefinite source of biological material for experimental purposes. Under the right conditions and with appropriate controls, authenticated cancer cell lines retain most of the genetic properties of the cancer of origin. During the last few years, comparing genomic data of most cancer cell lines has corroborated this statement and those that were observed studying the tumoral tissue equivalents included in the The Cancer Genome Atlas (TCGA) database. We are at the disposal of comprehensive open access cell line datasets describing their molecular and cellular alterations at an unprecedented level of accuracy. This aspect, in association with the possibility of setting up accurate culture conditions that mimic the in vivo microenvironment (e.g., three-dimensional (3D) coculture), has strengthened the importance of cancer cell lines for continuing to sustain medical research fields. However, it is important to consider that the appropriate use of cell lines needs to follow established guidelines for guaranteed data reproducibility and quality, and to prevent the occurrence of detrimental events (i.e., those that are linked to cross-contamination and mycoplasma contamination)
Background: Acridine and thiourea derivatives are important compounds in medicinal chemistry due to their diverse biological properties including anticancer and antimicrobial effects. However, literature reveals some side effects associated with use of acridines. It is suggested that hybrid molecules may reduce the side effects and enhance the beneficial properties due to synergistic activity. The objectives of the present study are to synthesize and evaluate the anticancer and antimicrobial properties of new hybrids of acridine thiosemicarbazides derivatives. Results: The structures of the synthesized compounds 4a–4e were elucidated by MS and NMR spectra. In antimicrobial assay, Compound 4c exhibited potent antimicrobial activity compared to the other four compounds. In anticancer studies, we observed that compounds 4a, 4b, 4d and 4e exhibited high cytotoxicity against the MT-4 cell line, with IC50 values of 18.42 ± 1.18, 15.73 ± 0.90, 10.96 ± 0.62 and 11.63 ± 0.11 μM, respectively. The evaluation of anticancer effects, and the associated mechanism reveals that, the anticancer activities may be related to Topo I inhibitory activity, apoptosis and cell-cycle. Molecular docking studies revealed that the presence of planar naphtho-fused rings and a flexible thiourea group together, could improve DNA-intercalation and inhibition of DNA-Topo I activity. Conclusions: The results of this study demonstrate that the rational design of target derivatives as novel antimicrobial or antitumor leads is feasible.
One of the key stages in the development of new therapies in the treatment of toxoplasmosis is the identification of new non-toxic small molecules with high specificity to Toxoplasma gondii. In the search for such structures, thiosemicarbazide-based compounds have emerged as a novel and promising leads. Here, a series of imidazole-thiosemicarbazides with suitable properties for CNS penetration was evaluated to determine the structural requirements needed for potent anti-Toxoplasma gondii activity. The best 4-arylthiosemicarbazides 3 and 4 showed much higher potency when compared to sulfadiazine at concentrations that are non-toxic to the host cells, indicating a high selectivity of their anti-toxoplasma activity.
A series of thiosemicarbazide derivatives was designed and synthesized by reaction of carboxylic acid hydrazide with isothiocyanates. The molecular structures of the investigated thiosemicarbazides were confirmed and characterized by spectroscopic analysis. The conformational preference of carbonylthiosemicarbazide chain and intra- and intermolecular interactions in the crystalline state were characterized using X-ray analysis. The antituberculosis activity of the target compounds were tested in vitro against four Mycobacterium strains: M. H37Ra, M. phlei, M. smegmatis, M. timereck. The most active compounds were those with 2-pyridine ring. They exhibited lower minimal inhibitory concentration (MIC) values in the range 7.81–31.25 μg/mL in comparison to the other isomers. Compound 5 had activity against M. smegmatis at a concentration of 7.81 μg/mL whereas compound 2 had activity against all tested strains at a concentration of 15.625 μg/mL. The molecular docking studies were performed for investigated compounds using the Mycobacterium tuberculosis glutamine synthetase MtGS as their molecular target.
The medical history of cancer began millennia ago. Historical findings of patients with cancer date back to ancient Egyptian and Greek civilizations, where this disease was predominantly treated with radical surgery and cautery that were often ineffective, leading to the death of patients. Over the centuries, important discoveries allowed to identify the biological and pathological features of tumors, without however contributing to the development of effective therapeutic approaches until the end of the 1800s, when the discovery of X-rays and their use for the treatment of tumors provided the first modern therapeutic approach in medical oncology. However, a real breakthrough took place after the Second World War, with the discovery of cytotoxic antitumor drugs and the birth of chemotherapy for the treatment of various hematological and solid tumors. Starting from this epochal turning point, there has been an exponential growth of studies concerning the use of new drugs for cancer treatment. The second fundamental breakthrough in the field of oncology and pharmacology took place at the beginning of the ‘80s, thanks to molecular and cellular biology studies that allowed the development of specific drugs for some molecular targets involved in neoplastic processes, giving rise to targeted therapy. Both chemotherapy and target therapy have significantly improved the survival and quality of life of cancer patients inducing sometimes complete tumor remission. Subsequently, at the turn of the third millennium, thanks to genetic engineering studies, there was a further advancement of clinical oncology and pharmacology with the introduction of monoclonal antibodies and immune checkpoint inhibitors for the treatment of advanced or metastatic tumors, for which no effective treatment was available before. Today, cancer research is always aimed at the study and development of new therapeutic approaches for cancer treatment. Currently, several researchers are focused on the development of cell therapies, anti-tumor vaccines, and new biotechnological drugs that have already shown promising results in preclinical studies, therefore, in the near future, we will certainly assist to a new revolution in the field of medical oncology.
