Use of the Allium cepa Model to Assess the Cytogenotoxicity of Luffariella herdmani Marine Sponge Extract.

Marine sponge extracts are known to contain potentially toxic compounds that have biological activities of possible pharmacological interest. Thus, it is vital that biological models are used for the preliminary toxicity screening of such extracts. The present study reports the use of Allium cepa, a low-cost plant-based in vivo model, to assess the cytotoxicity and genotoxicity of Luffariella herdmani marine sponge crude extract (SCE). Pre-germinated onion bulbs, exposed for 96 hours to different concentrations of SCE (ranging from 0.3125 to 20 µg/ml), were used to determine general cytotoxicity. Root length as well as morphological abnormalities were recorded. Genotoxicity was assessed by exposing the root tips to SCE (0.3125-20 µg/ml) and the appropriate controls for 48 hours, and then staining with acetocarmine. The Mitotic Index (MI), Mitotic Phase Indices (MPIs) and chromosomal aberrations were evaluated and recorded. SCE inhibited A. cepa root growth (EC50 = 10.34 µg/ml) and elicited a mitodepressive effect (LC50 = 1.95 µg/ml) in a dose-dependent and significant manner. In addition, macroscopic alterations as well as chromosomal aberrations were detected. Overall, our findings indicate that L. herdmani crude extract exhibits cytotoxic and genotoxic activity, suggesting that it might contain substances with anti-proliferative/anticancer potential that could be subject to further characterisation.

Cytogenotoxicity evaluation of a heavy metal mixture, detected in a polluted urban wetland: Micronucleus and comet induction in the Indian green frog (Euphlyctis hexadactylus) erythrocytes and the Allium cepa bioassay.

Heavy metal contamination in wetland ecosystems is a serious environmental and health concern. This study evaluated the cytogenotoxicity of a previously evidenced heavy metal contamination (Cd, Cr, Cu, Pb and Zn ∼5 ppm each) in a polluted urban wetland, the Bellanwila-Attidiya sanctuary (BAS) in Sri Lanka, using a battery of cytogenotoxic assays. Micronucleus and comet assays evaluated the genotoxicity in erythrocytes of a common amphibian, the Indian green frog (Euphlyctis hexadactylus), under natural metal exposure in the wetland, and in vitro exposure, respectively.The Allium cepa bioassay assessed the cytogenotoxicity of the heavy metal mixture and of the individual metals, under laboratory exposure. Although in vivo natural exposure showed no significant induction of micronuclei in frog erythrocytes (P > 0.1), a significant and dose dependent elevation of comets was evident with in vitro exposure to the metal mixture (P < 0.001). Field controls did not show significant impacts in the A. cepa bioassay, whereas individual exposure to heavy metals reported lower effects than their combined exposure under laboratory conditions; Pb2+was the most toxic metal, with the highest mitotic inhibition (Pb2+>Cd2+>Zn2+>Cr6 >Cu2+), mutagenic potential as evaluated in the percentage incidence of chromosomal aberrations (Pb2+> Zn2+> Cu2+> Cr6+> Cd2+) and cytotoxicity evaluated by the incidence of cell apoptosis and necrosis (Pb2+>Cr6+>Cu2+>Cd2+>Zn2+). Thus, the test battery of micronucleus, comet and A. cepa assays that reveal differential aspects of cytogenotoxicity may serve as a valuable tool in environmental monitoring, primarily to screen for complex environmental mixtures of heavy metals that may impact ecological health.

Water hardness influenced variations in reproductive potential of two freshwater fish species; Poecilia reticulata and Betta splendens.

OBJECTIVE: Hardness of water in the form of CaCO3 affects reproductive potential in various fish species, differently. This study evaluates the effect of water hardness on growth and reproduction of two aquarium fishes, Poecilia reticulata (Ovo-viviparous sp.) and Betta splendens (Oviparous sp.) by growing them under 150 (control), 320, 540 and 900 ppm CaCO3 levels in semi natural aquaria. RESULTS: Growth increased with increasing water hardness, reporting a significant progress of P. reticulata (p = 0.005) at 900 ppm. Similarly, the reproductive potential of P. reticulata was improved significantly, recording the highest fecundity (16.22 ± 3.90) and Gonadosomatic Index (GSI-2.48 ± 0.6) at 900 ppm. However, in B. splendens water hardness adversely affected the reproduction by resulting a significantly low hatchability and disturbed bubble nests at 900 ppm, compared to the largest bubble nest formed at the control condition (108.58 ± 16.19 cm2). Thus, the study revealed differential effects of water hardness on reproductive potential of the test species, by increasing the potential of P. reticulata while decreasing that of B. splendens. Though larval survival was affected in both species, larval growth was improved significantly in P. reticulata at 900 ppm level. Understanding reproductive potential of aquarium fishes in natural waters is crucial for their management purposes.

