Developmental toxicity assay using high content screening of zebrafish embryos

Typically, time‐consuming standard toxicological assays using the zebrafish (Danio rerio) embryo model evaluate mortality and teratogenicity after exposure during the first 2 days post‐fertilization. Here we describe an automated image‐based high content screening (HCS) assay to identify the teratogenic/embryotoxic potential of compounds in zebrafish embryos in vivo. Automated image acquisition was performed using a high content microscope system. Further automated analysis of embryo length, as a statistically quantifiable endpoint of toxicity, was performed on images post‐acquisition. The biological effects of ethanol, nicotine, ketamine, caffeine, dimethyl sulfoxide and temperature on zebrafish embryos were assessed. This automated developmental toxicity assay, based on a growth‐retardation endpoint should be suitable for evaluating the effects of potential teratogens and developmental toxicants in a high throughput manner. This approach can significantly expedite the screening of potential teratogens and developmental toxicants, thereby improving the current risk assessment process by decreasing analysis time and required resources. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

Long‐term exposure to bisphenol S damages the visual system and reduces the tracking capability of male zebrafish (Danio rerio)

Bisphenol S (BPS) is widely detected in aquatic environments and in human bodies. BPS has reproductive and thyroid disrupting effects, but its effect on the visual system remains unknown. In the present study, zebrafish embryos were exposed to BPS at concentrations of 1, 10, 100 and 1000 μg l^?1 until 120 days post‐fertilization in a semistatic system, and the effect of BPS on the visual behavior was examined using the optokinetic response and the optomotor response tests in male zebrafish. The retinal histology, mRNA expression of photoreceptor opsin genes (zfrho, zfblue, zfgr1, zfred and zfuv) and apoptosis‐related genes (bax and bcl‐2) were also assessed. Long‐term BPS exposure decreased the tracking capability of male zebrafish, consistent with structural damage to the retina. BPS induced different amounts of vacuoles in the retinal pigment epithelium, and 1000 μg l^?1 BPS exposure decreased the length of the inner plexiform layer, ganglion cell layer and retina, and induced an irregular arrangement of photoreceptor cells. The expression levels of the opsin genes (zfred, zfgr1 and zfrho) were significantly elevated, indicating an enhanced spectral sensitivity to red, green and dim light to compensate for the reduction of the optomotor response. Together, the results showed for the first time that long‐term exposure to BPS damaged the structure of male zebrafish retina and reduced their tracking capability.     

Hepatic oxidative stress,DNA damage and apoptosis in adult zebrafish following sub‐chronic exposure to BDE‐47 and BDE‐153

Polybrominated diphenyl ethers (PBDEs) are ubiquitous and prolific contaminant in both the abiotic and biotic environment because of the wide industrial applications of these chemicals. In the present study, the effects of 2,2′,4,4′‐tetrabrominateddiphenyl ether (BDE‐47) and 2,2′,4,4′,5,5′‐hexabromodiphenyl ether (BDE‐153) exposure on the induction of hepatic oxidative stress, DNA damage, and the expression of apoptosis‐related genes in adult zebrafish were investigated. The activities of antioxidant enzymes, such as catalase and superoxide dimutase, significantly increased when adult zebrafish was exposed to various concentrations of BDE‐47 and BDE‐153 for 7 and 15 days. BDE‐47 and BDE‐153 elicited significant alterations in zebrafish 7‐Ethoxyresorufin‐O‐deethylase activity at 3, 7, or 15 days of exposure. In addition, the significant increase in comet assay parameters of zebrafish hepatocytes in a concentration‐dependent manner indicated BDE‐47 and BDE‐153 induced DNA damage, probably due to observed oxidative stress. Furthermore, a monotonically upregulation of p53 and Caspase3, which are apoptotic‐regulated genes, and decreased expression ratio of the anti‐apoptotic B‐cell lymphoma/leukaemia‐2 and Bcl2‐associated X protein genes for all BDE‐47 and BDE‐153 treatments at 7 and 15 days indicated apoptosis induction in zebrafish liver. Our findings help elucidate the mechanisms of BDE‐47‐ and BDE‐153‐induced toxicity in zebrafish hepatocytes.     