Chemotherapeutic drugs have a common intent to activate apoptosis in tumor cells. However, master regulators of apoptosis (e.g., p53, p16/CDKN2A) are frequently genetically inactivated in cancers, resulting in multidrug resistance. An alternative, p53-independent method for terminating malignant proliferation is to engage terminal-differentiation. Normally, the exponential proliferation of lineage-committed progenitors, coordinated by the master transcription factor (TF) MYC, is self-limited by forward-differentiation to terminal lineage-fates. In cancers, however, this exponential proliferation is disengaged from terminal-differentiation. The mechanisms underlying this decoupling are mostly unknown. We performed a systematic review of published literature (January 2007–June 2018) to identify gene pathways linked to differentiation-failure in three treatment-recalcitrant cancers: hepatocellular carcinoma (HCC), ovarian cancer (OVC), and pancreatic ductal adenocarcinoma (PDAC). We analyzed key gene alterations in various apoptosis, proliferation and differentiation pathways to determine whether it is possible to predict treatment outcomes and suggest novel therapies. Poorly differentiated tumors were linked to poorer survival across histologies. Our analyses suggested loss-of-function events to master TF drivers of lineage-fates and their cofactors as being linked to differentiation-failure: genomic data in TCGA and ICGC databases demonstrated frequent haploinsufficiency of lineage master TFs (e.g., GATA4/6) in poorly differentiated tumors; the coactivators that these TFs use to activate genes (e.g. ARID1A, PBRM1) were also frequently inactivated by genetic mutation and/or deletion. By contrast, corepressor components (e.g., DNMT1, EED, UHRF1, and BAZ1A/B), that oppose coactivators to repress or turn off genes, were frequently amplified instead, and the level of amplification was highest in poorly differentiated lesions. This selection by neoplastic evolution towards unbalanced activity of transcriptional corepressors suggests these enzymes as candidate targets for inhibition aiming to re-engage forward-differentiation. This notion is supported by both pre-clinical and clinical trial literature.
The fathead minnow fish embryo toxicity (FET) test has been proposed as a more humane alternative to current toxicity testing methods, as younger organisms are thought to experience less distress during toxicant exposure. However, the FET test protocol does not include endpoints that allow for the prediction of sublethal adverse outcomes, limiting its utility relative to other test types. Researchers have proposed the development of sublethal endpoints for the FET test to increase its utility. The present study 1) developed methods for previously unmeasured sublethal metrics in fathead minnows (i.e., spontaneous contraction frequency and heart rate) and 2) investigated the responsiveness of several sublethal endpoints related to growth (wet weight, length, and growth‐related gene expression), neurodevelopment (spontaneous contraction frequency, and neurodevelopmental gene expression), and cardiovascular function and development (pericardial area, eye size and cardiovascular related gene expression) as additional FET test metrics using the model toxicant 3,4–dichloroaniline. Of the growth, neurological and cardiovascular endpoints measured, length, eye size and pericardial area were found to more responsive than the other endpoints, respectively. Future studies linking alterations in these endpoints to longer‐term adverse impacts are needed to fully evaluate the predictive power of these metrics in chemical and whole effluent toxicity testing. This article is protected by copyright. All rights reserved
As the most commonly occurring cancer in women worldwide, breast cancer poses a formidable public health challenge on a global scale. Breast cancer consists of a group of biologically and molecularly heterogeneous diseases originated from the breast. While the risk factors associated with this cancer varies with respect to other cancers, genetic predisposition, most notably mutations in BRCA1 or BRCA2 gene, is an important causative factor for this malignancy. Breast cancers can begin in different areas of the breast, such as the ducts, the lobules, or the tissue in between. Within the large group of diverse breast carcinomas, there are various denoted types of breast cancer based on their invasiveness relative to the primary tumor sites. It is important to distinguish between the various subtypes because they have different prognoses and treatment implications. As there are remarkable parallels between normal development and breast cancer progression at the molecular level, it has been postulated that breast cancer may be derived from mammary cancer stem cells. Normal breast development and mammary stem cells are regulated by several signaling pathways, such as estrogen receptors (ERs), HER2, and Wnt/β-catenin signaling pathways, which control stem cell proliferation, cell death, cell differentiation, and cell motility. Furthermore, emerging evidence indicates that epigenetic regulations and noncoding RNAs may play important roles in breast cancer development and may contribute to the heterogeneity and metastatic aspects of breast cancer, especially for triple-negative breast cancer. This review provides a comprehensive survey of the molecular, cellular and genetic aspects of breast cancer.
Thirty new derivatives of palmitic acid were efficiently synthesized. All obtained compounds can be divided into three groups of derivatives: Thiosemicarbazides (compounds1-10), 1,2,4-triazoles (compounds1a-10a) and 1,3,4-thiadiazoles (compounds1b-10b) moieties. ¹H-NMR,13C-NMR and MS methods were used to confirm the structure of derivatives. All obtained compounds were tested in vitro against a number of microorganisms, including Gram-positive cocci, Gram-negative rods andCandida albicans. Compounds4,5,6,8showed significant inhibition againstC. albicans. The range of MIC values was 50-1.56 μg/mL. The halogen atom, especially at the 3rd position of the phenyl group was significantly important for antifungal activity. The biological activity againstCandida albicansand selected molecular descriptors were used as a basis for QSAR models, that have been determined by means of multiple linear regression. The models have been validated by means of the Leave-One-Out Cross Validation. The obtained QSAR models were characterized by high determination coefficients and good prediction power.
In order to discover novel eco-friendly lead compounds for plant pathogenic fungi control, a series of benzaldehyde thiosemicarbazide derivatives with a piperidine moiety have been designed and synthesized. Fungicidal activities of all the synthesized compounds were evaluated in vitro. The results indicated that all the title compounds exhibited moderate to good fungicidal activities. Compound 3b displayed excellent activities against Pythium aphanidermatum, Rhizoctonia solani, Valsa mali, and Gaeu-mannomyces graminsis, with EC50 values lower than 10 μg/mL. Especially, in the case of Pythium aphanidermatum, its activity (EC50 = 1.6 μg/mL) is superior to the commercial azoxystrobin (EC50 = 16.9 μg/mL) and close to fluopicolide (EC50 = 1.0 μg/mL). Initial structure–activity relationship (SAR) analysis showed that the heterocyclic piperidine group can influence the biological activities of the title compounds significantly. The fungicidal activity of compounds with piperidine is better than that of compounds without piperidine. The highly-active compound 3b, with its simple structure and easy synthetic route, is worthy to be further studied as a new lead fungicide.