Toxicity Assessment of Herbal Medicine Using Zebrafish Embryos: A Systematic Review.

Herbal remedies have been practiced by humans over centuries and therefore possess time-proven safety. However, it is imperative to evaluate the toxic effects of herbal medicine to confirm their safety, particularly when developing therapeutic leads. Use of laboratory animals such as rats, mice, and rabbits was considered as gold standard in herbal toxicity assessments. However, in the last few decades, the ethical consideration of using higher vertebrates for toxicity testing has become more contentious. Thus, possible alternative models entailing lower vertebrates such as zebrafish were introduced. The zebrafish embryotoxicity model is at the forefront of toxicology assessment due to the transparent nature of embryos, low cost, short cycle, higher fecundity, and genetic redundancy to the humans. Recently, its application has been extended to herbal toxicology. The present review intends to provide a comprehensive assembly of studies that applied the zebrafish embryo model for the assessment of herbal toxicity. A systematic literature survey was carried out in popular scientific databases. The literature search identified a total of 1014 articles in PubMed = 12, Scopus SciVerse® = 623, and Google Scholar = 1000. After screening, 25 articles were included in this review, and they were categorized into three groups in which the zebrafish embryotoxicity assay has been applied to investigate the toxicity of (1) polyherbal formulae/medical prescription (2 full texts), (2) crude extracts (12 full texts), and (3) phytocompounds/isolated constituents (11 full texts). These studies have investigated the toxicity of 6 polyherbal formulae, 16 crude extracts, and more than 30 phytocompounds/isolated constituents using the zebrafish embryotoxicity model. Moreover, this model has explicated the teratogenic effects and specific organ toxicities such as the kidney, heart, and liver. Furthermore, in some studies, the molecular mechanisms underlying the toxicity of herbal medicine have been elucidated. This comprehensive collection of scientific data solidifies the zebrafish embryo model as an effective model system for studying toxicological effects of a broad spectrum of herbal remedies. Henceforth, it provides a novel insight into the toxicity assessment of herbal medicine.

Potential Use of Euphorbia hirta for Dengue: A Systematic Review of Scientific Evidence.

Euphorbia hirta commonly known as Tawa-Tawa is a plant used in folklore medicine in the Philippines for the treatment of dengue. Though, E. hirta has been extensively investigated for numerous bioactivities, limited studies have been conducted on the antidengue activity. Thus, the present study provides a comprehensive review of studies conducted on the antidengue activity of E. hirta. A systematic literature survey was carried out in scientific databases, PubMed®, Scopus, and Google Scholar, for research carried on the antidengue activity of E. hirta. The literature search identified a total of 867 articles: databases PubMed = 6, Scopus SciVerse® = 423, and Google Scholar = 437; one additional article was identified by searching reference lists. Eight full papers were entitled to the review; out of those, two studies focused on ethnobotanical surveys, three on animal experiments, one on human trial, and two on in vitro antiviral activities, and one was computational study. The available evidence conclusively demonstrates the potential of E. hirta against dengue as it holds significant antiviral and platelet increasing activities. However, the number of studies conducted to validate its antidengue activity was found to be inadequate. Hence, well-controlled clinical trials and contemporary pharmacological approaches including activity guided fractionation and elucidation of the mode of action are encouraged to establish the use of E. hirta for dengue.

Heavy metal mediated innate immune responses of the Indian green frog, Euphlyctis hexadactylus (Anura: Ranidae): Cellular profiles and associated Th1 skewed cytokine response.