Are zebrafish larvae suitable for assessing the hepatotoxicity potential of drug candidates?

Drug‐induced liver injury (DILI) is poorly predicted by single‐cell‐based assays, probably because of the lack of physiological interactions with other cells within the liver. An intact whole liver system such as one present in zebrafish larvae could provide added value in a screening strategy for DILI; however, the possible occurrence of other organ toxicities and the immature larval stage of the zebrafish might complicate accurate and fast analysis. We investigated whether expression analysis of liver‐specific fatty acid binding protein 10a (lfabp10a) was an appropriate endpoint for assessing hepatotoxic effects in zebrafish larvae. It was found that expression analysis of lfabp10a was a valid marker, as after treatment with hepatotoxicants, dose–response curves could be obtained and statistically significant abnormal lfabp10 expression levels correlated with hepatocellular histopathological changes in the liver. However, toxicity in other vital organs such as the heart could impact liver outgrowth and thus had to be assessed concurrently. Whether zebrafish larvae were suitable for assessing human relevant drug‐induced hepatotoxicity was assessed with hepatotoxicants and non‐hepatotoxicants that have been marketed for human use and classified according to their mechanism of toxicity. The zebrafish larva showed promising predictivity towards a number of mechanisms and was capable of distinguishing between hepatotoxic and non‐hepatotoxic chemical analogues, thus implying its applicability as a potential screening model for DILI. Copyright © 2015 John Wiley & Sons, Ltd.     

Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity and embryotoxicity using a training set of 27 compounds

To anticipate to increased testing needs for reproductive toxicity and 3R approaches, we studied zebrafish embryo/larva as an alternative for animal testing for developmental toxicity and embryotoxicity and evaluated a training set of 27 compounds with a standardized protocol. The classification of compounds in the zebrafish embryo/larva assay, based on a prediction model using a TI (teratogenic index) cut-off value of 2, was compared to available animal and human data. When comparing the classification of compounds in the zebrafish embryo/larva assay to available animal classification, a sensitivity of 72% and specificity of 100% were obtained. The predictive values obtained in comparison to a limited set of human data were 50, 60% respectively for teratogens, non-teratogens. Overall, we demonstrated that the zebrafish embryo/larva assay, may be used as screening tool for prioritization of compounds and could contribute to reduction of animal experiments in the field of developmental toxicology.     

Clinical trial: oral colon‐release parnaparin sodium tablets (CB‐01‐05 MMX®) for active left‐sided ulcerative colitis

Aliment Pharmacol Ther 31, 375‐386

Summary

Background The administration of parnaparin sodium as oral colon‐release tablets (CB‐01‐05 MMX^®) has been proposed as a novel approach for the treatment of ulcerative colitis (UC). Aim To assess the efficacy and the tolerability of 8 weeks’ oral daily administration of 210 mg of parnaparin sodium compared with placebo in subjects treated with stable‐doses of oral aminosalicylates. Methods This multicenter, randomized, double‐blind proof of concept trial compared the efficacy of CB‐01‐05 MMX^® 210 mg tablets to placebo in 141 subjects with mild to moderately active left‐sided UC treated with stable‐doses of aminosalicylates. The efficacy was assessed by clinical activity index (CAI), endoscopic index (EI) and histological score (HS). Results A total of 121 subjects (61 in test group and 60 in control group) formed the per protocol (PP) population. After 8 weeks of treatment, clinical remission was achieved in 83.6% of the CB‐01‐05 MMX^® group, and in 63.3% in the comparator group (P = 0.011). This effect was also significantly evident in the test group at week 4 (P = 0.028). A significant difference was also detected in rectal bleeding, (disappeared respectively in 75.4% and 55.0%; P = 0.018), and in mucosal friability (recovered respectively in 80.3% and in 56.7%; P = 0.005). Conclusions CB‐01‐05 MMX^® was safe and significantly effective in treating subjects with mild‐to‐moderate left‐sided UC treated with stable‐doses of aminosalicylates.     