Drinking mothers expose their fetuses to ethanol, which produces birth defects: craniofacial defects, cognitive impairment, sensorimotor disabilities and organ deformities, collectively termed as fetal alcohol spectrum disorder (FASD). Various congenital heart defects (CHDs) are present in FASD patients, but the mechanisms of alcohol-induced cardiogenesis defects are not completely understood. This study utilized zebrafish embryos and older larvae to understand FASD-associated CHDs. Ethanol-induced cardiac chamber defects initiated during embryonic cardiogenesis persisted in later zebrafish life. In addition, myocardial damage was recognizable in the ventricle of the larvae that were exposed to ethanol during embryogenesis. Our studies of the pathogenesis revealed that ethanol exposure delayed differentiation of first and second heart fields and reduced the number of early- and late-added cardiomyocytes in the heart. Ethanol exposure also reduced the number of endocardial cells. Together, this study showed that ethanol-induced heart defects were present in late-stage zebrafish larvae. Reduced numbers of cardiomyocytes partly accounts for the ethanol-induced zebrafish heart defects.
Lung cancer is the leading cause of cancer deaths worldwide, and this makes it an attractive disease to review and possibly improve therapeutic treatment options. Surgery, radiation, chemotherapy, targeted treatments, and immunotherapy separate or in combination are commonly used to treat lung cancer. However, these treatment types may cause different side effects, and chemotherapy-based regimens appear to have reached a therapeutic plateau. Hence, effective, better-tolerated treatments are needed to address and hopefully overcome this conundrum. Recent advances have enabled biologists to better investigate the potential use of natural compounds for the treatment or control of various cancerous diseases. For the past 30 years, natural compounds have been the pillar of chemotherapy. However, only a few compounds have been tested in cancerous patients and only partial evidence is available regarding their clinical effectiveness. Herein, we review the research on using current chemotherapy drugs and natural compounds (Wortmannin and Roscovitine, Cordyceps militaris, Resveratrol, OSU03013, Myricetin, Berberine, Antroquinonol) and the beneficial effects they have on various types of cancers including non-small cell lung cancer. Based on this literature review, we propose the use of these compounds along with chemotherapy drugs in patients with advanced and/or refractory solid tumours.
In vertebrates, carbonic anhydrases (CAs) play important roles in ion transport and pH regulation in many organs, including the eyes, kidneys, central nervous system, and inner ear. In aquatic organisms, the enzyme is inhibited by various chemicals present in the environment, such as heavy metals, pesticides, and pharmaceuticals. In this study, the effects of CA inhibitors, i.e., sulfonamides [ethoxyzolamide (EZA), acetazolamide (AZA), and dorzolamide (DZA)], on zebrafish embryogenesis were investigated. In embryos treated with the sulfonamides, abnormal development, such as smaller otoliths, an enlarged heart, an irregular pectoral fin, and aberrant swimming behavior, was observed. Especially, the development of otoliths and locomotor activity was severely affected by all the sulfonamides, and EZA was a consistently stronger inhibitor than AZA or DZA. In the embryos treated with EZA, inner ear hair cells containing several CA isoforms, which provide HCO3(-) to the endolymph for otolith calcification and maintain an appropriate pH there, were affected. Acridine orange/ethidium bromide staining indicated that the hair cell damage in the inner ear and pectral fin is due to apoptosis. Moreover, RNA measurement demonstrated that altered gene expression of cell cycle arrest- and apoptosis-related proteins p53, p21, p27, and Bcl-2 occurred even at 0.08 ppm with which normal development was observed. This finding suggests that a low concentration of EZA may affect embryogenesis via the apoptosis pathway. Thus, our findings demonstrated the importance of potential risk assessment of CA inhibition, especially regarding the formation of otoliths as a one of the most sensitive organs in embryogenesis.
In animals, hatching represents the transition point from a developing embryo to a free-living individual, the larva. This process is finely regulated by many endogenous and environmental factors and has been shown to be sensitive to a variety of chemical agents. It is commonly evaluated in bioassays in order to establish the effects of different agents on early development and reproductive capabilities in fish and other aquatic animals. In fish, the breakdown of the chorion is achieved by the secretion of choriolysin by hatching gland cells (HGCs) into the perivitelline space (PVS), coupled with spontaneous movements of the developing larva. In this work, we used zebrafish to assay the effects of a family of widely used agrochemicals—triazoles Triadimefon (FON), Triadimenol (NOL) and free triazole (1,2,4-T)—on hatching success. We found a strong inhibition of hatching by triazole exposure which was correlated with morphological changes and a reduction in the secretory function of the HGCs. As a consequence, the release of choriolytic enzymes by HGCs was reduced. We also found that HGC secretion reduction after exposure to FON can be rescued by co-incubation with a dopamine D2 receptor antagonist but not by antagonists of the D1-like receptors. This suggests a specific pathway through which this family of fungicides may be impairing a critical event in the fish life cycle.
Cancer is known as abnormal cell division and consisting of a group of diseases on various organ tissues. Many therapies are available in cancer treatment such as chemotherapy, radiotherapy etc. Without damaging normal tissue, there is a huge need for specified anticancer drugs which have effect only on abnormal cancer cells. Therefore, advances in anticancer drug discovery in treating cancer in the recent years, directed towards to the macromolecular targets. Heterocyclic molecules, such as fluconazole, acetazolamide, etc., have a significant role in health care and pharmaceutical drug design. Thiosemicarbazides (NH2-NH-CSNH2) are the simplest hydrazine derivatives of thiocarbamic acid and are not only transition compounds, but they are also very effective organic compounds. Thiosemicarbazides possess an amide and amine protons, carbonyl and thione carbons. These structures have attracted the attention of the researchers in the development of novel compounds with anticonvulsant, antiviral, anti-inflammatory, antibacterial, antimycobacterial, antifungal, antioxidant and anticancer activities. Recently, a number of thiosemicarbazides are available commercially as anticancer drugs for novel anticancer drug discovery. Antineoplastic or anticancer drugs prevent or inhibit the maturation and proliferation of neoplasms. These observations have been guiding the researchers for the development of new thiosemicarbazides that possess anticancer activity.