Immune cell and cytokine profiles in relation to metal exposure though much studied in mammals has not been adequately investigated in amphibians, due mainly to lack of suitable reagents for cytokine profiling in non-model species. However, interspecies cross reactivity of cytokines permitted us to assay levels of IFNγ, TNFα, IL6 and IL10in a common anuran, the Indian green frog (Euphlyctis hexadactylus), exposed to heavy metals (Cd, Cr, Cu, Zn and Pb, at ~5ppm each) under field and laboratory settings in Sri Lanka. Enumeration of immune cells in blood and melanomacrophages in the liver, assay of serum and hepatic cytokines, and Th1/Th2 cytokine polarisation were investigated. Immune cell counts indicated overall immunosuppression with decreasing total WBC and splenocyte counts while neutrophil/lymphocyte ratio increased with metal exposure, indicating metal mediated stress. Serum IL6 levels of metal exposed frogs reported the highest (~9360pg/mL) of all cytokines tested. Significantly elevated IFNγ production (P<0.05) was evident in heavy metal exposed frogs. Th1/Th2 cytokine ratio in both serum and liver tissue homogenates was Th1 skewed due to significantly higher production of pro-inflammatory cytokines, IFNγ in serum and TNFα in the liver (P<0.01).Metal mediated aggregations of melanomacrophages in the liver were positively and significantly (P<0.05) correlated with the hepatic expression of TNFα, IL6 and IL10 activity. Overall, Th1 skewed response may well be due to oxidative stress mediated nuclear factor κ-light chain enhancer of activated B cells (NFκB) which enhances the transcription of pro-inflammatory cytokines. Xenobiotic stress has recently imposed an unprecedented level of threat to wildlife, particularly to sensitive species such as amphibians. Therefore, understanding the interactions between physiological stress and related immune responses is fundamental to conserve these environmental sentinels in the face of emerging eco-challenges.

Combined Effects of Pesticides and Trematode Infections on Hourglass Tree Frog Polypedates cruciger.

The impact of widespread and common environmental factors, such as chemical contaminants, on infectious disease risk in amphibians is particularly important because both chemical contaminants and infectious disease have been implicated in worldwide amphibian declines. Here we report on the lone and combined effects of exposure to parasitic cercariae (larval stage) of the digenetic trematode, Acanthostomum burminis, and four commonly used pesticides (insecticides: chlorpyrifos, dimethoate; herbicides: glyphosate, propanil) at ecologically relevant concentrations on the survival, growth, and development of the common hourglass tree frog, Polypedates cruciger Blyth 1852. There was no evidence of any pesticide-induced mortality on cercariae because all the cercariae successfully penetrated each tadpole host regardless of pesticide treatment. In isolation, both cercarial and pesticide exposure significantly decreased frog survival, development, and growth, and increased developmental malformations, such as scoliosis, kyphosis, and also edema and skin ulcers. The combination of cercariae and pesticides generally posed greater risk to frogs than either factor alone by decreasing survival or growth or increasing time to metamorphosis or malformations. The exception was that lone exposure to chlorpyrifos had higher mortality without than with cercariae. Consistent with mathematical models that suggest that stress should increase the impact of generalist parasites, the weight of the evidence from the field and laboratory suggests that ecologically relevant concentrations of agrochemicals generally increase the threat that trematodes pose to amphibians, highlighting the importance of elucidating interactions between anthropogenic activities and infectious disease in taxa of conservation concern.

Malformations and mortality in the Asian Common Toad induced by exposure to pleurolophocercous cercariae (Trematoda: Cryptogonimidae).

Malformations and increased mortality due to infection by the digenetic trematode, Riberioa ondatrae have been reported for many species of amphibians. Severe malformations have also been reported in the Common Hourglass Tree Frog, Polypedates cruciger induced by pleurolophocercous cercariae in Sri Lanka in addition to the changes in the behaviour, development and survival of the host. We exposed pre-limb bud stage tadpoles (Gosner stages 25-26) of the Asian Common Toad, Duttaphrynus melanostictus to the same pleurolophocercous type cercariae under laboratory conditions. Molecular and morphological identification showed that these cercariae belonged Acanthostomum burminis infecting freshwater snakes as definitive hosts. These cercariae induced malformations (27.8%) and reduced survival to metamorphosis (53.8%). The magnitude of the effects increased with the dose of cercariae. Types of malformations were mainly axial, such as scoliosis and kyphosis. Severe limb malformations such as extra or missing limbs as reported for amphibians exposed to R. ondatrae were not observed in the D. melanostictus. Same authors reported a higher percentage of malformations previously when P. cruciger was exposed to the cercariae A. burminis compared to D. melanostictus. However, tadpoles of D. melanostictus, which are smaller compared to those of P. cruciger, experienced higher mortality than P. cruciger tadpoles. Trematode induced malformations and mortality in amphibians are highly variable and depend on multiple factors such as host species differences such as resistance to infection and tolerance, life-history characteristics such as size at metamorphosis and length of the metamorphosis period, and other factors such as size of the amphibian at the time of trematode exposure.