A matrix‐assisted laser desorption ionization–time‐of‐flight–time‐of‐flight–mass spectrometry‐based toxicoproteomic screening method to assess in vitro particle potencies

Knowledge of biological reactivity and underlying toxicity mechanisms of airborne particulate matter (PM) is central to the characterization of the risk associated with these pollutants. An integrated screening platform consisting of protein profiling of cellular responses and cytotoxic analysis was developed in this study for the estimation of PM potencies. Mouse macrophage (J774A.1) and human lung epithelial cells (A549) were exposed in vitro to Ottawa urban particles (EHC6802) and two reference mineral particles (TiO₂ and SiO₂). Samples from the in vitro exposure experiment were tested following an integrated classical cytotoxicity/toxicoproteomic assessment approach for cellular viability (CellTiter Blue®, lactate dehydrogenase) and proteomic analyses. Cellular proteins were pre‐fractionated by molecular weight cut‐off filtration, digested enzymatically and were analyzed by matrix‐assisted laser desorption ionization–time‐of‐flight–time‐of‐flight–mass spectrometry for protein profiling and identification. Optimization of detergent removal, pre‐fractionation strategies and enzymatic digestion procedures led to increased tryptic peptide (m/z) signals with reduced sample processing times, for small total protein contents. Proteomic analyses using this optimized procedure identified statistically significant (P < 0.05) PM dose‐dependent changes at the molecular level. Ranking of PM potencies based on toxicoproteomic analysis were in line with classical cytotoxicity potency‐based ranking. The high content toxicoproteomic approach exhibited the potential to add value to risk characterization of environmental PM exposures by complementing and validating existing cytotoxicity testing strategies.     

Endocrine disruption effects of p,p′‐DDE on juvenile zebrafish

The persistent organic pollutant p,p′‐DDE, the major metabolite of the insecticide DDT, has displayed evidence of endocrine disruption through the inhibition of androgen binding to androgen receptors in different species. Although p,p′‐DDE was continuously detected in wild fish with abnormal gonad development such as intersex, little is known about its mode of action during gonad development in fish. To elucidate the potential endocrine effects of this pollutant in zebrafish (Danio rerio), juveniles (30 days post hatch) were exposed to p,p′‐DDE during the critical window of sexual differentiation. Fish were exposed to sublethal concentrations ranging from 0.01 to 20 µg l^–1 over 14 days and were maintained in control water for an additional 4 months. As core endpoints, the vitellogenin (vtg) concentration was measured at the end of exposure, and sex ratio and the gonadosomatic index were assessed 4 months after the end of exposure. An increase in vtg production in whole body homogenate was observed in fish exposed to 0.2 and 2.0 µg l^–1 p,p′‐DDE. No significant differences were displayed in morphological parameters such as the gonadosomatic index of males and females or sex ratio. However, exposed females presented histopathological changes that include the reduction of the number of mature oocytes, which might impair their successful reproduction. These results demonstrate the ability of p,p′‐DDE to cause endocrine disruption in zebrafish exposed during gonad differentiation of juvenile specimens. Furthermore, vtg induction by p,p′‐DDE in juvenile zebrafish arises as a predictive marker for adverse effects of this DDT metabolite on the ovarian function of female zebrafish. Copyright © 2014 John Wiley & Sons, Ltd.     

Inter‐laboratory validation of the modified murine local lymph node assay based on 5‐bromo‐2′‐deoxyuridine incorporation

The murine local lymph node assay (LLNA) is a well‐established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of a drug, cosmetic material, pesticide or industrial chemical. Instead of radioisotope using in this method, Takeyoshi M. et al. ( 2001 ) has developed a modified LLNA based on the 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation (LLNA:BrdU‐ELISA). The LLNA:BrdU‐ELISA is practically identical to the LLNA methodology excluding the use of BrdU, for which a single intraperitoneal injection of BrdU is made on day 4, and colorimetric detection of cell turnover. We conducted the validation study to evaluate the reliability and relevance of LLNA:BrdU‐ELISA. The experiment involved 7 laboratories, wherein 10 chemicals were examined under blinded conditions. In this study, 3 chemicals were examined in all laboratories and the remaining 7 were examined in 3 laboratories. The data were expressed as the BrdU incorporation using an ELISA method for each group, and the stimulation index (SI) for each chemical‐treated group was determined as the increase in the BrdU incorporation relative to the concurrent vehicle control group. An SI of 2 was set as the cut‐off value for exhibiting skin sensitization activity. The results obtained in the experiments conducted for all 10 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA:BrdU‐ELISA against those of GPMT/BT were 7/7 (100%), 3/3 (100%), and 10/10 (100%), respectively. Copyright © 2010 John Wiley & Sons, Ltd.     