The developing fish heart is vulnerable to a diverse array of toxic chemical contaminants in freshwater, estuarine, and marine habitats. Globally occurring examples of cardiotoxic agents include dioxins, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs). The disruption of cardiac function during the process of heart morphogenesis can lead to adverse outcome pathways (AOPs) that can negatively affect fish survival at hatching as well as later life stages. Proximal impacts include cardiogenic fluid accumulation (edema) and defects of the body axis and jaw that preclude larval feeding. More subtle changes in heart development can produce permanent structural defects in the heart that reduce cardiac output and swimming performance in older fish. In recent decades, the presence of edema in fish embryos and larvae has been a very common bioindicator of cardiotoxicity. However, the different ways that edema forms in fish from different habitats (i.e., freshwater vs. marine, pelagic vs. demersal) has not been rigorously examined. Oil spills are an important source of PAHs in fish spawning areas worldwide, and research in this area is revealing how patterns of cardiogenic edema are shaped by species-specific differences in developmental anatomy and ionoregulatory physiology. Here we review the visible evidence for circulatory disruption across nine freshwater and marine fish species, exposed to crude oils from different parts of the world. We focus on the close interconnectedness of the cardiovascular and osmoregulatory systems during early development, and corresponding implications for fish in hyperosmotic and hyposmotic habitats. Finally, we suggest there may be poorly understood adverse outcomes pathways related to osmotic gradients and water movement within embryos, the latter causing extreme shifts in tissue osmolality.
In this study, the antibacterial, cytotoxic and antiproliferative activities of novel thiosemicarbazide derivatives were assessed. Our results demonstrated that some of the novel compounds possess good antibacterial properties against Staphylococcus epidermidis, Streptococcus mutans and Streptococcussanguinis and are only slightly cytotoxic; thus, they exhibit an excellent therapeutic index, which is higher than that of ethacridine lactate. Moreover, our data showed that compounds 2 and 4 have an antiproliferative activity against human breast adenocarcinoma and human hepatocellular carcinoma cell lines. We expect that the novel thiosemicarbazide derivatives can be used as agents for treatment of dental caries and also for chemotherapy support.
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The online version of this article (doi:10.1007/s00044-016-1599-6) contains supplementary material, which is available to authorized users.
Originally designed as an alternative for the acute fish toxicity test according to, e.g., OECD TG 203, the fish embryo test (FET) with the zebrafish (Danio rerio) has been optimized, standardized, and validated during an OECD validation study and adopted as OECD TG 236 as a test to assess toxicity of embryonic forms of fish. Given its excellent correlation with the acute fish toxicity test and the fact that non-feeding developmental stages of fish are not categorized as protected stages according to the new European Directive 2010/63/EU on the protection of animals used for scientific purposes, the FET is ready for use not only for range-finding but also as a true alternative for the acute fish toxicity test, as required for a multitude of national and international regulations. If-for ethical reasons-not accepted as a full alternative, the FET represents at least a refinement in the sense of the 3Rs principle. Objections to the use of the FET have mainly been based on the putative lack of biotransformation capacity and the assumption that highly lipophilic and/or high molecular weight substances might not have access to the embryo due to the protective role of the chorion. With respect to bioactivation, the only substance identified so far as not being activated in the zebrafish embryo is allyl alcohol; all other biotransformation processes that have been studied in more detail so far were found to be present, albeit, in some cases, at lower levels than in adult fish. With respect to larger molecules, the extension of the test duration to 96 h (i.e., beyond hatch) has-at least for the substances tested so far-compensated for the reduced access to the embryo; however, more research is necessary to fully explore the applicability of the FET to substances with a molecular weight >3 kDa as well as substances with a neurotoxic mode of action. An extension of the endpoints to also cover sublethal endpoints makes the FET a powerful tool for the detection of teratogenicity, dioxin-like activity, genotoxicity and mutagenicity, neurotoxicity, as well as various forms of endocrine disruption.
Drug-induced cardiotoxicity is a leading factor for drug withdrawals, and limits drug efficacy and clinical use. Therefore, new alternative animal models and methods for drug safety evaluation have been given great attention. Anthracyclines (ANTs) are widely prescribed anticancer agents that have a cumulative dose relationship with cardiotoxicity. We performed experiments to study the toxicity of ANTs in early developing zebrafish embryos, especially their effects on the heart. LC50 values for daunorubicin, pirarubicin, doxorubicin (DOX), epirubicin and DOX-liposome at 72 h post-fertilization were 122.7 μM, 111.9 μM, 31.2 μM, 108.3 μM and 55.8 μM, respectively. At the same time, zebrafish embryos were exposed to ANTs in three exposure stages and induced incomplete looping of the heart tube, pericardia edema and bradycardia in a dose-dependent manner, eventually leading to death. DOX caused the greatest heart defects in the treatment stages and its liposome reduced the effects on the heart, while daunorubicin produced the least toxicity. Genes and proteins related to heart development were also identified to be sensitive to ANT exposure and downregulated by ANTs. It revealed ANTs could disturb the heart formation and development. ANTs induced cardiotoxicity in zebrafish has similar effects in mammalian models, indicating that zebrafish may have a potential value for assessment of drug-induced developmental cardiotoxicity. Copyright © 2014 John Wiley & Sons, Ltd.
Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
Systematic toxicological study is still required to fully understand the hazard potentials of gold nanoparticles (AuNPs).
Because their biomedical applications are rapidly evolving, we investigated developmental toxicity of AuNPs in an in vivo embryonic zebrafish model at exposure concentration ranges from 0.08 to 50mg/l. Exposure of zebrafish embryos to 1.3nm AuNPs
functionalized with a cationic ligand, N,N,N-trimethylammoniumethanethiol (TMAT-AuNPs), resulted in smaller malpigmented eyes.
We determined that TMAT-AuNPs caused a significant increase of cell death in the eye, which was correlated with an increase
in gene expression of p53 and bax. Expression patterns of key transcription factors regulating eye development (pax6a, pax6b, otx2, and rx1) and pigmentation (sox10) were both repressed in a concentration-dependent manner in embryos exposed to TMAT-AuNPs. Reduced spatial localization of
pax6a, rx1, sox10, and mitfa was observed in embryos by whole-mount in situ hybridization. The swimming behavior of embryos exposed to sublethal concentrations of TMAT-AuNPs showed hypoactivity, and
embryos exhibited axonal growth inhibition. Overall, these results demonstrated that TMAT-AuNPs disrupt the progression of
eye development and pigmentation that continues to behavioral and neuronal damage in the developing zebrafish.
Abstract Prenatal alcohol exposure is known to have many profound detrimental effects on human fetal development (fetal alcohol spectrum disorders), which may manifest as lifelong disabilities. However, how alcohol affects the auditory/vestibular system is still largely unknown. This is the first study to investigate morphological effects of alcohol on the developing octavolateral system (the inner ear and lateral line) using the zebrafish, Danio rerio. Zebrafish embryos of 2 hours post fertilization (hpf) were treated in 2% alcohol for 48 hours and screened at 72 hpf for morphological defects of the inner ear and lateral line. Octavolateral organs from both alcohol-treated and control zebrafish were examined using light, confocal, and scanning electron microscopy. We observed several otolith phenotypes for alcohol-treated zebrafish including zero, one, two abnormal, two normal, and multiple otoliths. Results of this study show that alcohol treatment during early development impairs the inner ear (smaller ear, abnormal otoliths, and fewer sensory hair cells) and the lateral line (smaller neuromasts, fewer neuromasts and hair cells per neuromast, and shorter kinocilia of hair cells). Early embryonic alcohol exposure may also result in defects in hearing, balance, and hydrodynamic function of zebrafish.
The preliminary cytotoxic effect of 4-ethoxycarbonylmethyl-1-(piperidin-4-ylcarbonyl)-thiosemicarbazide hydrochloride (1)—a potent topoisomerase II inhibitor—was measured using a MTT assay. It was found that the compound decreased the number of viable cells in both estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231breast cancer cells, with IC50 values of 146 ± 2 and 132 ± 2 μM, respectively. To clarify the molecular basis of the inhibitory action of 1, molecular docking studies were carried out. The results suggest that 1 targets the ATP binding pocket.
Figure
4-ethoxycarbonylmethyl-1-(piperidin-4-ylcarbonyl)-thiosemicarbazide hydrochloride
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The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) assay is a classical method for screening cytotoxic anti-cancer agents. Candidate drugs from the MTT assay need in vivo models to test their efficiency and to assess the absorption, distribution, metabolism, excretion, and toxicity of the drugs. An in vivo screening model could increase the rate of development of anti-cancer drugs. Here, we used zebrafish to screen a library of 502 natural compounds and compared the results with those from an MTT assay of the MCF7 breast cancer cell line. We identified 59 toxic compounds in the zebrafish screen, 21 of which were also identified by the MTT assay, and 28 of which were already known for their anti-cancer and apoptosis-inducing effects. These compounds induced apoptosis and activated the p53 pathway in zebrafish within 3h treatment. Our results indicate that zebrafish is a simple, reliable and highly efficient in vivo tool for cancer drug screening, and could complement the MTT assay.
Halogenated dipeptides, 3, 5-di-I-tyrosylalanine (DIYA), have been identified as novel disinfection byproducts (DBPs), following chloramination of authentic water. However, little is known about their toxicity. Zebrafish embryos were used to assess the toxicity of novel iodinated DBPs (I-DBPs). Although DIYA did not exhibit high acute toxicity to embryonic zebrafish (LC50 > 2 mM), it significantly inhibited pigmentation of melanophores and xanthophores on head, trunk and tail at 500 μM as determined by photographic analysis. Whereas N-phenylthiourea (PTU) as a pigment inhibitor did not inhibit development of yellow pigments. Colorimetric detection of melanin further confirmed these results. Quantitative real time polymerase chain reaction (qRT-PCR) measurements indicated that genes (dct, slc24a5, tyr, tyrp1a, tyrp1b, silva) associated with the melanogenesis pathway were dramatically down-regulated following exposure to 500 μM DIYA. In addition, enzymatic activity of tyrosinase (TYR) decreased, also demonstrating that the underlying mechanism of hypopigmentation was attributed to the disruption of melanogenesis pathway. Transcription levels of xanthophore genes (gch2, bnc2, csf1a, csf1b, pax7a and pax7b) were also monitored by qRT-PCR assay. DIYA exposure up-regulated expression of gch2 and bnc2, but not csf1 and pax7. Tested DIYA analogues, brominated tyrosine was unlikely to inhibit pigmentation, indicating that the iodine substitution and dipeptides structure are of important structural feature for the inhibition of pigmentation. In this study, we observed that DIYA inhibited melanogenesis related genes, which might contribute to pigmentation defects. Moreover, as an emerging I-DBPs, the developmental toxicity of aromatic dipeptides should be further studied.
A major goal of translational toxicology is to identify adverse chemical effects and determine whether they are conserved or divergent across experimental systems. Translational toxicology encompasses assessment of chemical toxicity across multiple life stages, determination of toxic mode-of-action, computational prediction modeling, and identification of interventions that protect or restore health following toxic chemical exposures. The zebrafish is increasingly used in translational toxicology because it combines the genetic and physiological advantages of mammalian models with the higher-throughput capabilities and genetic manipulability of invertebrate models. Here, we review recent literature demonstrating the power of the zebrafish as a model for addressing all four activities of translational toxicology. Important data gaps and challenges associated with using zebrafish for translational toxicology are also discussed.