Triptolide‐induced hepatotoxicity via apoptosis and autophagy in zebrafish

Previous research about the development of triptolide (TP) as a natural active compound has often focused on hepatotoxicity. Among its various mechanisms, autophagy and apoptosis are two important signaling pathways. In this study, we used zebrafish to establish a TP‐induced hepatotoxicity model, and investigated the roles of autophagy and apoptosis in the progress of liver injury. Zebrafish exposed to TP showed increased mortality and malformation because of the increased drug dose and duration of exposure. Meanwhile, we found that TP induced liver injury in a time‐ and dose‐dependent manner, which was observed as a reduction in liver area, slow yolk absorption, upregulation of transaminase and local neurosis. With the application of the high‐content imaging system (HCIS) technique in liver 3D imaging in vivo, clear imaging of the zebrafish liver was achieved. The results showed a decrease in volume and location of necrosis in the liver after TP exposure. Increased expression of inflammatory cytokines genes tumor necrosis factor (Tnf)α, Il1β and Il6 were shown, particularly Tnfα. The Fas‐Caspase8 signaling pathway was activated. The apoptosis‐related gene Bcl‐2 was increased, and Bax, Caspase9 and Caspase3 were increased. However, autophagy related genes Beclin1, Atg5, Atg3 and Lc3 were increased more significantly, and the changes of Beclin1 and Atg5 were the most severe. This study successfully established a TP‐induced zebrafish hepatotoxicity model and applied the HCIS technique in a zebrafish hepatotoxicity study. The result indicated Fas might be the main target of TP‐induced hepatotoxicity. Autophagy played a more important role than apoptosis and was characterized by the overexpression of Beclin1 and Atg5.     

Mortality,teratogenicity and growth inhibition of three glyphosate formulations using Frog Embryo Teratogenesis Assay‐Xenopus

Ample evidence around the world exists suggesting a link between exposure to glyphosate, toxicity and perturbed physiological functions in non‐target organisms. Although glyphosate formulations are widely used for weed and alien plant management, their ecotoxicological information remain scanty. Using the 96‐hour Frog Embryo Teratogenesis Assay‐Xenopus protocol, embryotoxicity and teratogenicity of three glyphosate‐based formulations were assessed. Embryos of Xenopus laevis were exposed to Roundup, Kilo Max and Enviro Glyphosate at concentration of 0.3‐1.3, 130‐280 and 320‐560 mg acid equivalent (a.e.)/L respectively. The results showed Roundup to be more toxic than the other formulations with a 96‐hour LC₅₀ of 1.05 mg a.e/L. compared with 207 mg a.e./L, and 466 mg a.e./L for Kilo Max and Enviro Glyphosate respectively. Although, both Roundup and Kilo Max formulations show inhibition on growth of the embryo‐larva (P ? .05), the minimum concentration inhibiting growth ratios of the three formulations was >0.30 baseline, indicating no significant growth inhibiting effect in the formulations. For teratogenicity, Roundup and Enviro Glyphosate formulations exhibited increasing teratogenic traces, with the teratogenic index at 1.7 and 1.6 respectively. Kilo Max formulation shows low teratogenicity with the teratogenic index at 1.4. Characteristic malformation induced by these formulations included generalized edema, cardiac and abdominal edema, improper gut formation and axial malformations. This study confirms that these formulations could be a potential physiological and ecological health disruptor, particularly concerning teratogenicity and growth disruption. Further studies to characterize the contributions of their surfactants will be invaluable.     