Sumithion, a synthetic organophosphate, is widely used as an agricultural insecticide and for control of tiger bug (Cicindela spp.) in larval rearing for aquaculture. An experiment was conducted to examine the effects of su-mithion on embryological and larval development of zebrafish Danio rerio. Fertilized egg (n = 100) and larvae (n = 100) were exposed to six concentrations of sumithion (0, 0.1, 0.2, 0.4, 0.8 and 1.6 mg L −1) in three replicates. LC 50 values for embryos and larvae were calculated by probit analysis. The 24 h LC 50 value of sumithion for embryo was 0.235 (0.079-0.428) mgL −1. Increasing sumithion concentrations decreased hatching success and increased embryonic mortality. In embryos, sumithion induced several malformations including immature yolk sac, dark yolk sac, yolk sac bud, broken eggshell and notochord, unhatched eggs. Larval LC 50 values at 24, 48 and 72 h of various doses of sumithion exposure were 0.620 (0.436-0.963), 0.475 (0.302-0.801) and 0.341 (0.177-0.617) mgL −1 , respectively. Various physical deformities, including edema, notochord deformity, yolk sac damage, body arcuation, lordosis and black pigmentation on the yolk sac were evident in response to different concentrations of sumithion. The results of the current study indicate that sumithion exerts developmental toxicity to zebrafish embryos and larvae. It is expected that current findings will increase sensitivity about the toxic effect of sumithion in early development, as well the possibility of similar actions induced by other insecticides and pesticides.
To screen the tens of thousands of chemicals for which no toxicity data currently exists, it is necessary to move from in vivo rodent models to alternative models, such as zebrafish. Here, we used dechorionated Tropical 5D wildtype zebrafish embryos to screen a 91-compound library provided by the National Toxicology Program (NTP) for developmental toxicity. This library contained 86 unique chemicals that included negative controls, flame retardants, polycyclic aromatic hydrocarbons (PAHs), drugs, industrial chemicals and pesticides. Fish were exposed to five concentrations of each chemical or an equal amount of vehicle (0.5% DMSO) in embryo medium from 6 h post-fertilization (hpf) to 5 d post-fertilization (dpf). Fish were examined daily for mortality and teratogenic effects and photomotor behavior was assessed at 4 and 5 dpf. Of the five negative control compounds in the library, none caused mortality/teratogenesis, but two altered behavior. Chemicals provided in duplicate produced similar outcomes. Overall, 13 compounds caused mortality/teratology but not behavioral abnormalities, 24 only affected behavior, and 18 altered both endpoints, with behavior affected at concentrations that did not cause mortality/teratology (55/86 hits). Of the compounds that affected behavior, 52% caused behavioral abnormalities at either 4 or 5 dpf. Compounds within the same functional group caused different behavioral abnormalities, while similar behavioral patterns were caused by compounds from different groups. Our data suggest that behavior is a sensitive endpoint for developmental toxicity screening that integrates multiple modes of toxic action and is influenced by the age of the larval fish at the time of testing.
Epilepsy is a neurological disorder treated with antiepileptic drugs (AEDs). Since AEDs are administered in women in childbearing age, it is critical to study if drugs are capable of inducing developmental toxicity. Along the bibliography available, there is no research comparing teratogenicity and anticonvulsant effect within the same study.
In the present study, we evaluated the teratogenic and anticonvulsant effects of six different AEDs: carbamazepine, levetiracetam, lamotrigine, phenobarbital, phenytoin and valproic acid. Zebrafish was the selected animal model because of its small size, rapid external development and similar neurophysiology to mammals.
Zebrafish embryo and larvae were exposed to AEDs. Embryo development was monitored by their hatching and morphology. In larvae, locomotor activity was measured as a parameter of neurotoxicity. Finally, anticonvulsant effect was determined after exposure to AEDs in zebrafish larvae treated with the proconvulsant drug pentylenetetrazole.
Our results suggest that lamotrigine and phenytoin could be suitable non-teratogenic and efficient anticonvulsant options for epilepsy treatment.
Cadmium (Cd) is a naturally occurring trace metal that is widely considered to be highly toxic to aquatic organisms and a significant health hazard to humans (Amzal et al., 2009; Bernhoft 2013; Burger, 2008; Satarug et al., 2009). The zebrafish (Danio rerio) has been used as a model organism for toxicological studies with Cd (Banni et al., 2011; Blechinger et al., 2007; Chow et al., 2009; Chow et al., 2008; Favorito et al., 2011; Kusch et al., 2007; Matz et al., 2007; Wang and Gallagher, 2013). We asked what the lasting longitudinal effects would be from short early developmental Cd exposure (between 24–96 hours post-fertilization) in a range that larvae might experience living atop typical Cd-containing surface sediments (0, 0.01, 0.1, 1.0 and 10 μM CdCl2: 1.124, 11.24, 112.4 and 1124 μg Cd/L). The goal of this exposure window was to specifically target secondary neurogenesis, monoaminergic differentiation and cardiovascular development, without affecting earlier patterning processes. Developmental abnormalities in body size and CNS morphology increased with concentration, but were statistically significant only at the highest concentration used (10 μM). Heart rate for Cd-treated larvae increased with concentration, and was significant even at the lowest concentration used (0.01 μM). Longitudinal survival was significantly lower for fish developmentally exposed to the highest concentration. Except for brain weight, overall morphology was not affected by developmental Cd exposure. However, developmental exposure to lower concentrations of Cd (0.01, 0.1, and 1.0 μM) progressively lowered cocaine-induced conditioned place preference (CPP), used to measure function of the reward pathways in the brain. Baseline heart rate was significantly lower in longitudinal fish developmentally exposed to 1.0 μM Cd. Cardiovascular response to isoproterenol, a potent ß-adrenergic agonist, in longitudinal adults was also significantly affected by developmental exposure to Cd at low doses (0.01, 0.1 and 1.0 μM). Surviving longitudinal adult fish exposed to the highest concentration of Cd showed normal CPP and cardiovascular physiology. The data imply that even lower exposure concentrations can potentially result in fitness-affecting parameters without affecting survival in a laboratory setting.