High‐Content Analysis of Cancer‐Cell‐Specific Apoptosis and Inhibition of in Vivo Angiogenesis by Synthetic (−)‐Pironetin and Analogs

The natural product (?)‐pironetin is a structurally simple small molecule microtubule‐perturbing agent whose biological activities appear to be exquisitely dependent on defined stereochemistry and the presence of an eletrophilic α,β‐unsaturated lactone moiety. We used alkaloid‐catalyzed acyl halide‐aldehyde cyclocondensation reactions in asymmetric total syntheses of (?)‐pironetin and three synthetic analogs, and evaluated their biological activities by high‐content analysis in cell culture and in a zebrafish model. Synthetic (?)‐pironetin and 2,3‐dihydro‐3‐hydroxypironetin caused mitotic arrest and programmed cell death in human lung cancer cells but not in normal lung fibroblasts, had nanomolar growth inhibitory activity in multi‐drug resistant cells, and inhibited neovascularization in zebrafish embryos. Synthetic (?)‐pironetin delayed the onset but increased the extent of tubulin assembly in vitro. The data illustrate the power of acyl halide‐aldehyde cyclocondensation to generate biologically active synthetic analogs of stereochemically complex targets and suggest that (?)‐pironetin and 2,3‐dihydro‐3‐hydroxypironetin possess unique properties that may bestow them with advantages over existing microtubule‐perturbing agents in the context of a whole organism or under conditions of multi‐drug resistance.     

High‐Content Analysis in Toxicology: Screening Substances for Human Toxicity Potential,Elucidating Subcellular Mechanisms and In Vivo Use as Translational Safety Biomarkers

High‐content analysis (HCA) of in vitro biochemical and morphological effects of classic (small molecule) drugs and chemicals is concordant with potential for human toxicity. For hepatotoxicity, concordance is greater for cytotoxic effects assessed by HCA than for conventional cytotoxicity tests and for regulatory animal toxicity studies. Additionally, HCA identifies chronic toxicity potential, and drugs producing idiosyncratic adverse reactions and/or toxic metabolites are also identified by HCA. Mechanistic information on the subcellular basis for the toxicity is frequently identified, including various mitochondrial effects, oxidative stress, calcium dyshomeostasis, phospholipidosis, apoptosis and antiproliferative effects, and a fingerprinting of the sequence and pattern of subcellular events. As these effects are frequently non‐specific and affect many cell types, some toxicities may be detected and monitored by HCA of peripheral blood cells, such as for anticancer and anti‐infective drugs. Critical methodological and interpretive features are identified that are critical to the effectiveness of the HCA cytotoxicity assessment, including the need for multiple days of exposure of cells to drug, use of a human hepatocyte cell line with metabolic competence, assessment of multiple pre‐lethal effects in individual live cells, consideration of hormesis, the need for interpretation of relevance of cytotoxicity concentration compared to efficacy concentration and quality management. Limitations of the HCA include assessment of drugs that act on receptors, transporters or processes not found in hepatocytes. HCA may be used in a) screening lead candidates for potential human toxicity in drug discovery alongside of in vitro assessment of efficacy and pharmacokinetics, b) elucidating mechanisms of toxicity and c) monitoring in vivo toxicity of drugs with known toxicity of known mechanism.     

Estrogenic effects of fluorinated diiodine alkanes in MCF‐7 cells,H295R cells and zebrafish embryo assays

Fluorinated diiodine alkanes (FDIAs), important industrial intermediates in the synthesis of various perfluorinated compounds, which are distributed widely in wildlife and humans. Recent studies showed that FDIAs had in vitro estrogenic effects. However, to date, little information is available regarding the in vivo estrogenic effects of FDIAs and the mechanisms are unclear. In this study, a combination of in vitro and in vivo assays was used to investigate the estrogenic effects of FDIAs. We tested the in vitro estrogenic effects and estrogen receptor‐related gene expression via MCF‐7 cell assay. The hormone level of estradiol and the expression of estrogenic synthesis genes were measured in the H295R cell assay. Finally, the in vivo effects of FDIAs on development and estrogen‐related gene expression were assessed in the zebrafish embryos assay. The results demonstrated that FDIAs could exhibit estrogenic activity through inducing cell proliferation (1.6‐6.7‐fold of the control) and estrogen receptor alpha gene expression (1.07‐1.39‐fold of the control), altering estradiol production (1.14‐1.22‐fold of the control) and the major estrogenic synthesis gene expression of CYP19 (1.22‐1.31‐fold of the control), disrupting the estrogen‐related genes (esr1 and cyp19b) levels in zebrafish (1.52‐2.99‐fold and 2.95‐5.00‐fold of the control for esr1 and cyp19b, respectively). The current findings indicated the potential estrogenic effects of FDIAs and provided novel information for human risk assessment.     

Developmental toxicity and DNA damage to zebrafish induced by perfluorooctane sulfonate in the presence of ZnO nanoparticles

Perfluorooctane sulfonate (PFOS) and ZnO nanoparticles (ZnO–NPs) are frequently detected in the environment, but few studies have assessed their joint toxicity. In this study, the acute toxicity and chronic toxicity to zebrafish (Danio rerio) induced by PFOS in the presence of ZnO–NPs were investigated, including developmental toxicity and DNA damage. The embryos were exposed to PFOS (only) (0.4, 0.8, and 1.6 mg/L) and PFOS plus ZnO–NPs (0.4 + 50, 0.8 + 50, and 1.6 + 50 mg/L) solutions to evaluate mortality (96 h), body length (96 h), hatch rate (72 h), heart rate (48 h),and malformation rate (96 h). The results revealed that the co‐treatment could cause more severe developmental toxicity compared with the control and single‐treatments, and the toxic effects generally increased in a dose–response manner. In addition, adult zebrafish were exposed to single and mixed solutions of PFOS and ZnO–NPs (at the concentrations mentioned above) for 30 days. DNA damage to zebrafish was evaluated by the comet assay and micronucleus test. We found that the PFOS single‐treatment at all doses (0.4, 0.8, and 1.6 mg/L) could strongly induce DNA damage to peripheral blood cells and that ZnO–NPs could aggravate the formation of DNA damage in co‐treatments. Histological examination of liver, testicle, and ovary showed that the presence of ZnO–NPs (50 mg/L) could also cause more serious histological damage to adult zebrafish than PFOS alone. As a result, the synergistic effects played an important role during joint exposure. Our observations provide a basic understanding of the joint toxicity of PFOS and ZnO–NPs to aquatic organisms. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 360–371, 2016.     

Anti‐doping analytes in serum: A comparison of SST and SST‐II Advance blood collection tubes

Serum insulin‐like growth factor‐1 (IGF‐1), procollagen type III N‐terminal peptide (PIIINP), and human growth hormone (hGH) isoforms were analyzed in identical serum samples collected into BD Vacutainer^® SST and BD Vacutainer^® SST‐II Advance serum separator tubes. Comparing the serum collected into each tube, measurement correlation was high (R² > 0.83) and percent bias was minimal (<|3.2%|) for all analytes measured using World Anti‐Doping Agency (WADA)‐approved tests. As such, it is recommended that both SST and SST‐II Advance tubes can be used interchangeably for anti‐doping purposes.     

Zebrafish larva as a reliable model for in vivo assessment of membrane remodeling involvement in the hepatotoxicity of chemical agents

The easy‐to‐use in vivo model, zebrafish larva, is being increasingly used to screen chemical‐induced hepatotoxicity, with a good predictivity for various mechanisms of liver injury. However, nothing is known about its applicability in exploring the mechanism called membrane remodeling, depicted as changes in membrane fluidity or lipid raft properties. The aim of this study was, therefore, to substantiate the zebrafish larva as a suitable in vivo model in this context. Ethanol was chosen as a prototype toxicant because it is largely described, both in hepatocyte cultures and in rodents, as capable of inducing a membrane remodeling leading to hepatocyte death and liver injury. The zebrafish larva model was demonstrated to be fully relevant as membrane remodeling was maintained even after a 1‐week exposure without any adaptation as usually reported in rodents and hepatocyte cultures. It was also proven to exhibit a high sensitivity as it discriminated various levels of cytotoxicity depending on the extent of changes in membrane remodeling. In this context, its sensitivity appeared higher than that of WIF‐B9 hepatic cells, which is suited for analyzing this kind of hepatotoxicity. Finally, the protection afforded by a membrane stabilizer, ursodeoxycholic acid (UDCA), or by a lipid raft disrupter, pravastatin, definitely validated zebrafish larva as a reliable model to quickly assess membrane remodeling involvement in chemical‐induced hepatotoxicity. In conclusion, this model, compatible with a high throughput screening, might be adapted to seek hepatotoxicants via membrane remodeling, and also drugs targeting membrane features to propose new preventive or therapeutic strategies in chemical‐induced liver diseases. Copyright © 2016 John Wiley & Sons, Ltd.     