A series of thiosemicarbazides with 4-nitrophenyl group was obtained in the reaction of carboxylic acid hydrazides with isothiocyanates. All compounds were checked for their antibacterial and antiproliferative activity. Our results have shown that derivatives 6-8 possessed antibacterial activity against S. aureus, S. epidermidis, S. mutans and S. sanguinis, moderate cytotoxicity and good therapeutic safety in vitro. Additionally, compounds 1 and 4 significantly inhibited A549, HepG2 and MCF-7 cell division. Moreover, PASS software indicated that newly obtained compounds are potential α-glucosidase inhibitors. This was confirmed by in vitro studies. To investigate the mode of interaction with the molecular target compounds were docked to glucose binding site of the enzyme and exhibited a similar binding mode as glucose.
Single-wall carbon nanotubes functionalized with polyethylene glycol (SWCNT-PEG) are promising materials for biomedical applications such as diagnostic devices and controlled drug-release systems. However, several questions about their toxicological profile remain unanswered. Thus, the aim of this study was to investigate the action of SWCNT-PEG in Danio rerio zebrafish embryos at the molecular, physiological and morphological levels. The SWCNT used in this study were synthesized by the high-pressure carbon monoxide process, purified and then functionalized with distearoyl phosphatidylethanolamine block copolymer-PEG (molecular weight 2 kDa). The characterization process was carried out with low-resolution transmission electron microscopy, thermogravimetric analysis and Raman spectroscopy. Individual zebrafish embryos were exposed to the SWCNT-PEG. Toxic effects occurred only at the highest concentration tested (1 ppm) and included high mortality rates, delayed hatching and decreased total larval length. For all the concentrations tested, the alkaline comet assay revealed no genotoxicity, and Raman spectroscopy measurements on the histological slices revealed no intracellular nanotubes. The results shown here demonstrate that SWCNT-PEG has low toxicity in zebrafish embryos, but more studies are needed to understand what mechanisms are involved. However, the presence of residual metals is possibly among the primary mechanisms responsible for the toxic effects observed, because the purification process was not able to remove all metal contamination, as demonstrated by the thermogravimetric analysis. More attention must be given to the toxicity of these nanomaterials before they are used in biomedical applications. Copyright © 2016 John Wiley & Sons, Ltd.
Cadmium is known to cause developmental defects in a variety of vertebrate species, but relatively little is known about the underlying molecular mechanisms. In this study, we used zebrafish (Danio rerio) embryos as a model system to investigate cadmium-induced toxicities. Fertilized embryos collected at 5-h after fertilization were incubated for 18 h in culture media containing 1 to 1,000 muM CdCl2. The median embryolethal concentration (LC50) was 168 muM, whereas the median effect concentration (EC50) for total adverse effect (mortality and developmental defects) was 138 muM. Six major types of deformities were observed: head and eye hypoplasia, hypopigmentation, cardiac edema, yolk sac abnormalities, altered axial curvature, and tail malformations. The frequency of malformations increased with cadmium concentration. Somites of embryos with altered axial curvature were investigated using the antimyosin antibody MF-20. This study demonstrated, to our knowledge for the first time, reduced myotome formation in cadmium-induced spinal deformity. Embryos with head and eye hypoplasia were studied using the anti-neural tissue antibody zns-2, and a poorly developed central nervous system was revealed. Head and eye hypoplasia were associated with lack of expression of the sonic hedgehog gene, which controls the patterning of the neural tube and somites. Genes involved in tail formations, such as evenskipped 1 and no tail, were ectopically expressed in embryos with tail malformations. Our data support the hypothesis that fish embryonic malformations induced by cadmium might be mediated through ectopic expression of developmental regulatory genes.
P53 is a transcription factor highly inducible by many stress signals such as DNA damage, oncogene activation, and nutrient deprivation. Cell-cycle arrest and apoptosis are the most prominent outcomes of p53 activation. Many studies showed that p53 cell-cycle and apoptosis functions are important for preventing tumor development. p53 also regulates many cellular processes including metabolism, antioxidant response, and DNA repair. Emerging evidence suggests that these noncanonical p53 activities may also have potent antitumor effects within certain context. This review focuses on the cell-cycle arrest and apoptosis functions of p53, their roles in tumor suppression, and the regulation of cell fate decision after p53 activation. © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.
There have been tremendous development in the chemotherapy of cancer and researches are still developing new and more effective drugs to combat this disease. Thiosemicarbazides and thiosemicarbazone possess a wide range of biological applications. This key biological role is often related with their capability to inhibit the enzyme ribonucleotide reductase, similar to what is observed with potent anticancer drugs such as triapine and methisazone. Recent studies have revealed that thiosemicarbazones can inhibit topoisomerase II enzyme. This review discusses current advances of an emerging ‘new wave’ of thiosemicarbazide/thiosemicarbazone and their metal complexes as potent anticancer agents, mode of action and toxicity caused by them. © 2014, International Journal of Pharmacy and Pharmaceutical Sciences. All rights reserved.
Cadmium-caused head and eye hypoplasia and hypopigmentation has been recognized for a long time, but knowledge of the underlying mechanisms is limited. In this study, we found that high mortality occurred in exposed embryos after 24hpf, when cadmium (Cd) dosage was above 17.8μM. Using high-throughput in situ hybridization screening, we found that genes labelling the neural crest and its derivative pigment cells exhibited obviously reduced expression in Cd-exposed embryos from 24hpf, 2 days earlier than head and eye hypoplasia and hypopigmentation occurred. Moreover, based on expression of crestin, a neural crest marker, we found that embryos before the gastrula stage were more sensitive to cadmium toxicity and that damage caused by Cd on embryogenesis was dosage dependent. In addition, by phenotype observation and detection of neural crest and pigment cell markers, we found that BIO and retinoic acid (RA) could neutralize the toxic effects of Cd on zebrafish embryogenesis. In this study, we first determined that Cd blocked the formation of the neural crest and inhibited specification of pigment cells, which might contribute to the molecular mechanisms underlying the phenotype defects of head and eye hypoplasia and hypopigmentation in Cd-exposed embryos. Moreover, we found that compounds BIO or RA could neutralize the toxic effects of Cd.
Copyright © 2015 Elsevier B.V. All rights reserved.