A novel screening method for transition metal‐based anticancer compounds using zebrafish embryo‐larval assay and inductively coupled plasma‐mass spectrometry analysis

As novel metallodrugs continue to emerge, they are evaluated using models, including zebrafish, that offer unique sublethal endpoints. Testing metal‐based anticancer compounds with high‐throughput zebrafish toxicological assays requires analytical methods with the sensitivity to detect these sublethal tissue doses in very small sample masses (e.g., egg mass 100 μg). A robust bioanalytical model, zebrafish embryos coupled with inductively coupled plasma‐mass spectrometry (ICPMS) for measurement of delivered dose, creates a very effective means for screening metal‐based chemotherapeutic agents. In this study, we used ICPMS quantitation with the zebrafish embryo assays to detect metal equivalents at multiple response endpoints for two compounds, the chemotherapeutic agent cisplatin and ruthenium (Ru)‐based prospective metallodrug, PMC79. We hypothesized that cisplatin and PMC79 have different mechanisms for inducing apoptosis and result in similar lesions but different potencies following water‐borne exposure. An ICPMS method was developed to detect the metal in waterborne solution and tissue (detection limit: 5 parts per trillion for Ru or platinum [Pt]). The Ru‐based compound was more potent (LC₅₀: 7.8 μm ) than cisplatin (LC₅₀: 158 μm ) and induced disparate lesions. Lethality from cisplatin exposure exhibited a threshold (values >15 mg/L) while no threshold was observed for delayed hatching (lowest observed adverse effect level 3.75 mg/L cisplatin; 8.7 Pt (ng)/organism). The Ru organometallic did not have a threshold for lethality. Cisplatin‐induced delayed hatching was investigated further by larval‐Pt distribution and preferentially distributed to the chorion. We propose that zebrafish embryo‐larval assays coupled with ICPMS serve as a powerful platform to evaluate relative potency and toxic effects of metallodrug candidates.     

Divergent solid‐phase synthesis and candidacidal activity of MUC7 D1, a 51‐residue histidine‐rich N‐terminal domain of human salivary mucin MUC7

Abstract: Domain 1 of the low‐molecular‐weight human salivary mucin, designated MUC7 D1, spans the 51 N‐terminal amino acid residues. This domain contains a 15‐residue basic histidine‐rich subdomain (R3‐Q17) which has 53% sequence similarity to histatin 5 (Hsn‐5), a salivary molecule known to exert potent in vitro cidal activity against Candida albicans and many other medically important fungi. The MUC7 D1‐15mer and its derivatives have previously been synthesized in our laboratory and their candidacidal activities have been found to be inferior to that of Hsn‐5. We were therefore intrigued to explore the candidacidal potency of the full‐length MUC7 D1 (51‐mer). Linear solid‐phase synthesis of this domain has been accomplished following standard Fmoc chemistry. The problems of partial coupling, owing to the peptide chain length, at several stages of the solid‐phase step‐by‐step synthesis were circumvented either by double‐coupling techniques or efficient coupling procedures. The MUC7 D1 peptide was purified to homogeneity by conventional reverse‐phase HPLC using two columns connected in series. Secondary structure of the purified peptide was assessed by circular dichroism (CD) spectroscopy in phosphate buffer and trifluoroethanol and compared to that of MUC7 D1‐15mer and Hsn‐5. The MUC7 D1 candidacidal activity was assessed against azole‐sensitive and azole‐resistant C. albicans strains and was found, unlike that of the MUC7 D1‐15mer, to be comparable with that of Hsn‐5, indicating that in addition to Hsn‐5, MUC7 D1 could provide an attractive alternative to the classical antifungal agents. The candidacidal potency of MUC7 D1, like that of MUC7 D1‐15mer, and of Hsn‐5, appears to be largely dependent on peptide charge, irrespective of α‐helical structure.