We performed a combined study to determine the chronic toxicity and carcinogenicity of semicarbazide hydrochloride (SEM-HCl). Male and female Wistar Hannover GALAS rats were fed a diet containing SEM-HCl at 0, 10, 50, and 250 ppm for 52 weeks (10 rats/sex/group) or for 104 weeks (50 rats/sex/group). Enlargement of the knee joints was apparent in both sexes at 250 ppm. Reduced body weight was observed at 250 ppm from week 76 only in males. SEM-HCl exerted no toxic effects on hematology, serum biochemistry, or organ weights. Histopathologically, disarrangement of chondrocytes accompanied by increased connective tissues, and degeneration of articular cartilage were found in males at 50 ppm and above and in females at 250 ppm. Mild changes in the elastic laminae were observed at 250 ppm for both sexes in the chronic toxicity study. There were no significant intergroup differences in the incidences or types of any tumors. Taken together, toxicological effects of chronic exposure to SEM-HCI mainly occurred in the bone, cartilage, and aorta. Based on histopathological findings, the no-observed-adverse-effect-level was 10 ppm in males and 50 ppm in females (equal to 0.6 mg/kg/day in males and 3.9 mg/kg/day in females). SEM-HCl was not carcinogenic in rats.
Introduction:
Off-target effects represent one of the major concerns in the development of new pharmaceuticals, requiring large-scale animal toxicity testing. Faster, cheaper and more reliable assays based on zebrafish embryos (ZE) are being developed as major tools for assessing toxicity of chemicals during the drug-discovery process.
Areas covered:
This paper reviews techniques aimed to the analysis of in vivo sublethal toxic effects of drugs on major physiological functions, including the cardiovascular, nervous, neuromuscular, gastrointestinal and thyroid systems among others. Particular emphasis is placed on high-throughput screening techniques (HTS), including robotics, imaging technologies and image-analysis software.
Expert opinion:
The analysis of off-target effects of candidate drugs requires systemic analyses, as they often involve the complete organism rather than specific, tissue- or cell-specific targets. The unique physical and physiological characteristics of ZE make this system an essential tool for drug discovery and toxicity assessment. Different HTS methodologies applicable to ZE allow the screening of large numbers of different chemicals for many diverse and relevant toxic endpoints.
Cardiovascular toxicity is a major challenge for the pharmaceutical industry and predictive screening models to identify and eliminate pharmaceuticals with the potential to cause cardiovascular toxicity in humans are urgently needed. In this study, taking advantage of the transparency of larval zebrafish, Danio rerio, we assessed cardiovascular toxicity of seven known human cardiotoxic drugs (aspirin, clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride) and two non-cardiovascular toxicity drugs (gentamicin sulphate and tetracycline hydrochloride) in zebrafish using six specific phenotypic endpoints: heart rate, heart rhythm, pericardial edema, circulation, hemorrhage and thrombosis. All the tested drugs were delivered into zebrafish by direct soaking and yolk sac microinjection, respectively, and cardiovascular toxicity was quantitatively or qualitatively assessed at 4 and 24 h post drug treatment. The results showed that aspirin accelerated the zebrafish heart rate (tachycardia), whereas clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride induced bradycardia. Quinidine and terfenadine also caused atrioventricular (AV) block. Nimodipine treatment resulted in atrial arrest with much slower but regular ventricular heart beating. All the tested human cardiotoxic drugs also induced pericardial edema and circulatory disturbance in zebrafish. There was no sign of cardiovascular toxicity in zebrafish treated with non-cardiotoxic drugs gentamicin sulphate and tetracycline hydrochloride. The overall prediction success rate for cardiotoxic drugs and non-cardiotoxic drugs in zebrafish were 100% (9/9) as compared with human results, suggesting that zebrafish is an excellent animal model for rapid in vivo cardiovascular toxicity screening. The procedures we developed in this report for assessing cardiovascular toxicity in zebrafish were suitable for drugs delivered by either soaking or microinjection. Copyright © 2013 John Wiley & Sons, Ltd.
Cadmium is known to cause developmental defects in a varietyof vertebrate species, but relatively little is known about the underlying molecular mechanisms. In this study, we used zebrafish (Danio rerio) embryos as a model system to investigate cadmium-induced toxicities. Fertilized embryos collected at 5-h after fertilization were incubated for 18 h in culture media containing 1 to 1, 000 μM CdCl2. The median embryolethal concentration (LC50) was 168 μM, whereas the median effect concentration (EC50) for total adverse effect (mortality and developmental defects) was 138 μM. Six major types of deformities were observed: head and eye hypoplasia, hypopigmentation, cardiac edema, yolk sac abnormalities, altered axial curvature, and tail malformations. The frequency of malformations increased with cadmium concentration. Somites of embryos with altered axial curvature were investigated using the antimyosin antibody MF-20. This study demonstrated, to our knowledge for the first time, reduced myotome formation in cadmium-induced spinal deformity. Embryos with head and eye hypoplasia were studied using the anti-neural tissue antibody zns-2, and a poorly developed central nervous system was revealed. Head and eye hypoplasia were associated with lack of expression of the sonic hedgehog gene, which controls the patterning of the neural tube and somites. Genes involved in tail formations, such as evenskipped 1 and no tail, were ectopically expressed in embryos with tail malformations. Our data support the hypothesis that fish embryonic malformations induced by cadmium might be mediated through ectopic expression of developmental regulatory genes.
We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis—the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts. ©1995 Wiley-Liss, Inc.
A series of novel chalcone thiosemicarbazide derivatives (4a-4x) have been designed, synthesized, structurally determined, and their biological activities were also evaluated as potential EGFR kinase inhibitors. All the synthesized compounds are first reported. Among the compounds, compound 4r showed the most potent biological activity (IC(50) = 0.78 ± 0.05 μM for HepG2 and IC(50) = 0.35 μM for EGFR), which is comparable to the positive controls. Docking simulation was also performed to position compound 4r into the EGFR active site to determine the probable binding model. Antiproliferative assay results demonstrated that some of these compounds possessed good antiproliferative activity against HepG2. Compound 4r with potent inhibitory activity in tumor growth inhibition may be a potential anticancer agent.